SESSION 1 JOINT OPENING SESSION

C1 END OF LIFE DECISIONS: THE PALLIATIVE CARE VIEWPOINT

FINLAY IG^1,2

¹Cardiff University, Cardiff, United Kingdom,²Velindre Cancer Centre, Cardiff, United Kingdom,³House of Lords, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: quality of life, symptom control, patient-centred

Palliative care for patients with neurological disease begins at the time of diagnosis; cure is not an option. Patients are in turmoil, facing their world disintegrating in front of them. Care needs to focus around the physical, improving their environment and controlling symptoms, as well as focusing on the emotional adjustment and on social and spiritual concerns. Patients will be grieving for their lost hopes and fears, for the life that they realise that they will not be able to enjoy and for unfulfilled dreams.

At the same time, the family needs support to come to terms with the difficult situation that they face. Family members are often the key carers, fearful of what lies ahead, not knowing who to call and fearing that they will be abandoned in their hour of greatest need.

Decision making in palliative care has to always way up the benefits against the risks and burdens to the patient in the broader context of their social environment as well as in the wider context of any co-morbidity. Improving quality-of-life requires an adjustment of hopes and expectations so that they are more achievable within the reality of disease, simultaneously with an improvement in all aspects of the patient's reality of a lived daily experience on a day-to-day basis.

Children in the family may often bear much of the burden of caring. When the patient is a young parent, children can feel culpable for the disease itself. Children need clear explanations of what is happening and have their questions answered, knowing that they can ask more questions. Their school needs to be aware of a parent's illness and may need a supportive letter from the clinical team to explain the stresses that the child is under. Many families overlook the profound bereavement that occurs as the child sees a parent's health failing. Children pushed away, excluded from caring, can feel increased isolation and rejection. They often want to be involved in care; in bereavement this can be a source of support as much adjusting will have happened prior to the parent's death.

If the decision is made to cease treatment, such as discontinuing ventilation, the patient must receive full support and care until death occurs. It is the patient's decision to consent to ongoing treatment, or to withdraw that consent. Such decisions need to be fully informed and every effort must be made to help the patient communicate their decision. In the event of the patient lacking capacity to take a decision, the clinician carries responsibility for a ‘best interest' decision, taken in the best interest of the patient as an individual person.

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C2 THE WINDING ROAD TO TREATING ALS

ROBBERECHT W^1,2

¹Flanders Institute for Biotechnology, Leuven, Belgium,²University of Leuven, Leuven, Belgium

E-mail address for correspondence: [email protected]

Keywords: disease models, modifying factors, treatment

Progress in the understanding of the biology of amyotrophic lateral sclerosis (ALS) has been significant thanks to a large set of data gathered in genetic, cellular and mouse studies. Several ALS causing genes have been identified and common themes through which they damage motor neurons are beginning to emerge. The cause of sporadic ALS however remains unknown. Therefore, animal models based on the familial forms of ALS remain essential to identify factors that modify motor neuron degeneration in all forms of the disease, and which could represent targets for therapeutic intervention. For this purpose, models based on rodents, fish, flies and worms are being used, all with intrinsic strengths and weaknesses.

In spite of the exciting results obtained using these approaches over the last decade, translation into a therapeutic strategy for ALS patients has proven to be difficult. The reason for this is multifactorial and certainly not only due to the validity of the models used. Confirmation of effects found should keep us from investing in false positive results, while rigorous pharmacodynamic and pharmacokinetic studies should prevent discarding potentially interesting molecules. Collaborations between academic labs with each a specific expertise and between academia and the private sector will increase our chances of being successful.

Although the absence of a therapeutic breakthrough so far is a reality we have to face, numerous accomplishments in ALS are very encouraging, especially compared to other similar fields. Mice that overexpress mutant SOD1, a cause of dominant ALS, develop a late onset, fatal and quantifiable motor neuron disease. The discovery of mutations in unrelated proteins allows testing of compounds independent of the “type” of ALS. Knockdown of a specific mutant protein becomes feasible. New methods for the delivery of compounds are being explored. Trial methodology is well established and remains the topic of constructive criticism. An intense interaction between patients and clinicians has established a network that is able to randomize a large number of patients in a very short time, and at the same time has tremendously increased the quality of care for ALS patients, as is clear from the changing natural history of the disease. The number of research groups and their output exponentially rises. Interest from the private sector in ALS increases rather than fades.

It is very likely that a combination of drugs will be used based on general rules but also on the “type” of ALS and characteristics of the patient. This will require fast and reliable testing in an individual patient. The availability of a biomarker, easily accessible and truly reflecting the evolution of the disease, will be pivotal.

SESSION 2A PROTEINOPATHIES

C3 PROTEIN AGGREGATES IN NEURODEGENERATIVE DISEASES

SPILLANTINI MG

University of Cambridge, Cambridge, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Neurodegenerative diseases, protein aggregates, Tau alpha-synuclein

Several neurodegenerative diseases of the nervous system are characterised by the presence of extracellular and/or intracellular protein aggregates. Deposits of ß-amyloid and microtubule-associated protein tau are characteristic of Alzheimer's diseases (AD) but tau deposits can also be found in diseases such as progressive supranuclear palsy, corticobasal degeneration, Pick's diseases/frontotemporal dementia (FTLD-T) that are now known as “tauopathies” (1). More recently insoluble tau aggregates have been also identified in progressive forms of multiple sclerosis (2). The major component of the inclusions present in Parkinson's diseases, (PD) dementia with Lewy bodies and multiple system atrophy is instead alpha-synuclein and these diseases are now named “alpha-synucleinopathies” (3). Forms of FTLD characterized by the presence of ubiquitin inclusions (FTLD-U) can have as major component of the aggregates TDP-43 that is also present in protein deposits in motor neuron diseases (MND) (4). The identification of similar protein aggregates in FTLD and MND has shown that these disorders are at the extreme ends of a disease spectrum containing FTLD-MND. Interestingly, while it was believed that protein aggregates were disease specific it is now clear that in some cases different protein can aggregate in the same disease, for example in AD besides ß-amyloid and tau also alpha-synuclein and TDP-43 deposits can be present. Furthermore, epidemiological and genetic studies have shown that even if protein deposits do not co-exist proteins can still interact as in PD where no tau deposits are found with alpha-synuclein inclusions but a certain tau gene haplotype increases susceptibility for the disease (5). The relevance of protein aggregates for disease pathogenesis has become clear with the discovery that mutations in the genes encoding these proteins such as amyloid precursor protein, tau, alpha-synuclein, TDP-43 and FUS cause rare familial forms of the diseases (6–9). The findings in familial cases indicate that also in sporadic diseases a direct link exists between the aggregated protein and disease pathogenesis. Understanding how and why these aggregates form and cause cell death will lead to the design of mechanism-based therapies for the cure of neurodegenerative diseases.

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C4 ROLE OF TDP-43 AND FUS IN DISEASE PATHOGENESIS

NEUMANN M

Institute of Neuropathology, Zurich, Switzerland

E-mail address for correspondence: [email protected]

Keywords: TDP-43, FUS, pathogenesis

In 2006, the transactive response (TAR) DNA binding protein with Mr 43 kD (TDP-43) was identified as the pathological protein in a common subtype of frontotemporal lobar degeneration (now referred to as FTLD-TDP) and amyotrophic lateral sclerosis (ALS), thereby providing strong evidence that FTLD, ALS with dementia and classical ALS are all part of a clinicopathological spectrum of disease. Subsequently, mutations in TARDBP, the gene encoding TDP-43, were identified in familial ALS and FTLD, emphasizing that TDP-43 dysfunction is directly involved in disease pathogenesis and neurodegenerative processes in these conditions. TDP-43 is a DNA/RNA-binding protein which becomes abnormally phosphorylated, cleaved and ubiquitinated in disease process. The development of novel antibodies against disease-specific modifications of TDP-43, such as phopshorylation-specific antibodies, or epitope specific antibodies are not only valuable tools for diagnostic purposes, but have already improved our knowledge on the TDP-species composition in abnormal inclusions. Thus, cortical inclusions are selectively enriched for hyperphosphorylated C-terminal fragments over full-length TDP-43 compared to spinal cord inclusions thereby suggesting that TDP-43 is differentially processed in brain versus spinal cord. It is expected that elucidating the functional consequences of disease-associated TARDBP mutations, e. g. by generation of transgenic mouse models, will provide important insights into the disease mechanisms underlying ALS and FTLD-TDP.

Only recently, missense mutations in the gene encoding the fused in sarcoma (FUS) protein (also known as translated in liposarcoma, TLS), another DNA/RNA binding protein with striking structural and functional similarities to TDP-43, were identified as a cause of familial ALS. Because of the recognized overlap between ALS and FTLD we investigated the possible role of FUS in FTLD cases. While no FUS pathology was identified in cases of FTLD with TDP-43 or tau pathology, or TDP-43-positive ALS, we were able to demonstrate FUS as the pathological protein in neuronal cytoplasmic and intranuclear inclusions in cases of tau/TDP-43-negative FTLD, a FTLD subtype accounting for up to 10% of FTLD cases. Immunoblot analysis confirmed the presence of insoluble FUS in post-mortem brain tissue from these cases. No mutations in the FUS gene were identified in any of our FTLD patients with FUS pathology. These findings suggest that FUS is also the pathological protein in a significant subgroup of sporadic FTLD thereby reinforcing the concept that FTLD and ALS are closely related conditions.

In summary, the identification of two DNA/RNA binding proteins, TDP-43 and FUS, as pathological proteins in ALS and FTLD, implies alterations in RNA processing as a key event in the pathogenesis of these diseases.

C5 INTERACTION PARTNERS FOR MISFOLDED SOD1

ZETTERSTRÖM P, GRAFFMO KS, ANDERSEN PM, BRÄNNSTRÖM T, MARKLUND SL

Umeå University, Umeå, Sweden

E-mail address for correspondence: [email protected]

Keywords: Misfolding, Protein interaction, proteomics

Background: Mutations in the free radical scavenging enzyme Superoxide dismutase-1 (SOD1) were linked to ALS in 1993 and for the last 15 years there has been intense research regarding the role of SOD1 in ALS. Still the precise mechanism by which mutant SOD1 is toxic to motor neurons is unknown.

One of the dominating hypotheses about SOD1 toxicity concerns protein misfolding. A role for SOD1 misfolding in ALS is indicated by several lines of evidence. At autopsy, large intraneuronal inclusions that stain for SOD1 are found in patients with SOD1 mutations, rodents that overexpress mutant SOD1 generate similar inclusions at endstage and soluble misfolded SOD1 is enriched in spinal cords of different transgenic ALS model mice. Also, studies on recombinant SOD1 have shown that mutations in SOD1 destabilize the precursor monomers and weaken the dimer interface.

Objectives: When SOD1 misfolds, internal structures normally hidden in the hydrophobic core become exposed. These sticky parts of the molecule might form non-native interactions and in this way be cytotoxic to motor neurons. Here we set out to identify novel interaction partners for misfolded SOD1.

Methods: We have created a set of anti-peptide antibodies that are highly specific for misfolded SOD1. For this study, an antibody directed towards the C-terminal of SOD1 was used since this part of the molecule is missing in ALS-linked truncation mutants. Thus the C-terminal antibody will not interfere with any interaction relevant for ALS pathogenesis. The antibody was coupled to Sepharose and used to immunocapture misfolded SOD1 in spinal cord extracts from transgenic mice. The misfolded SOD1 together with interacting proteins is then released by addition of the peptide used to raise the antibody. With this paradigm, only interaction partners to the supposedly toxic misfolded SOD1 are isolated. Interacting proteins are identified by proteomic techniques including 2D-PAGE and mass spectrometry, but also in a candidate approach by western immunoblots to verify or rule out interesting conceivable binding partners.

Results: Misfolded SOD1 interacts with several heat shock proteins and chaperones including Hsc70 and Grp78 and also to cytoskeleton proteins like actin and actin related proteins.

Discussion: Identification of binding partners for misfolded SOD1 might lead to new hypothesis about the cytotoxic mechanism exerted by mutant SOD1s.

C6 TEMPLATE-DIRECTED MISFOLDING OF SOD1 IN VITRO: A MODEL FOR ALS PROPAGATION?

CASHMAN N, YANAI A, GRAD L

University of British Columbia, Vancouver, British Columbia, Canada

E-mail address for correspondence: [email protected]

Keywords: prion, protein misfolding, propagation

Background: Protein misfolding diseases probably comprise several different classes of pathogenesis. Prion diseases are thought to propagate through template-directed misfolding (TDM) of the cellular prion protein PrP^C by the disease-misfolded prion protein PrP^Sc. Recent discoveries suggested that a TDM mechanism might participate in the pathogenesis of Alzheimer's disease (AD), and seeded aggregation in vitro has been observed for many proteins and peptides implicated in AD, PD, and ALS. Mutations in SOD1 are associated with ~20% of familial amyotrophic lateral sclerosis (ALS). When mutated and/or oxidized, superoxide dismutase 1 (SOD1) is subject to misfolding and aggregation, and is efficiently exported from the cell. We have hypothesized that ALS propagates via the template-directed misfolding mechanism similar to that thought to underlie the prion diseases.

Objectives: To establish whether mutant misfolded SOD1 can induce misfolding and aggregation of natively structured wild-type SOD1 intracellularly, and if propagation of SOD1 misfolding can be transmitted from cell-to-cell.

Methods: We exploited G127X, a natural familial ALS frameshift mutation, in transfection and immunoprecipitation (IP)/immunocytochemistry (ICC) paradigms in which the mutant, wild-type (wt) and misfolded wtSOD1 species could be unambiguously identified by antibodies specifically directed against these molecular species, including monoclonal antibodies directed against disease-specific epitopes of misfolded SOD1. Characterization studies of misfolded/aggregated SOD1 species were also obtained by solubility in non-denaturing detergents, and protease resistance using proteinase K (PK).

Results: wtSOD1 misfolding-specific antibody immunoreactivity is induced by contact with G127X mutant SOD1 as determined by IP and ICC. Misfolded wtSOD1 acquires PK sensitivity, but not detergent insolubility. Misfolded SOD1 (mutant and wt) acquire non-native inter-chain disulfide bonds, although the induction of misfolding of wtSOD1 is preserved with G127X mutants in which all remaining cysteines were mutated to serine residues by in vitro mutagenesis. G127X SOD1-induced misfolding of wtSOD1 is not observed in mouse cell lines. We have detected misfolded mutant and wtSOD1 in the media of G127X SOD1-transfected cells; non-transfected cells incubated in the presence of this medium can induce SOD1 misfolding intracellularly. Incubation of G127X SOD1-transfected cells with poly-specific SOD1 antibodies reduces wtSOD1 misfolding in vitro.

Discussion: Misfolded SOD1 can induce wtSOD1 misfolding by non-covalent interaction in human neural and mesenchymal cell lines. G127X-induced wtSOD1 acquires PK sensitivity consistent with conformational loosening. Non-native disulfide bonds are a consequence (not a cause) of wtSOD1 misfolding, but may “lock in” wtSOD1 misfolding, and/or stabilize G127X interactions for co-immunoprecipitation. Misfolding of wtSOD1 respects a “species barrier” between human and mouse proteins. Furthermore, misfolding of SOD1 propagates in cell culture by intercellular exportation and uptake of antibody-accessible misfolded molecular species.

Conclusion: Misfolded SOD1 can engage in template-directed misfolding, a possible explanation for the prion-like propagation of ALS in the neuroaxis.

C7 KINETIC ANALYSIS OF SOD1 MISFOLDING RESULTING FROM OXIDATIVE DAMAGE: POTENTIAL RELEVANCE TO FAMILIAL AND SPORADIC ALS

MULLIGAN VK¹, CHAKRABARTTY A^1,2

¹Department of Biochemistry,²Department of Medical Biophysics; University of Toronto, Ontario, Canada

E-mail address for correspondence: [email protected]

Keywords: superoxide dismutase, misfolding, oxidative damage

Background: Mutations in the gene encoding the Cu, Zn superoxide dismutase (SOD1) are known to cause approximately 20–25% of familial ALS cases, as well as a considerable number of apparently sporadic ALS cases (1,2). Mutation is believed to promote SOD1's entry into a cytotoxic misfolded conformation (3). It is possible that oxidative damage to the protein or other stresses may also trigger SOD1 misfolding, suggesting a potential mechanism by which wild-type SOD1 could cause sporadic ALS (4). We previously developed kinetic assays for examining the release of SOD1's bound metals, unfolding of the SOD1 beta-barrel, and dissociation of the SOD1 dimer (5). Using these assays, we were able to elucidate the unfolding mechanism for wild-type SOD1 denatured in guanidine hydrochloride (5).

Objectives: This study aims to apply the previously developed techniques to elucidate the mechanism of SOD1 misfolding triggered by oxidative damage.

Methods: SOD1 was treated with millimolar hydrogen peroxide (H₂O₂) in the presence of buffer and 100 µM 4-(2-pyridylazo)resorcinol (PAR). Kinetic measurements of PAR absorbance allowed determination of rates of copper and zinc release resulting from H₂O₂-induced damage. Kinetic tryptophan fluorescence measurements were used to examine beta-barrel conformational changes. Data were analyzed via model-free Laplace methods using a custom algorithm, and were also fit to various sequential release models by least-squares methods.

Results: H₂O₂ appeared to induce damage selectively at the SOD1 active site, inducing release of both copper and zinc. No change in tryptophan fluorescence was observed, suggesting that global structural rearrangements do not result from H₂O₂ treatment. Both metals were released via three-state sequential mechanisms, each step involving one molecule of hydrogen peroxide, suggesting that two metal-binding histidine residues must be oxidatively modified to trigger release of a metal atom. Copper release was total but zinc release was not, consistent with a model in which the catalytic copper's presence is necessary to convert H₂O₂ to superoxide (O₂⁻), which then damages histidine residues.

Discussion and Conclusions: SOD1's role as a scavenger of free radical species comes with the occupational hazard of damage caused by those species. The preliminary findings presented here suggest that this damage can result in partial or total loss of copper and zinc without global destabilization of the protein, resulting in native-like metal-deficient/misfolded SOD1 species. These are candidates for cytotoxic species that may play a causal role in ALS.

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C8 MODIFICATIONS OF SUPEROXIDE DISMUTASE IN HUMAN ERYTHROCYTES: A POSSIBLE ROLE IN ALS

WILCOX K, ZHOU L, JORDON J, HUANG Y, YU Y, REDLER R, CHEN X, CAPLOW M, DOKHOLYAN N

University of North Carolina at Chapel Hill, Chapel Hill, United States

E-mail address for correspondence: [email protected]

Keywords: biophysics, molecular modeling, superoxide dismutase

Background: Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified.

Objectives: Our objective was to test the hypothesis that SOD1 is modified in humans, determine these modifications, and understand their role in promoting SOD1 dissociation, which is the first step in the SOD1 aggregation pathway.

Methods: We used size-exclusion chromatography for separation of modified SOD1 species from the unmodified ones. We used a combination of “bottom-up” and “top-down” mass spectrometry approaches to search and to identify the modifications. To measure Kd we used SOD1 activity as an assay: We tested for a reduction in the rate of 6-hydroxydopamine.

Results: Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315nm rather than 212nm at physiological SOD1 concentrations.

Discussion: The link between SOD1 mutations, protein aggregation and FALS is not fully understood, but there are multiple reports showing that dimer dissociation is an early event during SOD1 aggregation. Our finding that modifications can facilitate SOD1 dimer dissociation suggests a possible link between the normal characteristics of SOD1 and its role in FALS. Although relatively modest, a 2-fold increase in Kd resulting from SOD1 modification translates to nearly a 70% increase in SOD1 monomer concentration. Because nucleation of SOD1 aggregation is dependent on at least the square of the monomer concentration, we expect a 70% increase in monomer concentration to have a marked effect on the nucleation of SOD1 aggregates. For example, the formation of a hypothetical nucleus made up of 3 glutathionylated SOD1 monomers becomes roughly 5 times more likely and a nucleus of 6 monomers 24 times more likely relative to unmodified SOD1.

Conclusions: Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation.

C9 PROTEIN DISULPHIDE ISOMERASE IS S-NITROSYLATED IN ALS

WALKER A^1,2, TURNER B^1,2, FARG M¹, MCLEAN C³, HORNE M^1,4, ATKIN J^1,2

¹Howard Florey Institute, Florey Neuroscience Institutes,²Centre for Neuroscience; University of Melbourne, Parkville, Victoria, Australia,³Department of Anatomical Pathology, The Alfred Hospital, Prahran, Victoria, Australia,⁴Department of Neurology, St Vincent's Hospital, Fitzroy, Victoria, Australia

E-mail address for correspondence: [email protected]

Keywords: protein disulphide isomerase, ER stress, S-nitrosylation

Background: Endoplasmic reticulum (ER) stress is a key feature of ALS pathology, occurring early in disease in transgenic mutant SOD1 ALS models and also in spinal cords of human patients. ER stress occurs when misfolded proteins accumulate in the ER, triggering a homeostatic mechanism known as the unfolded protein response (UPR). Activation of the UPR causes up-regulation of chaperones such as protein disulphide isomerase (PDI), which modulates disulphide bond formation. However, prolonged activation of the UPR leads to apoptotic signaling. In ALS, PDI is highly up-regulated throughout disease course and in mutant SOD1 mice prior to the onset of symptoms. Over-expression of PDI is protective against mutant SOD1 inclusion formation and toxicity, suggesting that protein aggregation and ER stress are involved in ALS pathogenesis. Recently, S-nitrosylated PDI (SNO-PDI) was detected in brains of Alzheimer's and Parkinson's disease patients, providing a further link between PDI function and protein misfolding in neurodegeneration. S-nitrosylation of PDI involves aberrant post-translational modification whereby nitrosative stress triggers the transfer of nitric oxide groups to critical active site cysteine residues, resulting in functional inactivation of the enzyme.

Objectives: The aim of this study was to determine whether or not PDI modification by S-nitrosylation occurs in spinal cord of human ALS patients and transgenic mutant SOD1 mice.

Methods: Lumbar spinal cord tissues from ALS patients and controls without evidence of neurological disease were extracted at post-mortem. Spinal cord tissues from SOD1^G93A transgenic mice, along with non-transgenic littermate controls, were also used. SNO-PDI was detected using a biotin switch assay in which S-nitrosylated cysteine residues were specifically biotinylated, followed by precipitation of samples with streptavidin-agarose and immunoblotting. Levels of SNO-PDI were normalised to the amount of total PDI in each sample by densitometry.

Results: SNO-PDI was highly increased in lumbar spinal cord tissue of ALS patients compared to controls, even when normalised to total PDI to account for the significantly increased total PDI levels in ALS patients. S-nitrosylated PDI was also detected in SOD1^G93A mouse spinal cords but not in non-transgenic littermate controls.

Discussion and conclusions: S-nitrosylation of critical cysteine residues has previously been demonstrated to prevent the normal protective function of PDI. Although there is a large up-regulation of PDI in ALS, the detection of SNO-PDI in spinal cords of ALS patients and SOD1^G93A mice in this study suggests that inactivation of PDI could contribute to disease pathogenesis. Therapeutics targeting PDI or ER stress may therefore be beneficial in ALS.

SESSION 2B TRANSLATING EVIDENCE INTO PRACTICE

C10 PERFORMANCE MEASURES AND ACCOUNTABILITY

RINGEL S

University of Denver, Denver, CO, United States

E-mail address for correspondence: [email protected]

Keywords: quality of care, accountability, performance measures

The quality and safety of today's medical care are quite variable and there is growing concern about runaway health care costs. Studies have demonstrated poor adherence to guidelines, indefensible variations in care, high cost and waste. As a result, there is growing distrust of the medical profession and calls for greater accountability by consumers, payers and regulators. A variety of methods are used to achieve accountability including competition, transparency, differential payment, accreditation, regulation and litigation. All of these approaches rely on performance measures even though measuring quality of care is in its infancy. Today's emphasis on information technology to assure accountability has created high societal expectations that can only be met if greater emphasis and resources are applied to measure quality of care.

Common measures of health care quality include: (1) how health care is organized (structure); (2) what is done (process); and (3) what happens to the patient (outcome). Because outcomes are the most difficult to obtain, many systems rely heavily on structure and process measures to hold physicians accountable. Successful approaches require system thinking, a culture of process improvement, and the development of performance data that truly reflect cost-effective patient care. Barriers that have to be overcome include: insufficient evidence, the cost of information technology, a perceived threat to physician autonomy, a traditional silo approach to health care delivery, and competing priorities.

Increasingly, academic medicine must support fundamental research in quality, safety and efficiency. Successful models for quality assessment and improvement will be presented and include creating and implementing data registries, developing and promoting cross department collaboration and fostering a no-blame culture of accountability. Future medical practice must extend beyond the quest for medical innovation to incorporate systems and contexts for using medical discoveries cost-effectively.

C11 AAN PRACTICE PARAMETER UPDATE: THE CARE OF THE PATIENT WITH AMYOTROPHIC LATERAL SCLEROSIS (AN EVIDENCE-BASED REVIEW)

MILLER RG¹, JACKSON CE², KASARSKIS EJ³, ENGLAND JD⁴, FORSHEW DA¹, JOHNSTON W⁵, KALRA S⁵, KATZ JS¹, MITSUMOTO H⁶, ROSENFELD J⁷, SHOESMITH C⁸, STRONG MJ⁸, WOOLLEY SC¹, AND THE QUALITY STANDARDS SUB-COMITTEE OF THE AMERICAN ACADEMY OF NEUROLOGY THE ALS PRACTICE PARAMETER TASK FORCE⁹

¹California Pacific Medical Center, San Francisco, CA, United States,²University of Texas Health Science Center, San Antonio, TX, United States,³University of Kentucky, Lexington, KY, United States,⁴Louisana State University Health Sciences Center, New Orleans, LA, United States,⁵University of Alberta, Edmonton, AB, Canada,⁶Neurological Institute(NI-9), New York, NY, United States,⁷UCSF Fresno, Freson, CA, United States,⁸London Health Sciences Center, London, ON, Canada,⁹American Academy of Neurology, St. Paul, MN, United States

E-mail address for correspondence: [email protected]

Keywords: evidence-based medicine, management of ALS, practice parameters

Background: The American Academy of Neurology (AAN) issued an evidence-based report on managing patients with amyotrophic lateral sclerosis (ALS) in 1999.

Objective: To systematically review evidence bearing on the management of patients with ALS and update the 1999 AAN practice parameter.

Methods: The authors completed a systematic literature review from 1998 to 2008. Topics included breaking the news, symptom management, slowing disease progression, nutrition, respiratory management, palliative care, cognitive and behavioral impairment, multidisciplinary clinics, and communication for patients with ALS.

Results: The authors identified 10 Class I studies, 13 Class II studies, and 73 Class III studies in ALS. More studies are clearly needed to examine the best tests of respiratory function in ALS, the optimal time for starting PEG, the impact of PEG on quality of life and survival, the effect of vitamins and supplements, symptomatic therapies and palliative care. The following recommendations are made based on the studies analyzed: Riluzole should be offered to slow disease progression (Level A). Percutaneous endoscopic gastrostomy (PEG) should be considered to stabilize weight and to prolong survival (Level B). Noninvasive ventilation (NIV) should be considered to treat respiratory insufficiency in order to lengthen survival (Level B), and may be considered to slow the decline of forced vital capacity (Level C) and improve quality of life (Level C). Early initiation of NIV may increase compliance (Level C), and insufflation/exsufflation may be considered to help clear secretions (Level C). Multidisciplinary clinic referral should be considered to optimize health care delivery and prolong survival (Level B) and may be considered to enhance quality of life (Level C). For the treatment of refractory sialorrhea, botulinum toxin B should be considered (Level B) and low-dose radiation therapy to the salivary glands may be considered (Level C). For treatment of pseudobulbar affect, the combination therapy of dextromethorphan with quinidine should be considered, though side effects are not uncommon and the treatment is currently not approved by the U.S. Food and Drug Administration (Level B). For patients who develop fatigue while taking riluzole, withholding the drug may be considered (Level C). Because many patients with ALS demonstrate cognitive impairment, which in some cases meets criteria for dementia, screening for cognitive and behavioral impairment should be considered in patients with ALS (Level B). Other management strategies all lack strong evidence.

Discussion: There are many treatments available for patients with ALS that can alleviate suffering. NIV, PEG, riluzole, and multidisciplinary clinics are the most important and have the best evidence.

Conclusions: More high-quality, controlled studies are needed to guide management and to assess outcomes in patients with ALS.

C12 MEDICATION IN THE LAST DAYS OF LIFE OF MND/ALS – A STUDY FROM SPECIALIST PALLIATIVE CARE PROVIDERS IN THE UK

OLIVER D¹, CAMPBELL C², O'BRIEN T³, SLOAN R⁴, SYKES N⁵, TALLON C⁶, TAYLOR-HORAN J¹

¹Wisdom Hospice, Rochester, United Kingdom,²St Catherine's Hospice, Scarborough, United Kingdom,³Marymount Hospice, Cork, Ireland,⁴Weldmar Hospice, Dorchester, United Kingdom,⁵St Christopher's Hospice, London, United Kingdom,⁶Cynthia Spencer Hospice, Northampton, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: opioids, palliative care, end of life

Background: Specialist palliative care providers (SPC) are often involved in the care of people with ALS/MND in the UK, particularly in the management symptoms at the end of life. Despite this experience in the use of medication in the management at the end of life many patients and their families and professionals fear the last days of life with ALS/MND and the use of medication at this time.

Objectives: This study aimed to show the medication used in the last 72 hours of life within six specialist palliative care units in the UK and Ireland.

Methods: Six SPC units provided details of the last 10 patients who had died under their care. Patient information was collected together with the details of medication used in the last 72 hours before death.

Results: 60 patient records were audited −63% male and 37% female with a mean age of 67 years. The mean time from first symptom to death was 32 months. The majority of patients received medication in the last 72 hours of life, primarily:

Morphine-23 patients in the last 24 hours, commonly (38%) by subcutaneous infusion with a mean dose of 80mg (oral equivalent) over 24 hours

Midazolam-35 patients, commonly by subcutaneous infusion with a mean dose of 31mg/24 hours

Anticholinergic medication-as glycopyrronium bromide or hyoscine hydrobromide- 35 patients

All patients were reported as dying peacefully, without distress.

Discussion and Conclusion: This study showed that medication is commonly given within SPC units for the management of symptoms at the end of life. The doses used are similar to those in other studies and in surveys of cancer patients-for ALS/MND the studies showed a mean oral equivalent dose of morphine of 98mg/24hours (1) and 90mg/24 hours (2), and for cancer patients a mean dose of 166mg/24 hours (3).

The results show that professionals can feel secure in the administration of medication at the end of life and the doses used are not large and similar or less than for other terminal care groups. Patient and families can also be reassured that with good symptom management and the best use of medication dying form ALS/MND is peaceful.

Reference:

• Oliver D. Palliative Med 1998; 12: 113–115
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C13 FACTORS UNDERLYING END OF LIFE-DECISIONS IN ALS PATIENTS

SORG S¹, NONNENMACHER S², LULÉ D^3,1, KÜBLER A⁴, LUDOLPH AC¹

¹University of Ulm, Ulm, Germany,²Eberhard-Karls University, Tübingen, Germany,³University of Liége, Liége, Belgium,⁴University of Würzburg, Würzburg, Germany

E-mail address for correspondence: [email protected]

Keywords: end-of-life decision, depression, cognitive processes

Background: Over the course of the disease, ALS patients have to make decisions with regards to life-sustaining treatment (non-invasive ventilation, NIV; percutaneous endoscopic gastrostomy, PEG; invasive ventilation, IV). Depression and quality of life were found to be independent of the progression of the disease but to be predictors of the wish to die and physician-assisted suicide (PAS). Data on the processes underlying these end-of life decisions are sparse.

Objectives: Our investigation aimed to identify factors underlying the end-of-life decision by a longitudinal approach. The course of depression, subjective quality of life, attitudes toward life sustaining treatment and hastened death, as well as the development of the decisional process itself were investigated.

Methods: Patients with definite ALS and no cognitive impairment were eligible to participate in the study. The time between interviews was 6 months. Depression, subjective quality of life and attitudes toward hastened death were assessed by standardized instruments. Attitudes and other cognitive factors regarding the end-of-life decision were assessed by the Life Sustaining Treatment Questionnaire (Häcker, 2008, unpublished).

Results: T1: The sample consisted of 61 patients. Depression was neither related to demographic or disease related variables (time since diagnosis, bulbar symptoms, pain, ventilatory status) nor associated with the wish to die. Level of depression was related to quality of life (r = − 0.513), fear of death (r = 0.324), and the wish for legalizing PAS (r = 0.542). Quality of life was highest in IV-patients and was not associated with disease progression or bulbar symptoms. Low quality of life was related to stronger approval of the legalisation of PAS (r = − 0.438) and a stronger wish to die. About half of the sample had not made a decision regarding life sustaining treatment yet. Individuals reporting a positive decision toward life sustaining treatment and those who had not made a decision yet, reported a low wish to die. The highest wish to die was found in patients who decided against life sustaining treatment.

T2: Present results suggest that depression, quality of life and the wish to die do not change over time. Only the fear of death decreased significantly.

Discussion/conclusion: Most strikingly, attitudes toward life sustaining/shortening treatment might not be related to the disease itself. Rather, they seem to be mediated by psychological processes that could be subject to intervention. During all stages of the disease, a high quality of life and low levels of depression are possible. The results can contribute to the general debate over the legalisation of PAS. Focus of this debate should lie on the necessity to ensure every possible support to ALS patients to improve their well being towards the end-stage of the disease.

C14 HOW PATIENTS WITH ALS, MAKING ADVANCED DECISIONS, CAN INFLUENCE OUTCOMES IN WHERE THEIR END OF LIFE CARE IS RECEIVED.

CALLAGHER P, MITCHELL D, ADDISON-JONES R, BENNETT W, GARDHAM J

Preston MND Care and Research Centre, Preston, Lancashire, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: preferred priorities of care (PPC), choices end of life care, place of death

Objectives: The Preferred Priorities of Care document (PPC) gives the terminally ill patient an opportunity to think, talk about and write down preferences and priorities for end of life care. Our aim was to establish if patients had specified a choice of where their care would be received at their end of life, that this had been achieved and if not what had happened to prevent this.

Methods: Comparisons were made between three groups of patients with ALS, those who completed a PPC, those who had had discussions with the MND specialist nurse with regard to their PPC but had not formally recorded their wishes and those who did not wish or had not had the opportunity to complete a PPC. Data recorded included their preferred place of terminal care

Results: Of the 44 people who chose to complete preferred priorities of care, thirty-nine stated they would prefer to die at home. Twenty-five of these achieved their death at home, three in a hospice, nine in hospital, two in nursing homes. Two patients wished to die in a hospice as their second choice and this was achieved. Two chose to die in hospital and one in a nursing home.

Of the 45 patients who did not complete a PPC eighteen died at home, sixteen in hospital, eight in a nursing home and two in hospice. Fourteen patients had had discussions about PPC but had not made decisions on place of terminal care. Six of these died in hospital, five at home, two in a nursing home and one in hospice.

The main reasons for not achieving their desired PPC included carers being unable to cope and sudden change in medical condition.

Discussion: It is suggested that in the UK between about 80–90% of those with a terminal illness expressed a preference for death at home but 60% die in hospital. The results of our audit suggest that patients have a higher chance of achieving their preferred place of end of life care if their wishes have been recorded on a PPC. Further investigations should be made to establish causes of breakdown in care leading to hospital admission in the last few days or hours of life and how these admissions can be prevented

C15 CARING FOR THE CAREGIVER PART 1: USING EVIDENCE BASED PRACTICE TO DESIGN INTERVENTIONS TO SUPPORT ALS CAREGIVERS

STEPHENS HE¹, WALSH S², BREMER B³, SIMMONS Z¹

¹Penn State Hershey Medical Center, Hershey, Pennsylvania, United States,²ALS Association, Greater Philadelphia Chapter, Harrisburg, Pennsylvania, United States,³Penn State University, Harrisburg, Pennsylvania, United States,

E-mail address for correspondence: [email protected]

Keywords: caregiving, quality of life, evidence based practice

Background: Information on how to support caregivers of ALS patients is sparse. An Evidenced Based Practice (EBP) approach was used to identify potential interventions to support caregivers. EBP is the process of systematically searching for the best available evidence to support a clinical decision or clinical intervention.

Objectives: To report on the process by which we used EBP to develop a questionnaire which can be used to design interventions to support ALS caregivers.

Methods: A multidisciplinary team used EBP to gather and analyze available information on what can be done to support ALS caregivers. Information on caregiving was obtained from three sources: 1) published literature; 2) expert opinions; 3) caregiver focus groups. The information was reviewed for potential intervention development.

Results: Published literature: 70 relevant articles were retrieved, 21 of which were applicable to intervention development: Level 1 (systematic review or multicenter controlled studies)-5 articles; Level II (single-center controlled studies)-4 articles; Level III (case-control studies)-9 articles; Level IV (qualitative reviews or studies)-3 articles. The study population in the articles included 393 ALS caregivers, 4154 Alzheimer's caregivers, and 2062 other caregivers. The literature revealed the following: early interventions are most effective; high risk-factors and protective functions for caregivers can be identified; interventions should be multifocal; strategies exist to decrease caregiver perceptions of burden; extra supports aid well-being. Expert opinions generated by clinicians from an interdisciplinary ALS clinic supplemented the literature: educate about all aspects of the disease at 3 month intervals; identify sources of stress and coping; identify available support systems; caregiving experiences differ depending on the relationship to the patient; caregivers need encouragement to call the ALS team for assistance. Caregiver focus groups reinforced services that are helpful (hospice; repeating information about services; emotional/social supports from friends/community) and identified areas in which more attention is needed (finding good in-home care; providing information and options about equipment; removing physical signs after the loved one dies). The three sources of information were reviewed by the EBP team, who determined that the design and administration of a Caregiver Assessment Form in ALS clinic would potentially be a useful intervention. The assessment form includes demographics, assessment for caregiver risks (concern with tasks, health and well-being, stress), and protective functions (optimism, confidence, spirituality).

Discussion and Conclusions: EBP can be used to foster collaboration between clinicians and researchers to design and construct an instrument for the assessment of ALS caregivers. Such a tool can form the basis for interventions to maximize the quality of life of caregivers of patients with ALS.

SESSION 3A BIOMARKERS

C16 DISCOVERY AND VERIFICATION OF PROTEIN BASED BIOMARKERS FOR ALS

RYBERG H¹, GOPALAKRISHNAN V¹, CUDKOWICZ M², LACOMIS D¹, BOWSER R¹

¹University of Pittsburgh School of Medicine, Pittsburgh, PA, United States,²Harvard Medical School, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: biomarkers, proteomics, diagnostics

Background: Rapid diagnostic tests for motor neuron disease are not currently available but would assist neurologists in making a clinical diagnosis and more rapidly initiate proper patient treatments to extend quality of life and/or initiate patients into clinical trials at early stages of disease progression. Many groups have discovered candidate biomarkers for ALS, though most have utilized small numbers of test subjects and few proper disease mimics. Continued verification and follow-up validation studies are lacking for all candidate biomarkers and therefore represent an important next step in the ultimate translation of biomarkers from the bench to the clinic.

Objectives: Our goal was to further discover and verify candidate protein based biomarkers for motor neuron disease using mass spectrometry based proteomics and antibody based methodologies using a large cohort of samples.

Methods: Cerebrospinal fluid (CSF) from 85 patients with sporadic ALS, 15 with familial ALS, 18 with multiple sclerosis, 53 with Alzheimer's disease, 29 other disease control, and 41 healthy control subjects was examined by Surface Enhanced Laser Dissociation/Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS). Mass spectral data were analyzed by multiple computer based algorithms to identify potential biomarker peaks. The mass peaks with high predictive value were validated by enzyme-linked immunosorbent assay (ELISA).

Results: A total of 68 mass spectral peaks were found to be differently expressed between the ALS and healthy control groups. Mass peaks for cystatin C and transthyretin were reduced in ALS patients, whereas mass peaks for a post-translational modified form of transthyretin and C-reactive protein (CRP) were increased in ALS. CRP levels were 5.84±1.01 mg/L for controls and 11.24±1.52 mg/L for ALS subjects as measured by ELISA. Increased levels of free hemoglobin were observed in 56% of ALS patients. The total level of cystatin C correlated to ALS patient survival.

Conclusions: This is the largest ALS biomarker discovery and verification study performed to date. Our study validated our prior mass spectrometry results demonstrating decreased levels of cystatin C and increased levels of specific modified forms of transthyretin in the CSF of ALS patients. CRP levels are increased in the CSF of ALS patients, and cystatin C levels in the CSF correlate to survival of sporadic ALS patients with limb onset disease. We also verified other previously published candidate biomarkers in our study. Our proposed biomarker panel predicted ALS with a high level of specificity (94%) and 82% overall accuracy.

C17 VALIDATION OF A CSF BIOMARKER PANEL FOR AMYOTROPHIC LATERAL SCLEROSIS

MITCHELLE R, SIMMONS Z, CONNOR J

Pennsylvania State University College of Medicine, Hershey, PA, United States

E-mail address for correspondence: [email protected]

Keywords: CSF, biomarker, cytokines

Objective: To determine biomarkers associated with ALS in CSF samples obtained from multiple clinics.

Background: Multiple studies have sought biomarkers associated with ALS using a variety of techniques and control groups for comparison. We have previously reported a panel of CSF biomarkers distinguishing ALS patients from a group of neurological disease control subjects. All subjects included in our previous study were evaluated at the ALS Clinic at Penn State Milton S. Hershey Medical Center. The current study expands upon our earlier study to include samples collected at other clinics through multiple studies.

Methods: CSF samples were collected through the Northeast Amyotrophic Lateral Sclerosis (NEALS) Consortium. In total, 30 CSF samples were obtained from ALS patients and 30 CSF samples were obtained from healthy control subjects. Subjects were approximately matched for age, ethnicity and sex. We performed multiplex analysis on the CSF samples using the Bio-Plex Human 27-plex panel of cytokines and growth factors (Bio-Rad, Hercules, CA).

Results: The two comparison groups were approximately matched for age (median: 54.0 (ALS) vs. 40.0 (control) years, p = 0.07), sex (Chi-square = 1.11, p = 0.29), and ethnicity (Chi-square = 2.50, p = 0.47). Similar to the preliminary study, ALS patients demonstrated significantly elevated CSF levels of IL-6 (median: 6.07 vs. 4.58 pg/mL, p < 0.01), MCP-1 (218.9 vs. 140.6 pg/mL, p < 0.001), and VEGF (29.78 vs. 20.46 pg/mL, p < 0.05) compared to the healthy control group, and IFN-γ (12.53 vs. 19.13 pg/mL, p < 0.05) was significantly decreased in the ALS patient group. In addition, ALS patients had elevated CSF levels of IL-1 receptor antagonist (9.81 vs. 6.98 pg/mL, p < 0.05) and IL-8 (25.19 vs. 19.02 pg/mL, p < 0.001), and decreased levels of platelet-derived growth factor (6.14 vs. 10.60 pg/mL, p < 0.05), and IL-9 (26.20 vs. 34.11 pg/mL, p < 0.01). Analysis revealed a negative correlation between duration of symptoms and MCP-1 (r = − 0.515, p < 0.01).

Discussions and conclusions: In general agreement with our previous study comparing ALS patients with a group of neurological disease control patients, a number of biomarkers are associated with ALS suggesting inflammatory changes within the CNS. These biomarkers may have utility in allowing earlier diagnosis of ALS, elucidating disease pathogenesis, monitoring disease progress, and determining disease prognosis.

C18 PROTEIN DISULPHIDE ISOMERASE IN CSF- A NOVEL THERAPEUTIC AND/OR BIOMARKER FOR ALS?

ATKIN J, FARG M, WALKER A, TURNER B, HORNE M

Howard Florey Institute, Victoria, Australia

E-mail address for correspondence: [email protected]

Keywords: biomarker, protein disulphide isomerase, ER stress

Background: Protein Disulphide Isomerase (PDI) is responsible for the formation and isomerisation of native protein disulphide bonds. Mutant SOD1 forms high molecular weight aggregates containing aberrant, non-native disulfide bonds, which are linked to neuronal toxicity and disease progression. Hence, modulation of disulphide bond formation could be important in pathogenesis. We showed recently that PDI (a) was upregulated in motor neurons from SOD1^G93A mice and human patient tissues (b) located within cytoplasmic inclusions of motor neurons and (c) prevented the aggregation of SOD1 in vitro. These results suggest that PDI is a novel therapeutic target for ALS and because a good biomarker should be close to the fundamental pathology, possibly also an effective biomarker. At present there is no biochemical diagnostic test for ALS, so a biomarker that could detect early disease or could predict the onset of symptoms and burden of disease would be an excellent diagnostic tool. CSF is an excellent source of biomarkers due to its proximity to brain parenchyma.

Objective: To determine if PDI in CSF has potential as a biomarker for ALS.

Methods: CSF from SOD1^G93A and non-transgenic control rats was collected at p60, p90 and p120. CSF was obtained from 7 human patients with early disease and 8 patients with late stage disease, and from 5 controls without neurological disease. Western blotting was performed to detect the expression level of PDI in CSF.

Results: We detected markedly elevated levels of PDI in CSF from both SOD1^G93A rats and human patients compared to controls (over 5 fold; p < 0.01). Furthermore, there was a two fold elevation of PDI in CSF taken from patients at the diagnosis of sporadic MND (p < 0.001). However, by late disease (taken as close as possible to the date of death), this elevation of PDI increased to three-fold (p < 0.01) suggesting that PDI increases with disease progression. Importantly, PDI was elevated in the CSF (4 fold, p < 0.01) in animals 30 days prior to the onset of signs.

Discussion and Conclusions: PDI was greatly elevated in the pre-symptomatic phase of disease in the CSF of SOD1^G93A rats. In human patient CSF, its expression levels increased with disease course. These data suggest that PDI has potential as novel biomarker for ALS.

C19 NITRATED PROTEINS IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF ALS PATIENTS AND A RAT MODEL ARE USEFUL BIOMARKERS OF THE DISEASE

POZZI S^1,2, NARDO G^1,2, LAURANZANO E^1,2, PASETTO L^1,2, MANTOVANI S³, GARBELLI S³, MARINOU K⁴, MORA G^3,4, BENDOTTI C², BONETTO V^1,2

¹Dulbecco Telethon Institute, Milano, Italy,²Mario Negri Institute, Milano, Italy,³Fondazione S. Maugeri, Pavia, Italy,⁴Fondazione S. Maugeri, Milano, Italy

E-mail address for correspondence: [email protected]

Keywords: nitrotyrosine, biomarkers, lymphocytes

Background: Peroxynitrite formation has been largely considered as one of the possible mechanisms involved in amyotrophic lateral sclerosis (ALS) pathogenesis. Once formed peroxynitrite can exert its toxic effect converting tyrosine to 3-nitrotyrosine (NT) leading to protein nitration and oxidation. Increased levels of NT and NT-modified proteins have been found in the central nervous system (CNS) of patients and mouse models of familial ALS (fALS), suggesting a possible use of nitrated proteins as biomarkers. In ALS there is increasing evidence that alterations that occur in motor neurons are also present in peripheral cells such as lymphocytes.

Objectives: In search for protein biomarkers of ALS from accessible samples, we decided to analyze whether tyrosine nitrated proteins were increased in peripheral blood mononuclear cells (PBMC) of sporadic ALS (sALS) patients, at two degrees of disease severity, and of the mutant SOD1 transgenic rat model at different disease stages.

Methods: We analyzed nitrated proteins in PBMC samples by a proteomic approach, using two-dimensional gel electrophoresis in combination with MALDI-TOF/TOF mass spectrometry. The level of protein nitration was evaluated by immunoblot with the anti-NT antibody or with a specific anti-nitrated actin antibody.

Results: We identified for the first time the major over-nitrated proteins in PBMC from patients and rats at different disease stages. In the rats, the increased level of nitrated proteins was detected already at a presymptomatic stage. Among them, actin, ATP synthase and vinculin overlap between sALS patients and the rat model. Interestingly, in a previous study, we have found that actin and ATP synthase were over-nitrated in the spinal cord of a mouse model of fALS before disease onset, suggesting the possible involvement of these proteins in motor neuron degeneration. We are now developing and testing specific antibodies for over-nitrated proteins and in particular for actin that we found nitrated in patients and animal models.

Discussion and Conclusions: This work indicates that nitrative stress is not restricted to spinal cord, the tissue specifically affected by the disease, but extends to the periphery and is found in SOD1-linked animal models and sporadic cases. On the basis of these observations it is possible to speculate that protein nitration could be the effect of converging pathogenetic mechanisms and therefore worthwhile to be studied further from a mechanistic point of view. Moreover, nitrated proteins can be considered as promising candidate biomarkers for early diagnosis of ALS and an anti-NT-actin antibody may be an interesting tool for clinical evaluations.

C20 ALSBANK™ VIRTUAL BIOREPOSITORY PLATFORM WITH DYNAMIC SAMPLE SEARCHES FROM HETEROGENEOUS DATA SOURCES

SHERMAN A, ROYCE-NAGEL G, JAFFA M, CUDKOWICZ M, WALLACE K, GRASSO D

Massachusetts General Hospital (MGH), Charlestown, MA, United States

E-mail address for correspondence: [email protected]

Keywords: BioRepository, heterogeneous data sources, biomarkers

Background: Controlled and secure access to biosamples and ability to conduct dynamic searches based on clinical information from multiple databases is crucial to translational research. Most BioRepository platforms to not support association of existing inventory with clinical and phenotypic information collected during clinical trials and biomarker studies while meeting regulatory requirements.

Objective: To build a distributed Web-based platform for virtual BioRepository to manage sample collection and support dynamic search criteria capabilities across heterogeneous data sources from clinical trials and biomarker studies to ALS.

Methods: The ALSBank™ platform has been built on top of the existing PharmaENGINE™ system utilizing the “state machine” model for workflow configuration and customization; it provides the following capabilities to researchers:

Manages BioRepository operations and inventory across collection sites

Provides role-based access to samples inventory information

Manages accessibility to multiple clinical and trial databases

Queries information from multiple clinical sources utilizing their data dictionaries

Enables online sample requests across multiple BioRepositories

Results: The ALSBank™ Virtual BioRepository platform that enables secure and controlled Web access for investigators to search, request and receive biosamples and de-identified clinical data from virtual multiple BioRepositories is currently deployed at the Neurology Clinical Trials Unit of MGH and is available to all member sites of the NEALS consortium.

Repositories of biological samples from three research institutions, MGH, Emory University and University of Pittsburgh, are virtually connected

Individual vial bar-coding schema with unique clinical trials identifier is implemented

Individual vial's location is known down to the cell number in a storage box

Bar-coded storage boxes are tracked to freezers at their respective institutions

The ALSBank™ system allows to perform dynamic searches across multiple clinical databases with heterogeneous data dictionaries

Ad hoc variables could be created and linked to the databases

Searches could be saved and re-used for new samples as needed

Conclusion: The ALS researchers from different institutions may join the ALSBank™ community to create a virtual BioRepository with secure controlled access to biosamples and clinical information collected during multiple clinical trials and biomarker studies

SESSION 3B CLINICAL PHENOTYPES AND DISEASE PROGRESSION

C21 MRI-BASED NEUROIMAGING AS A SURROGATE MARKER IN ALS AND OTHER MOTOR NEURON DISORDERS: PROSPECTS AND PITFALLS

KASSUBEK J, LUDOLPH AC

Department of Neurology, University of Ulm, Ulm, Germany

E-mail address for correspondence: [email protected]

Keywords: magnetic resonance imaging, surrogate marker, computational neuroanatomy

Computerised magnetic resonance imaging (MRI)-based techniques are increasingly used to analyse the structural and functional pathoanatomy of the brain in neurodegenerative diseases such as ALS and other MND in vivo. Besides volumetric approaches in regions-of-interest and magnetic resonance spectroscopy, especially whole brain-based techniques are accepted advanced neuroimaging tools for cross-sectional or longitudinal investigations, such as morphometric 3-D T1-weighted MRI analysis (e.g. voxel-based morphometry) and diffusion tensor imaging (DTI) which provides insights into white matter microstructure at group or individual level. Most promising for the in vivo mapping of structural MRI-based computational neuropathology are multiparametric protocols with multiple neuroimaging techniques in combination. For a correlational or direct analysis of the functional impact, it has been shown that the inclusion of covariance analysis with clinical parameters or the co-registration with task-specific functional mapping (functional MRI) might give complementary information.

The application to ALS but also to other MND entities with different clinical phenotypes of upper or lower MN affectation has successfully been used to identify lesion patterns of central motor and extra-motor areas and thus provided the option to map the alterations of MND patients′ brains in vivo-at least at group level. That way, it has been possible to define pathomorphological grey matter or white matter ‘fingerprints‘ of various types of MND. Furthermore the post-processing techniques allow for a quantitative comparison of MRI data from MND patients with controls, including tract-based statistics of DTI data. It has to be considered, however, that with respect to being used as a diagnostic tool at the individual patient′s level, the above-named techniques face severe limitations in applicability, sensitivity and specificity. One important aim in MND beyond gaining a deeper understanding of the disturbed neuroanatomical and functional networks will be the establishment of (combined) MRI protocols as a (‘dry‘) biomarker (i) in natural history studies with MND patients who are well-defined in all aspects of their clinical presentation and (ii) in potential disease-modifying multi-center trials. For that purpose, there is not only a need for a higher number of longitudinal studies but also for more uniformity in the acquisition and especially post-processing of computer-based neuroimaging data in order for the methods to be both reproducible and valid. This aim is to be realised in large quality-controlled multi-national databases.

C22 NEW DIAGNOSTIC CRITERIA FOR PRIMARY LATERAL SCLEROSIS: A PROSPECTIVE VALIDATION STUDY

BRUGMAN F¹, VELDINK JH¹, FRANSSEN H², DE VISSER ME³, DE JONG JMB VIANNEY³, FABER CG⁴, KREMER BHP⁵, SCHELHAAS HJ⁵, VAN DOORN PA⁶, VERSCHUUREN JJGM⁷, BRUYN RPM⁸, KUKS JBM⁹, ROBBERECHT W¹⁰, WOKKE JHJ¹, VAN DEN BERG LH¹

¹Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Netherlands,²Department of Clinical Neurophysiology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Netherlands,³Department of Neurology, Academic Medical Center, Amsterdam, Netherlands,⁴Maastricht University Medical Center, Maastricht, Netherlands,⁵Radboud University Medical Center, Nijmegen, Netherlands,⁶Erasmus University Medical Center, Rotterdam, Netherlands,⁷Leiden University Medical Center, Leiden, Netherlands,⁸Diakonessenhuis Utrecht, Netherlands,⁹University Medical Center Groningen, Groningen, Netherlands,¹⁰University Hospital Leuven and Flanders Institute for Biotechnology, Leuven, Belgium

E-mail address for correspondence: [email protected]

Keywords: PLS, HSP, prognosis

Background: Diagnosis of primary lateral sclerosis (PLS) is by exclusion and current diagnostic criteria include a disease duration of ≥4 years to exclude ALS and a negative family history to exclude hereditary spastic paraparesis (HSP). However, sporadic presentation of HSP is not rare while genetic testing for many HSP forms is currently not available. We have previously shown that differentiation of sporadic HSP from PLS based on clinical characteristics is unreliable, although bulbar region UMN symptoms may support diagnosis of PLS.

Objectives: To prospectively assess the diagnostic and prognostic value of new criteria for clinical diagnosis of PLS, incorporating different levels of certainty of diagnosis of PLS instead of sporadic HSP.

Methods: 91 patients were included and classified according to our new PLS diagnostic criteria. Inclusion criteria were a gradually progressive, adult-onset (≥18 years), UMN syndrome, of ≥6 months duration. Exclusion criteria were a positive family history, clinical or electrophysiological evidence of generalized LMN involvement, and evidence of other causes.

Results: Included were 22 patients with ‘suspected PLS' (duration <4 years), 30 with ‘possible PLS' (duration ≥4 years, UMN signs only in legs), 13 with ‘probable PLS' (duration ≥4 years, UMN signs in arms and legs), and 26 with ‘definite PLS' (duration ≥4 years, UMN signs in at least bulbar region), according to our criteria. After a median follow-up of 3.0 years (range 0.1–4.3), 27 (30%) had shifted to a more certain PLS diagnosis, 7 (8%) fulfilled criteria for ALS, and 3 patients (3%; 2 ‘possible PLS', 1 ‘probable PLS') were re-diagnosed with another disorder (2 HSP, 1 corticobasal degeneration (CBD)). Conversion to ALS did not occur in ‘possible PLS'. In 3 ‘suspected PLS' patients conversion to ALS was associated with clinical LMN signs and with typical short survival (median 2.7 years, range 2.3–3.7). Overall, rate of disease progression was variable and not related to EMG findings. At last verification 21 patients had died: 4 died of ALS, 1 of CBD, 11 died indirectly related to PLS, and 5 died of unrelated causes.

Discussion: Our new PLS diagnostic criteria help to identify patients in whom PLS diagnosis is less certain, either because they may still evolve to typically progressive ALS (in ‘suspected PLS'), or because they may still have sporadic HSP instead of PLS (in ‘possible PLS' and ‘probable PLS'). Criteria for ‘definite PLS' identify patients very unlikely to have sporadic HSP, although some of these patients may develop mild LMN signs or even fulfill EMG criteria for ALS, which in our study was not clearly associated with more rapid clinical deterioration.

Conclusions: These new PLS criteria have diagnostic and prognostic value in the differentiation between sporadic HSP, typical ALS, and PLS.

C23 A RETROSPECTIVE ANALYSIS COMPARING FAST AND SLOW ALS PROGRESSION AND STUDYING FUNCTIONAL DECLINE AFTER INITIATING PEG AND BIPAP

ATASSI N^1,2, NELSON K^1,2, PALIWAL Y^1,2, CUDKOWICZ M^1,2

¹Massachusetts General Hospital, Boston, United States,²Harvard Medical School, Boston, United States

E-mail address for correspondence: [email protected]

Keywords: ALSFRS-R, PEG, BiPAP

Background: ALS Functional Rating Scale-Revised (ALSFRS-R) is a commonly used outcome measure that may predict survival in ALS clinical trials. Determining which of the 12 ALSFRS-R questions best predicts survival is valuable information for both trials and clinical practice. Bi-level Positive Airway Pressure (BiPAP) and Percutaneous Endoscopic Gastrostomy (PEG) improve ALS survival but their impact on patients' functional status is unclear.

Objectives: To determine patient characteristics and early ALSFRS-R answers that best predict future disease progression, and to compare the rate of functional (ALSFRS-R) and respiratory (FVC) decline before and after BiPAP and PEG placement.

Methods: This is a retrospective study of (519) ALS patients who participated in one of the following trials: celebrex (n = 300), creatine (n = 104), arimoclomol (n = 84), and CoQ10 (n = 31). A linear mixed effects model was used to distinguish fast progressing (FP) and slow progressing (SP) patients compared to mean ALSFRS-R decline over time. A t-test was used to compare continuous variables and a Chi-Squared test was used to compare categorical baseline variables. A Cochran-Armitage test was used to test for trends of baseline ALSFRS-R questions. A stepwise-Cox regression model was used to investigate the relationship between ALSFRS-R answers and survival. A linear mixed effects model was used to compare ALSFRS-R and FVC slopes before and after starting BiPAP and PEG. Only subjects with at least two ALSFRS-R and FVC measurements before and after BiPAP or PEG were included in this analysis.

Results: There were 41% fast progressors (FP) and 59% slow progressors (SP). Site of onset and time from symptom onset to diagnosis were different between the two groups (p = 0.03) and (p = 0.004), respectively. There was no significant difference in other baseline characteristics including age, gender, family history, baseline weight, or riluzole intake. Answers to ALSFRS-R questions at baseline visit were all significantly different between SP and FP except for handwriting (p = 0.41) and respiratory insufficiency (p = 0.17). Trial dropout rate of FP (41%) was 3.2 times more than SP (13%). Out of the 12 ALSFRS-R questions, climbing (p = 0008), writing (p = 0.04), and salivation (p = 0.04) were the only predictors of survival. ALSFRS-R rate of decline was 0.35 units/month faster after BiPAP onset (p < 0.0001) and 0.32 units/month faster after PEG placement (p < 0.0004). These results remained significant after omitting ALSFRS-R “Swallowing” and “Respiratory insufficiency” questions for PEG (p = 0.01) and BiPAP (p = 0.001) analysis. The rate of FVC decline was not significantly different after BiPAP (p = 0.09) or PEG (p = 0.94).

Discussion and Conclusion: Slow ALS progressors are more likely to enroll in and complete clinical trials. Most ALSFRS-R questions are good early predictors of disease progression. Climbing, writing, and salivation ALSFRS-R questions are good predictors of survival and none of the “respiratory” questions are. Functional decline measured by ALSFRS-R is faster after PEG or BiPAP use.

C24 TARDBP GENE MUTATION IN ALS PATIENTS: A GENOTYPE-PHENOTYPE CORRELATION STUDY

LIONNET C¹, KABASHI E², VALDMANIS P², MORALES R¹, PAGEOT N¹, DANEL-BRUNAUD V³, VANDENBERGHE N⁴, CORCIA P⁵, ROULEAU G², CAMU W¹

¹ALS Center, CHU and UM1, Montpellier, France,²Center for the Study of Brain Diseases, Montreal, Canada,³ALS Center, CHU Salengro, Lille, France,⁴ALS Center, CHU Wertheimer, Lyon, France,⁵ALS Center, CHU Bretonneau, Tours, France

E-mail address for correspondence: [email protected]

Keywords: familial ALS, TARDBP, phenotype

Objectives: To describe the phenotype of ALS patients with TARDBP gene mutations and present genotype-phenotype correlations.

Background: In approximately 10 to 20% of ALS cases, at least 2 cases are present within the pedigree, defining it as familial ALS (FALS). In 25% of those FALS cases a SOD1 mutation has been described as responsible for the disease. Recently, we and other have described TARDBP gene mutations in both sporadic and familial cases. While SOD1 mutations may modulate phenotype with rapid or slow evolution and with lower limb onset in the majority of the cases, the phenotype of ALS patients with TARDBP mutations has not been described, to date.

Methods: We describe phenotype and genotype of 9 ALS patients presenting with TARDBP gene mutations and their phenotype-genotype correlations.

Results: The Five men and four women studied had the following characteristics: mean age of onset 58±10 (range 46–78), 7 out of 9 had a upper limb onset, the disease was sporadic in 5 and within the 4 FALS cases (a mother and her son carried the same TARDBP mutation), mean disease duration was 71 months±53 (range 10–158), two patients are alive after 8 years and one of them had a tracheostomy after 5 years of ALS. Clinically all patients had both upper and lower motor neuron signs in 2 or 3 regions, with typical EMG feature, without sensory conduction abnormalities. Three patients had transcranial magnetic stimulation (TMS) and single fibre EMG (SFEMG). In all cases these examinations were abnormal. Out of the 9 patients, 6 different mutations were found. Comparison between mutations, FALS and sporadic cases suggested that FALS cases have an earlier onset and a much longer course of ALS (103 months vs 39).

Discussion: Compared with classical ALS, patients carrying TARDBP mutation have few different characteristics. The apparently most significant difference lies in the site of onset as an upper limb onset is present in 7 out the 9 patients. However, mutated patients cannot be clinically distinguished from classical ALS cases. ALS is more slowly evolving in familial cases with TARDBP mutations, as already suggested in other FALS cases with or without SOD1 mutations. To date, our series is too limited to conclude that some specific TARDBP mutations are associated with a more rapid or slow ALS in opposition with that shown with SOD1 mutations.

C25 TACKLING THE CHALLENGES OF ALS FROM A FAMILIAL PERSPECTIVE

GRONKA S, WUU J, USHER S, POLAK M, BENATAR M

Emory University, Atlanta, GA, United States

E-mail address for correspondence: [email protected]

Keywords: SOD1, pre-symptomatic, biomarkers

Background: Amyotrophic lateral sclerosis (ALS) is a disease that remains shrouded in mystery. With the exception of rare mutations in genes such as superoxide dismutase (SOD1), the etiology of ALS is unknown. Little is known about environmental risk factors for ALS. Other than electromyography and motor unit number estimation, there are no biomarkers for ALS that might permit early diagnosis or be useful in monitoring disease progression or therapeutic response. Apart from riluzole there are no effective therapies. We contend that the systematic study of asymptomatic SOD1 positive (SOD1 + ) individuals, the only population known to be at risk for developing ALS and SOD1+ ALS patients, offers several unique opportunities to unravel many of the mysteries of this disease.

Objectives: To (1) characterize the pre-symptomatic phase of ALS, (2) identify environmental factors that may modify the age of disease onset and which may be relevant to the risk of sporadic ALS, (3) develop biomarkers for early diagnosis, and (4) evaluate the safety and efficacy of arimoclomol in SOD1+ familial ALS patients.

Methods: We address these objectives through two parallel studies. The first, the Pre-familial ALS (Pre-fALS) study, is a prospective observational study. The second is a phase II/III randomized controlled trial (RCT) of arimoclomol, which targets a relevant pathophysiological mechanism and is effective in the SOD1 mouse when administered even after symptom onset. For Pre-fALS we offer genetic testing and counseling in order to identify and recruit presymptomatic SOD1+ individuals from familial ALS pedigrees. Study participants are evaluated annually using clinical, neurophysiological and neuroimaging techniques and environmental exposure assessment; biospecimens are also collected. For the RCT, we recruit individuals with recently diagnosed SOD1+ ALS. These individuals are randomized to receive arimoclomol or placebo and are followed serially over a 12-month period. The trial employs a novel ‘remote evaluation’ approach whereby most study procedures are performed in the study participant's home.

Results: As of May 2009, 68 subjects have been enrolled in Pre-fALS. Most participants who were previously unaware of their SOD1 gene mutation status, have elected to undergo genetic counseling and to learn their results. Over half of these subjects have completed their initial set of evaluations and some have already returned for their first annual follow-up visit. Twelve subjects have been screened for the arimoclomol trial and so far two subjects have been randomized.

Discussion and conclusion: Pre-fALS is unique insofar as it prospectively follows asymptomatic people at risk for ALS using a broad range of investigative modalities. The arimoclomol RCT employs a novel ‘remote evaluation’ approach and focuses exclusively on the SOD1+ population. Progress to date is evidence of the feasibility of these studies. Although currently in their early stages, they hold promise to provide unique insights into disease biology and therapy.

SESSION 4A FUNCTIONAL GENOMICS

C26 WHOLE-GENOME EXPRESSION PROFILING IN MOTOR CORTEX AND SPINAL CORD OF PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS

PARATORE S¹, ARONICA E², FRADALE F¹, CASULA M², TROOST D², CAVALLARO S¹, BAAS F²

¹Institute of Neurological sciences, Catania, Italy,²AMC, Amsterdam, Netherlands

E-mail address for correspondence: [email protected]

Keywords: gene expression, microarray

Amyotrophic lateral sclerosis (ALS) is a fatal disorder caused by the progressive degeneration of upper and lower motor neurons. Despite identification of disease-linked mutations, the etiology and pathogenesis of ALS are still elusive. By whole-genome expression profiling using Agilent 44k whole genome expression arrays and stringent significance tests we have characterized genes and pathways de-regulated in the motor cortex (43 samples) and spinal cord (31 samples) of patients with sporadic ALS. We used a two-sided one-way ANOVA using Welch's t-test and Benjamini and Hochberg False Discovery Rate procedure as a multiple testing correction. For pathway analysis we used the Gene Map Annotator and Pathway Profiler (GenMAPP) 2.0 software package. Mutations in SOD1, Ang, Tardbp, Fig4 and Chmp2b were excluded. Gene and sample numbers investigated allowed to associate genomic changes not only to disease status and tissues examined, but also to other phenotypic characteristics, such as gender and length of survival. Our findings highlight the importance of immune responses (e.g. the complement cascade and HLA), cytoskeletal (tubulin and neurofilament subunits) and signal transduction dysfunction (cell proliferation and vesicle processing), offering suggestions for new potential ALS therapies.

C27 CHROMOGRANIN VARIANTS AS RISK FACTOR AND MODIFIER OF DISEASE ONSET FOR AMYOTROPHIC LATERAL SCLEROSIS

GROS-LOUIS F¹, ANDERSEN P², DUPRE N¹, CAMU W³, MENINGER V⁴, BOUCHARD J-P¹, ROULEAU G⁵, JULIEN J-P¹

¹Laval University, Quebec, QC, Canada,²Umea University Hospital, Umea, Sweden,³Institute of Biology, Montpellier, France,⁴Hôpital Pitié Salpetrière, Paris, France,⁵Universite de Montreal, Montreal, QC, Canada

E-mail address for correspondence: [email protected]

Keywords: risk factor, modifier gene, chromogranin

Background: Recently, chromogranins were reported to interact specifically with mutant forms of superoxide dismutase (SOD1) that are linked to amyotrophic lateral sclerosis (ALS). Chromogranins can also interact with oxidized WT SOD1 but not with intact WT SOD1. This interaction is further supported by the recent report of a colocalization of chromogranins with SOD1-immunopositive aggregates in motor neurons of sporadic ALS cases.

Objective: Our objective was to screen the chromogranin B gene (CHGB) for the possible existence of sequence variations associated with ALS.

Methods: We have analyzed the frequencies of sequence variants of the CHGB gene in a total of 610 ALS patients and 561 controls from three different countries, France, Canada (French Canadian origin) and Sweden. PCR primer pairs were designed from genomic DNA to amplify each exon of the CHGB genes including the flanking splice sites. Products were PCR-amplified, checked on agarose gels, and then sequenced using the forward primer for all of the amplicons.

Results: Two novel missense variants not present in controls have been identified in the CHGB gene. Moreover, two other variants were overrepresented in ALS cases compared with controls. The combined CHGB variants were present in 11.2% of total ALS patients as compared to 4.1% in controls (odds ratio 2.9; 95% confidence interval 1.7–4.9) and conferred a 2.7 folds greater relative risk to develop the disease.

Of particular interest was the finding of a common CHGB variant that was significantly increased in ALS patient chromosomes when compared to the control population (P < 0.0001). This missense variation is predicted to have a functional effect and to alter the protein structure. The frequency of this particular variant in ALS patients of French origin (France and Quebec) was around 17% as compared to 4% in controls, conferring a relative risk of 4.1 times greater to develop disease. Furthermore, this CHGB variant lowers the age at onset by almost a decade in both SALS and FALS cases.

Discussion: These results suggest that some CHGB variants are important risk factors for ALS as well as modifiers of disease onset. The risk conferred by CHGB variants is quite robust being comparable to the over-representation by 3-folds of APOE-ε4 isoform in Alzheimer's disease patients. Our study reveals for the first time the existence of sequence variants in CHGB that increase the risk for ALS and that can associate with earlier onset of disease.

C28 CELLULAR IMPACT OF HFE H63D ALLELIC VARIANT ON ALS

LIU Y¹, LEE S¹, MITCHELL R¹, SIMMONS Z², CONNOR J¹

¹Department of Neurosurgery,²Department of Neurology; Penn State University, M.S. Hershey Medical Center, Hershey, PA, United States

E-mail address for correspondence: [email protected]

Keywords: HFE, cellular stress, SOD1

Background: In 2004, we were the first to report that a specific polymorphism in the hemochromatosis gene HFE, H63D, was over-represented in ALS. The prevalence (30%) of the H63D allele that we and the other groups reported was the second most frequent genetic variation reported in ALS. However, how H63D HFE might increase the vulnerability to ALS is as yet unknown.

Objectives: Here we continue our efforts in this new line of research to understand the mechanisms for how the presence of the H63D polymorphic allele of the HFE gene impacts ALS.

Methods: We developed a cell model by introducing tetracycline inducible expression system in human neuroblastoma SH-SY5Y cell line to determine the cellular effects of HFE H63D allele.

Results: The expression of H63D HFE mutant protein initially triggered the unfolded protein response (UPR), as revealed by the elevated level of the major UPR sensor, BiP/GRP78. This response was followed by down-regulation or cleavage of some UPR sensors, such as IRE1α and ATF4, and the activation of Caspase 3, indicating apoptosis was being promoted as a result of persistent ER stress. In addition, H63D HFE protein expression was associated with a higher level of protein oxidation, compared to wild-type protein. In the tetracycline inducible HFE expressing SH-SY5Y cell line, the presence of H63D HFE also resulted in the decreased expression and activity of Cu/Zn superoxide dismutase 1 (SOD1). Consistent with the cell culture model, we found that in ALS patients, individuals carrying H63D HFE allele have 40% less SOD1 expression in the muscle tissue compared to those with WT HFE.

Discussion and Conclusions: Together, our data suggest that the H63D HFE gene variant activates UPR, promotes apoptosis and increases oxidative stress. The presence of H63D HFE was associated with the decreased expression and activity of Cu/Zn SOD1. These results demonstrated that H63D HFE had multiple cellular consequences which might induce neuronal vulnerability. This novel cell model harboring H63D HFE may be instrumental for clarifying the relationship of this allelic variant to the disease mechanisms in ALS.

C29 HFE POLYMORPHISMS AFFECT GLUTAMATE REGULATION

MITCHELL R, LEE S, NANDAR W, NEELY E, SIMMONS Z, CONNOR J

Pennsylvania State University College of Medicine, Hershey, PA, United States

E-mail address for correspondence: [email protected]

Keywords: HFE, glutamate regulation, mouse model

Background: Glutamate neurotoxicity is one of the leading theories for neurodegeneration in ALS and the H63D HFE gene variant appears with increased frequency in patients with ALS. We developed cell and animal models that express different HFE polymorphisms to explore the mechanisms behind the relationship between HFE H63D and ALS.

Objective: To determine if the presence of the HFE H63D genetic allele, a putative disease modifier in ALS, can alter glutamate homeostasis.

Methods: Human neuroblastoma cells (SHSY5Y) were stably transfected with either wildtype (wt), H63D or C282Y variants of the HFE gene. Human astrocytoma cells were selected based on expression of HFE polymorphisms. A knock out/knock in mouse model was generated to express the H67D gene variant (mouse equivalent of the H63D variant). A series of analyses were performed to determine the effect of HFE polymorphisms on the relative amounts of glutamate secretion and uptake, and intracellular calcium levels that could impact glutamate release.

Results: Cells expressing H63D HFE have 57% greater calcium-induced glutamate secretion over a 24 hour period. Expression of wt HFE in the SH-SY5Y cells was associated with glutamate uptake 2.5-fold higher than cells expressing H63D HFE. Minocycline was examined because of its antioxidant properties and to test our hypothesis that HFE genotype will influence treatment outcomes. Minocycline treatment significantly increased glutamate uptake in vector cells and significantly decreased glutamate uptake in wt HFE cells but had no effect on glutamate uptake in the H63D cells. HFE-dependent effects on glutamate uptake were confirmed in astrocytoma cell lines. Because the cellular analyses indicated abnormalities in the glutmatergic system in the H63D cells, we determined the expression of the cystine-glutamate antiporter (xCT) which is associated with glutamate release and oxidative stress in the mouse model. At 6 months of age, the brains from wt, heterozygous and homozygous H67D mice were harvested. There is an increase in brain iron levels in both heterozygotic and homozygotic mice compared to wt. xCT is two-fold higher in the H67D mouse brains compared to age and sex matched controls. These data are consistent with increased glutamate release from cells and are suggestive of increased oxidative stress.

Discussion: This study demonstrates HFE H63D may promote glutamate toxicity in both cell and animal models. The ability of minocycline to affect glutamate uptake differed by HFE genotype and implicate oxidative stress in the HFE effects. These results have significant implications with respect to the underlying pathogenic relationship between HFE gene variants and ALS. Furthermore, the data suggest that response to Riluzole or other therapeutic agents which alter glutamate homeostasis could be influenced by HFE genotype and argue for stratification of clinical outcomes data according to HFE genotype.

C30 THE ROLES OF RNA EDITING AND CALCIUM-PERMEABLE AMPA RECEPTORS IN MOTOR NEURON DISEASE

SUN H¹, HEATH P², NOBLE B³, SHAW P², INCE P², WALKER R⁴, O'CONNELL M¹, KEEGAN L¹

¹MRC Human Genetics Unit, Edinburgh, United Kingdom,²Academic Unit of Neurology and Neuropathology, University of Sheffield, Sheffield, United Kingdom,³MRC Center for Regenerative Medicine, Edinburgh, United Kingdom,⁴Neuropathology Unit, The University of Edinburgh, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: ALS, RNA editing, GluR2

Background: One plausible hypothesis for selective neuronal death in sporadic ALS is AMPA receptor- mediated excitotoxicity. The Ca^{2 +}-permeability of AMPA receptors is largely determined by RNA editing at the GluR2 Q/R site, which is catalyzed by the ADAR2 enzyme. Professor Shin Kwak and colleagues have reported that there is deficient RNA editing at the GluR2 Q/R site in individual spinal motor neurons in Japanese sporadic ALS patients, suggesting that this is tightly linked to the etiology of sporadic ALS.

Objectives: To confirm the results in Japanese patients we obtained spinal cords (T6) of seven ALS and seven normal control subjects from the Sheffield Brain Tissue Bank in UK.

Methods: We dissected single spinal motor neurons using the PALM Laser Microdissection System and then extracted total RNA, generated RT-PCR products covering the GluR2 Q/R editing site and an adjacent intron, digested with restriction enzyme BbvI which cuts at the unedited but not the edited Q/R site and calculated the editing efficiency by measuring the relative efficiency of restriction digestion.

Results: The editing efficiency at the GluR2 Q/R site varied between 0% and 100% in 82 single spinal motor neurons of ALS patients and also in 96 from normal control subjects. There is no significant difference between the ALS and control sample sets.

Discussion: We confirm that incomplete RNA editing at the GluR2 Q/R site occurs in single spinal motor neurons of ALS patients as reported. However incomplete RNA editing was also found in normal control subjects stored in the Sheffield Brain Tissue Bank in UK. Our results for control subjects differ dramatically from the Japanese group who reported the only direct data that complete editing of the GluR2 Q/R site occurs in normal human motor neurons. We expect that reduced Q/R site editing retards splicing at the proximal splice junction of the GluR2 pre-mRNA-this may have led to a biased failure pattern in RT-PCR amplifications from single motor neurons in earlier work. The control samples from Sheffield were from, patients who died after hospitalization and there may have been some hypoxia in these patients. Therefore we obtained one spinal cord (C1 or C2) from the Edinburgh Sudden Death Bank in UK. However these motor neurons also showed incomplete RNA editing at GluR2 Q/R site.

Conclusions: We have verified the Japanese report that RNA editing at the GluR2 Q/R site is not complete in ALS motor neurons but our data so far suggests that less-than-complete RNA editing is normal in human motor neurons.

C31 MOTONEURON-SPECIFIC CHANGES IN THE EXPRESSION OF THE DRUG EFFLUX TRANSPORTER P-GLYCOPROTEIN THROUGHOUT DISEASE PROGRESSION IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS: IMPLICATIONS FOR ALS THERAPY

JACOB D, BOGUSH A, PARKAR A, PASINELLI P, TROTTI D

Thomas Jefferson University, Philadelphia, PA, United States

E-mail address for correspondence: [email protected]

Keywords: drug resistance, efflux transporter, p-glycoprotein

Background: Despite numerous animal drug trials to interfere with different pathogenic pathways, pharmacological approaches to cure the mouse that models amyotrophic lateral sclerosis (ALS) have so far failed. This suggests either yet undefined crucial pathogenic mechanisms or a frank pharmacoresistance to treatments in these mice. Multi-drug transporters mediate drug responses by influencing pharmacokinetics and pharmacodynamics and, among different drug transporter systems, the multi-drug resistance efflux transporter P-glycoprotein (P-gp; mdr1; ABCB1) extrudes a broad range of xenobiotics from cells and confers chemoresistance resulting in a poor clinical outcome. Physiologically, P-gp is predominantly localized to capillary endothelial cells in the CNS and, to a lesser extent, in parenchymal and perivascular astrocytes. Although kainate-triggered seizures lead to P-gp expression in neurons and upregulation in the cell types mentioned above, little is known about P-gp localization and distribution under pathological conditions of the spinal cord.

Objectives: Our objective is to examine P-gp distribution and expression in the spinal cord of SOD1-G93A mice during ALS disease progression.

Methods: At multiple disease stages, spinal cords of SOD1-G93A and age-matched control mice were removed, cryosectioned, and immunofluourescently labeled with an antibody that recognizes the C terminus of P-gp (H241; Santa Cruz) and the motoneuron specific antibody SMI-32. Transcript levels of P-gp from whole spinal cord samples of a separate SOD1-G93A cohort were also quantified at these disease stages using qRT-PCR.

Results: Here we report the first evidence that P-gp is expressed in motoneurons. In controls, ~60% of lumbar motoneurons express P-gp. Further, P-gp expression increases in lumbar motoneurons throughout disease progression, with ~70% and ~76% of motoneurons expressing P-gp in symptomatic and endstage animals, respectively. P-gp mRNA levels increase throughout the course of the disease, starting at onset, by ~20% and progressing to a ~50% increase at a later stage of disease.

Discussion and Conclusions: Since P-gp extrudes xenobiotics from cells, our results imply that spinal cord motoneurons may actively shunt pharmacological agents into the extracellular fluid of the spinal cord to facilitate drug extrusion by active transport systems in the blood cerebrospinal fluid barrier. Thus, P-gp localization to motoneurons may act as a secondary barrier to drug penetration into the nervous system. In pathological conditions, upregulation of efflux transporters in CNS parenchyma may provide a cellular compensatory response to extrude increased levels of endogenous toxins resulting from the disease. These combined mechanisms could constitute a powerful barrier that impedes drug delivery to the diseased spinal cord in ALS.

SESSION 4B CLINICAL ELECTROPHYSIOLOGY

C32 UNDERSTANDING THE PATHOPHYSIOLOGY OF ALS

KIERNAN M^1,2

¹Prince of Wales Medical Research Institute, University of New South Wales, Sydney, Australia,²Multidisciplinary Motor Neurone Disease Clinical Service, Prince of Wales Hospital, Sydney, Australia

E-mail address for correspondence: [email protected]

Keywords: hyperexcitability, excitotoxicity, transcranial magnetic stimulation

Establishing the presence of a combination of upper and lower motor neurone abnormalities affecting the same region is critical for the diagnosis of ALS. As such, a clear focus of current ALS research relates to the identification of the pathophysiological processes that may induce such an environment.

In terms of the lower motor neurone involvement, recent studies have identified widespread dysfunction of axonal membrane, with increased persistent Na⁺ conductances, and abnormalities of fast paranodal and internodal slow K⁺ channel function. Together, these changes contribute to the peripheral hyperexcitability typical of ALS, leading to the almost inevitable symptoms of cramps and fasciculations.

When considering upper motor neuronal involvement, ALS is characterised by progressive degeneration of the corticospinal tract, running from the brain through the spinal cord, to anterior horn cells that control all voluntary movements. Of relevance, most ALS aetiologies have been linked to an excitoxicity cascade, with excessive activation of glutamate receptors triggering destruction of motor neurones.

Recent studies that assessed cortical excitability using novel threshold tracking transcranial magnetic stimulation (TMS), demonstrated that cortical hyperexcitability was an early feature in sporadic ALS. Cortical hyperexcitability was also evident in familial patients with mutations in superoxide dismutase (SOD-1), suggesting similar pathophysiological processes operate in both sporadic and familial ALS patients.

Some have argued that reduction of short interval intracortical inhibition in ALS, and thereby the development of cortical hyperexcitability, was not a primary event, and merely reflected downregulation of inhibitory control compensating for spinal motor neurone loss. To consider this possibility, threshold tracking TMS studies undertaken in patients with spinobulbar muscular atrophy (SBMA) revealed normal cortical excitability when compared to ALS patients. Further comparison of SBMA to a pure lower motor neurone flail-arm ALS variant, established the presence of cortical hyperexcitability in the latter cohort of patients. Together, these findings indicate that the development of cortical hyperexcitability does not simply represent downregulation of intracortical inhibitory processes in ALS.

Longitudinal studies in asymptomatic SOD-1 mutation carriers recently established that cortical hyperexcitability may develop prior to the clinical onset of ALS. Reduction of short interval intracortical inhibition in ALS appears to be determined by a combination of a loss of inhibitory cortical interneurone and glutamate-mediated downregulation. These findings in SOD-1 mutation carriers were further supported in the G93A SOD-1 mouse model, where degeneration of spinal cord motor neurones occurred secondary to dysfunction within central nervous system motor pathways. Taken in total, these more recent findings may lend further support for the ‘dying forward' hypothesis, with corticomotoneurones inducing anterograde excitotoxic motoneuron degeneration. From a therapeutic perspective, neuroprotective strategies aimed at preserving the integrity of intracortical inhibitory circuits, as well as antagonizing excitatory cortical circuits, may provide novel therapeutic targets in ALS.

C33 THE NEW ELECTRODIAGNOSTIC CRITERIA FOR ALS: A RETROSPECTIVE STUDY

BOEKESTEIN W¹, KLEINE B², HAGEMAN G¹, VAN DEN BERG L³, SCHELHAAS H², ZWARTS M²

¹MST Twente, Enschede, Netherlands,²UMC Nijmegen, Nijmegen, Netherlands,³UMC Utrecht, Utrecht, Netherlands

E-mail address for correspondence: [email protected]

Keywords: Awaji, diagnostic, criteria

Background: Recently, a modification of the electrodiagnostic criteria for ALS was proposed by the Awaji commission in order to improve the sensitivity without diminishing the specificity. The commission did so, mainly by introducing 2 new concepts: 1) To accept neurophysiological evidence of lower motor neuron involvement as equivalent to clinical evidence. 2) To accept fasciculation potentials as an equivalent to fibrillation potentials or positive sharp waves. As a consequence ‘probable-laboratory-supported ALS' was no longer accepted as a separate category.

Objective: To evaluate the effect of the new diagnostic neurophysiological Awaji criteria for the diagnosis of ALS.

Methods: In a retrospective study we reviewed both clinical and neurophysiological data of 213 consecutive patients, that visited our outpatient clinic for motor neuron diseases, fromOctober 2006 until December 2008.

Results: Using the original criteria 93 patients were diagnosed with ALS: 13 definite, 38 probable, 14 probable-laboratory-supported, 28 possible. An alternative diagnosis was present in 120 patients. Using the new criteria 93 patients were diagnosed with ALS: 13 definite, 53 probable, 27 possible. Seven patients changed from possible to probable. Of the patients diagnosed as probable-lab-supported, 8 changed to probable, and 6 changed to possible. No patients with an ALS-mimic changed to the diagnosis ALS.

Discussion: The new criteria for ALS do not result in a loss of specificity and can potentially improve the sensitivity by 16% percent (15 patients). However, the fact that not all patients with ‘probable-lab-supported ALS' (requiring UMN signs in one region) fullfilled the new criteria for probable ALS (requiring two regions), nearly eliminated the diagnostic yield. For this, we propose an adaptation to the new criteria in the sense that patients, fulfilling the presence of LMN in two regions and UMN in one region are also categorized as probable ALS.

Conclusions: The Awaji modifications of the El Escorial Criteria do not result in a loss of specificity and might substantially improve the sensitivity.

C34 MODIFIED INCREMENTAL MOTOR UNIT ESTIMATION IN A LONGITUDINAL NATURAL HISTORY STUDY OF SUBJECTS WITH ALS

SHEFNER J¹, RUTKOVE S², DAVID W³, MARAGAKIS N⁴, SIMIONESCU L¹, BENATAR M⁵, WARDER J⁶, CARESS J⁷, SHARMA K⁸

¹SUNY Upstate Medical University, Syracuse, NY, United States,²Beth Israel Deaconess Medical Center, Boston, MA, United States,³Massachusetts General Hospital, Boston, MA, United States,⁴Johns Hopkins University, Baltimore, MD, United States,⁵Emory University, Atlanta, GA, United States,⁶University of Virginia, Charlottesville, VA, United States,⁷Wake Forest University, Winston-Salem, NC, United States,⁸University of Miami, Miami, FL, United States

E-mail address for correspondence: [email protected]

Keywords: motor unit number estimation, physiology, biomarker

Background: Motor unit number estimation (MUNE) has been used in previous ALS trials to assess the progress of motor axon loss and consequent re-ennervation of denervated muscle fibers by surviving neurons. A number of different MUNE methods have been described, but each method has limitations that have limited utility. Recently, we developed a method of MUNE that incorporates elements of both the incremental and multiple point stimulation technique, is rapidly performed, and has excellent test-retest variability. This method (called modified incremental MUNE) has been employed in a multicenter natural history study of electrical impedance myography and MUNE in ALS subjects at 8 participating sites.

Objectives: To determine test retest variability in normal subjects across multiple data collection sites, and assess the changes in MUNE and associated measures in subjects with ALS.

Methods: After informed consent was obtained, subjects underwent MUNE evaluation of a distal hand muscle that was felt to be moderately affected by axon loss. Either the ulnar or median nerve was stimulated at three sites that were rigidly defined, and three incremental responses were obtained at each site. The response of the third increment was added together for all three sites and divided by nine to obtain an estimate of single motor unit potential (SMUP) amplitude. This value was divided into the maximum compound motor unit action potential to yield the MUNE value. Subjects were studied approximately every 2–3 months for up to one year.

Results: 33 normal subjects were tested twice at 8 centers, after rigorous training of the evaluators. Average MUNE for normal subjects was 229 (SD: 93) for 33 median nerves, and 211 (SD: 68) for 14 ulnar nerves. Average test retest variability was 13% for median nerve studies, and 17% for ulnar nerve. Fifty one subjects with ALS have been studied longitudinally at the 8 participating sites. At onset average MUNE was 50 (range 3–192), and declined consistently over the course of the study. Average SMUP amplitude was 210 uV at study onset, and increased as disease progressed.

Discussion and Conclusions: Modified incremental MUNE can be reliably performed in a multicenter trial and yields results that show a consistent decline of MUNE over time, with corresponding increase in SMUP amplitude. This technique can be easily performed on any EMG machine, and requires no specialized software. These attributes make this method attractive for use in multicenter therapeutic trials.

C35 A PROSPECTIVE AND BLIND STUDY OF THE TRIPLE STIMULATION TECHNIQUE IN THE DIAGNOSIS OF ALS

KLEINE BU¹, SCHELHAAS HJ^1,2, VAN ELSWIJK G¹, DE RIJK MC³, STEGEMAN DF^1,4, ZWARTS MJ¹

¹Radboud University Medical Centre, Neurology and Clinical Neurophysiology, Nijmegen, Netherlands,²Netherlands ALS Centre, Amsterdam/Utrecht/Nijmegen, Netherlands,³Catharina Hospital Eindhoven, Neurology, Eindhoven, Netherlands,⁴VU University Amsterdam, Human Movement Sciences, Amsterdam, Netherlands

E-mail address for correspondence: [email protected]

Keywords: magnetic stimulation, upper motor neuron, prospective diagnostic study

Background: Demonstration of upper motor neuron (UMN) signs in several regions of the body increases the accuracy of the diagnosis of ALS, but validated tests are lacking. The importance of demonstrating subclinical UMN involvement in another region may even increase with the Awaji criteria. The triple stimulation technique (TST) combines transcranial magnetic stimulation of the motor cortex with nerve stimulation at intervals appropriate for collision. In open studies TST has a high sensitivity.

Objective: To study the utility TST in the diagnosis of ALS in a prospective study.

Methods: Fifty-nine patients were recruited to undergo TST in addition to the standard work-up for suspected motor neuron disease. The neurologist making the diagnosis was blinded to the TST results. Primary outcome was the number of abnormal TST results in patients with possible ALS. The positivity rate was also converted to the number needed to test with TST (NN-TST) for one extra diagnosis of ALS.

Results: Fifty patients underwent TST. In 19 of these patients, the amplitude ratio was abnormal. In the total patient group (n = 59), 18 patients had a motor neuron disorder but did not fulfill criteria for ‘probable' or ‘definite' ALS, and in 4 of these patients the TST was abnormal (NN-TST: 4.5). Most TST abnormalities were found in patients with clinically evident central motor disorders, resulting in an overall NN-TST of 15. In one patient the TST was erroneously interpreted as abnormal. TST findings were normal in all patients with inclusion body myositis and peripheral nerve disorders.

Conclusion: This prospective and blind study confirms open studies of TST in the evaluation of ALS. We suggest that abnormal TST can be used to arrive at a diagnosis of ‘probable' or ‘definite' ALS in patients lacking UMN signs in the upper extremities. We suggest that the equivalence of neurological signs and neurophysiological abnormalities, one of the major principles of the Awaji consensus, is valid for the upper and lower motor neuron.

C36 THE UTILITY OF SINGLE FIBER ELECTROMYOGRAPHY IN THE DIFFERENTIAL DIAGNOSIS OF AMYOTROPHIC LATERAL SCLEROSIS AND CERVICAL SPONDYLOSIS

CUI L, LIU M, LI X, GUAN Y, CHEN L

Peking Union Medical College Hospital, Beijing, China

E-mail address for correspondence: [email protected]

Keywords: single fiber electromyography, cervical spondylosis, diagnosis

Background: In the EI Escorial revised criteria for diagnosis of amyotrophic lateral sclerosis (ALS), single fiber electromyography (SFEMG) is regarded as a technique which can demonstrate the chronic partial denervation.

Objective: To assess the utility of single fiber electromyography (SFEMG) in the differential diagnosis of amyotrophic lateral sclerosis (ALS) and cervical spondylotic radiculopathy and myelopathy.

Methods: SFEMG was performed in extensor digitorum communis muscles (EDC) of three groups of patients, including 61 ALS patients with findings of cervical spondylosis on MRI, 59 ALS patients with normal MRI of cervical spine, and 55 patients with cervical spondylotic radiculopathy and myelopathy. The parameters of SFEMG between different groups were compared.

Results: The mean jitter were (81.2±25.9)µs, (91.6±32.4)µs, (40.9±11.8)µs in ALS patients with finding of cervical spondylosis on MRI, ALS patients with normal MRI of cervical spine, and patients with cervical spondylosis respectively, P₅₀ of the percentage of jitter > 55µs were 73%, 80% and 5% in the three groups respectively, P₅₀ of the percentage of block were 10%, 20% and 0% in the three groups respectively, Fiber Density were 2.9±0.5, 2.9±0.6 and 2.4±0.6 in the three groups respectively. There was no significant difference in those parameters of SFEMG between the ALS with findings of cervical spondysosis and those with normal MRI. There was significant difference in those parameters of SFEMG between the patients with ALS and those with cervical spondylosis (P < 0.01). In 18 patients with ALS, Conventional EMG studies showed active and chronic denervation in only one region at the first visit, including 10 in lower limbs, 5 in one upper limb, 3 in bulbar region. SFEMG was performed in EDC which had normal MRC and EMG. SFEMG showed Increased FD in 16 patients, increased jitter in 13 patients and impulse block in 6 patients.

Discussion and Conclusions: SFEMG showed significantly increased jitter and block in ALS whether there is MRI findings of cervical spondysosis or not, which can help to differentiate ALS from cervical spondysotic radiculopathy and myelopathy.

SESSION 6A MOTOR NEURON BIOLOGY

C37 SONIC HEDGEHOG SIGNALING AND THE CONTROL OF MOTOR NEURON AND VENTRAL INTERNEURON GENERATION

BRISCOE J

MRC National Institute for Medical Research, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Sonic Hedgehog, embryonic spinal cord, MN development

Neuronal subtype specification in the vertebrate neural tube is one of the best-studied examples of embryonic pattern formation. Distinct neuronal subtypes are generated in a precise spatial order from progenitor cells according to their location along the anterior-posterior and dorsal-ventral axes. Underpinning this organization is a complex network of extrinsic and intrinsic factors. Particularly well understood is the mechanism that determines the generation of motor neurons and interneurons that control motor output in ventral regions of the spinal cord. In these regions of the nervous system, the secreted protein Sonic Hedgehog (Shh) acts in graded fashion to organize the pattern of neurogenesis. This is a dynamic process in which increasing concentrations and durations of exposure to Shh generate neurons with successively more ventral identities. Interactions between the receiving cells and the graded signal underpin the mechanism of Shh action. Of note, the transcriptional regulation of genes induced or repressed by Shh signaling plays an essential role in shaping the graded readout. Thus the accurate patterning of the neural tube and the specification of motor neurons and the other neuronal subtypes characteristic of this region relies on the continuous processing and constant refinement of the cellular response to graded Shh signaling.

C38 FROM MOTOR CORTEX TO MUSCLE: SPECIES DIFFERENCE IN MOTOR PATHWAYS

LEMON R

Sobell Department for Motor Neuroscience and Movement Disorders, Institute of Neurology, London, United Kingdom

E-mail address for correspondence: [email protected]

Understanding the comparative functions of the corticospinal tract is of direct relevance to our understanding of how animal models can advance knowledge of the human motor system and its disorders, including ALS/MND. I will present different lines of evidence to demonstrate significant species differences in both the organisation and function of the corticospinal tract. There are also some important differences in corticospinal projections from different cortical regions within a particular species and these projections are known to support different functions. Therefore any differences in the organisation of corticospinal projections across species may well reflect differences in their functional role. These differences have probably evolved to support key elements of sensorimotor behaviour. It is now widely recognised that the corticospinal tract, in addition to its classical role as a descending motor pathway, also subserves a number of other functions. One of these is in descending control of somatosensory afferent input: this appears to be one the earliest corticospinal functions to have evolved. Another essential feature is the modulation of spinal reflex activity and interaction with central pattern generators. The capacity for direct influence over motoneurons is a relatively late evolutionary feature: direct cortico-motoneuronal (CM) projections are unique to primates, are well developed in the most dexterous non-human primates and most highly elaborated in humans. The excitatory input from these CM projections varies widely across muscle groups, being strongest to the peri-oral muscles and the muscles acting on the digits of the upper and lower extremities fingers (and especially the thumb). The highly developed nature of the CM system in humans and its pattern of projections to different muscle groups have implicated this system in some forms of ALS/MND. Studies in non-human primates will continue to be needed for understanding some special features of the human motor system, including the control of skilled hand movements and higher capacities such as tool use. These movements are often particularly vulnerable to neurological disease such as ALS/MND.

Reference:

• Lemon RN & Griffiths J Comparative anatomy of the motor system: differences in the organisation of corticospinal control in different species. In: Eisen A, editor. Clinical Neurophysiology of Motor Neuron Diseases; Handbook of Clinical Neurophysiology. Amsterdam: Elsevier, 2004. (Vol. 4, pp. 7–25)
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SESSION 6B CLINICAL TRIALS AND TRIAL DESIGN

C39 CEFTRIAXONE IN ALS: RESULTS OF STAGES 1 AND 2 OF AN ADAPTIVE DESIGN SAFETY, PHARMACOKINETIC AND EFFICACY TRIAL

CUDKOWICZ M¹, GREENBLATT D², SHEFNER J³, SCHOENFELD D¹, DELANEY K¹, SWARTZ A¹, PESTRONK A⁴, YU H¹, SHERMAN A¹, CHEN M¹, FELSENSTEIN D¹, KEROACK M¹, ROSENFELD J⁵, SIMPSON E⁶, PASCUZZI R⁷, VRIESENDORP F³, DAVID W¹, GLASS J⁸, KATZ J⁹, CARESS J¹⁰

¹Massachusetts General Hosptial, Boston, MA, United States,²Tufts Medical School, Boston, MA, United States,³SUNY at Upstate, Syracuse, NY, United States,⁴Washington University, St Louis, MO, United States,⁵UCSF Fresno, Fresno, CA, United States,⁶Methodist, Houston, TX, United States,⁷Indiana University, Indiannapolis, IN, United States,⁸Emory University, Atlanta, GA, United States,⁹CPMC, San Francisco, CA, United States,¹⁰Wake Forest, Winston Salem, NC, United States

E-mail address for correspondence: [email protected]

Keywords: clinical trials, ceftriaxone, pharmacokinetics

Background: The study aim is to determine the efficacy and safety of treatment with ceftriaxone in ALS. Ceftriaxone increases expression of the astrocytic glutamate transporter, EAAT2 and protects from glutamate, and superoxide dismutase mediated toxicity.

Objectives: We propose a novel study design strategy of nonstop drug development. We will apply data from an intermediate analyses of cerebrospinal fluid (CSF) penetration and safety toward the develoment of an efficacy study. The CSF pharmacokinetics of ceftriaxone in subjects with ALS (STAGE 1) will be followed by a safety and tolerability study for 20 weeks (STAGE 2), and then a full efficacy trial (STAGE 3). The first two STAGES are complete.

Methods: In STAGE 1, 66 subjects at ten clinical sites were enrolled, equally divided into groups receiving intravenous placebo, ceftriaxone 2g or ceftriaxone 4g daily. A plasma and CSF pharmacokinetic study was conducted at day 7. All subjects continued treatment after the STAGE 1 study and entered STAGE 2. The DSMB and part of the Steering Committee reviewed the data and decided that study should proceed to STAGE 3, at 4g/day, studying a total of 600 randomized research participants at 62 centers in US and Canada. New subjects will be randomized in a 2:1 fashion to receive treatment of (1) ceftriaxone or (2) placebo. Participants will remain in study until 52 weeks after the last participant is randomized. The co-primary outcome measures are survival and rate of change in ALSFRS-R. Secondary outcome measures include change in vital capacity, evaluation of upper and lower extremity muscles using hand-held dynamometry and quality of life and the long-term safety of ceftriaxone.

Results: STAGES 1 and 2 have been successfully completed. Ceftriaxone was found to have a volume of distribution of 13.8 liters and a plasma half-life of 8.6 hours. Plasma and CSF concentrations were closely correlated. CSF trough levels at both doses (2g and 4g) exceeded the prespecified target trough level of 1µM. At the 4g daily dosage, modeling predicted that CSF levels would stay above 1µM for 72 hours, enabling consideration of drug holidays for subjects if needed. At 20 weeks of treatment, subjects from both active treatment dose levels met the pre-specified criteria for tolerability.

Discussion: The complexity of this study has required a team of clinical investigators from several fields, toxicologists, pharmacokineticists, project managers, data managers, and biostatisticians. We successfully achieved the goals of the first two STAGES of the trial and now are proceeding with STAGE 3.

Conclusion: The novel activities of cephalosporins provide a unique opportunity to evaluate a single agent aimed at several pathways relevant to the pathophysiology in patients with ALS.

C40 KNS-760704-CL201, PART 1: A 12-WEEK PHASE 2 STUDY OF THE SAFETY, TOLERABILITY, AND CLINICAL EFFECTS OF KNS 760704 IN ALS SUBJECTS

BOZIK M¹, INGERSOLL E¹, VOLLES L¹, MATHER J¹, AMBURGEY C¹, MORITZ J¹, ARCHIBALD D¹, SULLIVAN M¹, GRIBKOFF V¹, MILLER R², MITSUMOTO H³, MOORE D², SCHOENFELD D⁴, SHEFNER J⁵, CUDKOWICZ M⁶

¹Knopp Neurosciences Inc., Pittsburgh, PA, United States,²California Pacific Medical Center, San Francisco, CA, United States,³Columbia University, New York, NY, United States,⁴Harvard University School of Public Health, Boston, MA, United States,⁵SUNY Upstate Medical Center, Rochester, NY, United States,⁶Massachusetts General Hospital, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: KNS-760704, clinical, neuroprotection

Background: KNS-760704 is a novel drug being developed for the treatment of ALS. This was a 12-week, double-blind, randomized, placebo-controlled study to evaluate the safety and tolerability of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine dihydrochloride (KNS-760704) in ALS patients. Subjects received 50, 150, or 300mg KNS-760704, or placebo daily for 12 weeks. A secondary objective was to evaluate the effects of KNS-760704 on clinical function measures including the ALS Functional Rating Scale-Revised (ALSFRS-R) and vital capacity (VC).

Methods: Key eligibility criteria were: possible to definite ALS diagnosis, ≤2 years since ALS symptom onset, and vital capacity ≥ 65% of predicted. Concomitant riluzole at a stable dose was permitted. Safety evaluations were conducted at Baseline and Weeks 1, 2, 4, 8, and 12. Clinical function was assessed at Baseline and Weeks 4, 8, and 12. Blood samples were collected in a subset of subjects after at least 1 week of dosing for a pharmacokinetic (PK) sub-study. Subjects completing the 12-week placebo-controlled study period (Part 1) were eligible to enter a double-blind, randomized active safety extension phase (Part 2). Reported here are Part 1 study results; Part 2 of the study will be completed by Nov 2009.

Results: A total of 102 subjects were enrolled at 20 US centers; 98 subjects (96%) completed the study. Twenty-three subjects participated in the PK sub-study. There were no deaths or treatment-related serious adverse events over 12 weeks. Two subjects (1 placebo; 1 300mg) discontinued due to adverse events (AEs). There were no per-treatment-group differences in the overall incidences of AEs, treatment-related AEs, or pre-specified clinically significant vital sign, ECG, or laboratory abnormalities. Two subjects (300mg) had reversible CTC grade II neutropenia, one of whom restarted treatment with KNS-760704 in Part 2 without recurrence. Mean/median changes from baseline to endpoint in ALSFRS-R total scores were −3.6/ − 4.0 (placebo), −5.0/ − 3.0 (50mg), −3.3/ − 2.5 (150mg), and −2.2/ − 2.0 (300mg). Relative to the decline in the placebo group, the 300mg group showed a 39% improvement in mean ALSFRS-R change from baseline to endpoint and a 50% improvement in median ALSFRS-R change from baseline to endpoint. Mean changes from baseline to study endpoint in upright VC (% predicted) were −13.1 (placebo), −10.8 (50mg), −6.4 (150mg) and −10.7 (300mg). Pharmacokinetics were linear over the range of doses tested and t_1/2 was ~8 hours.

Discussion: KNS-760704 was safe and well-tolerated in this study. Encouraging dose-related and time-dependent improvements in ALSFRS-R total scores relative to the placebo group observed in this study suggest that KNS-760704 may slow the rate of motor function loss in ALS subjects. Further evaluation of KNS-760704 in larger and longer studies is merited.

C41 STAR TRIAL: SUB-ANALYSIS OF AN INTERNATIONAL, MULTI-CENTER, PLACEBO-CONTROLLED STUDY OF AVP-923 (DEXTROMETHORPHAN/QUINIDINE) FOR THE TREATMENT OF PSEUDOBULBAR AFFECT (PBA) IN AMYOTROPHIC LATERAL SCLEROSIS (ALS) PATIENTS

BROOKS BR¹, CUMMINGS J², PIORO EP³, SCHIFFER R³, WYNN D⁴, HEPNER A⁵ & THE STAR TRIAL INVESTIGATORS

¹Carolinas Medical Center, Charlotte, NC, United States,²David Geffen School of Medicine, UCLA, Los Angeles, CA, United States,³Cleveland Clinic, Cleveland, OH, United States,⁴Consultants in Neurology, Northbrook, IL, United States,⁵Avanir Pharmaceuticals, Aliso Viejo, CA, United States

E-mail address for correspondence: [email protected]

Background: Pseudobulbar affect (PBA, also known as pathological laughing and crying) is characterized by exaggerated/involuntary emotional outbursts and occurs in patients with degenerative and traumatic neurological conditions including ALS. Incidence of PBA in ALS patients is particularly high (up to 50%). Dextromethorphan (DM), an NMDA-receptor antagonist and sigma-receptor agonist, reduces excitatory neurotransmission and may improve PBA. Quinidine (Q) inhibits CYP2D6 isoenzyme, slowing DM metabolism producing higher and sustained DM plasma levels. The STAR Trial has been designed to evaluate the safety and efficacy of AVP-923 for the treatment of PBA in ALS and MS patients. We report here the sub-analysis for ALS patients.

Methods: Eligible ALS patients were randomized in a 1:1:1 ratio into one of the three blinded treatment arms, to receive AVP-923-30/10 (30 mg DM/10mg Q), AVP-923-20/10 (20mg DM/10mg Q) or placebo b.i.d. for a 12-week period. Patients who completed the blinded phase of the study were eligible to participate in an open-label safety extension receiving the highest dose (AVP-923-30/10 b.i.d.) for an additional 12-week period. The main criterion for eligibility was a score of 13 or greater in the CNS-LS (Center for Neurologic Studies-Lability Scale). Primary efficacy endpoint was the number of PBA episodes recorded daily in a patient's diary; secondary efficacy endpoints were CNS-LS, NPI-Q (Neuro-Psychiatric Inventory), BDI-II (Beck Depression Inventory), SF-36 (Health Status Survey) and CSI (Caregiver Strain Index). Safety was assessed by physical examination, vital signs, clinical laboratory tests, ECGs, and oxygen saturation. Patients were asked to record any adverse experience and all medications taken in their diaries.

Results: From a total of 251 screened ALS patients, 197 (78.5%) were enrolled over a 15-month period at 68 clinical sites in USA, Argentina and Brazil. Frequent reasons for ineligibility were concomitant depression and disallowed medications. Interim demographic and baseline data are available for 148 randomized ALS patients. Gender distribution: 42% females, 58% males. Median age was 57.2 years old (Min: 25.3; Max: 75.12). Median time from ALS diagnosis at baseline was 11 months (1; 111). Median time from PBA diagnosis in ALS patients was 6 months (1; 111). CNS-LS median score at baseline was 20 (13; 35). The last patient was enrolled on 31 March 2009 with last follow-up scheduled at the end of June, 2009.

Conclusions: The STAR trial represents the largest and longest double-blind, randomized AVP-923 study to date for the symptomatic treatment by dextromethorphan/quinidine of pseudobulbar affect in ALS patients. Safety and efficacy data on primary and secondary endpoints will be available upon presentation.

C42 REGULATORY DOCUMENTS MANAGEMENT PLATFORM: EFFICIENCY, SCALABILITY, REGULATORY COMPLIANCE AND STANDARDIZATION IN CLINICAL TRIALS IN ALS

SHERMAN A, WALLACE K, KEARNEY M, BELOUIN F, CUDKOWICZ M

Massachusetts General Hospital, Charlestown, MA, United States

E-mail address for correspondence: [email protected]

Keywords: clinical trials management, project management, TREAT ALS

Background: The Neurology Clinical Trials Unit (NCTU) at MGH serves as Coordination Centre for The Northeast ALS consortium's Clinical Trials Network with the goal to “translate research advances into clinical trials for patients with ALS.” Several multi-site clinical trials and biomarker studies in ALS are coordinated simultaneously

Objective: To design and develop a comprehensive and customizable Web-based platform that brings efficiency, scalability, regulatory compliance and standardization in conducting clinical trials and biomarker studies.

Methods: Web-based TREAT ALS Platform was designed and built to manage clinical studies in ALS. It is comprised of several modules that allow Coordination Centre team to:

• Collect and maintain real-time information on: site equipment and capabilities and site members qualifications

• Follow Regulatory Documents (RD) during the life cycle of a clinical trial: from trial initiation to FDA submission.

The Platform allows users to:

• Define trial-specific roles and corresponding required RD

• Identify site member participants for trial-specific roles

• Provide RD for approval and regulatory compliance

• Verify Sites' RD completion and readiness

• Generate reports for missing and expired documents

• View site and trial participants readiness in Virtual Regulatory Binder

• Flag missing and expired documents

• Accelerate trials' initiation and subjects' enrollment

• Track IRB Submissions across multiple sites and protocol versions

• Reduce time spent on IRB submissions

• Track protocol deviations

• Increase accuracy of information

• Improve regulatory compliance

Results: The Regulatory Documents Management platform:

• Deployed at the Neurology Clinical Trials Unit at MGH

• Available to all member sites of the NEALS consortium

• Site and member information collected and maintained for 130 sites

• Sites notified via e-mail to update and upload missing & expired RD

• Collected site information utilized in sites selection for future trials

• Improved regulatory compliance

• Ability to track site metrics

• Faster trial startup and enrollment

• Utilized for Phase III trial of Ceftriaxone in ALS (58 sites) and Phase II trial of Lithium in ALS (36 sites)

Conclusion: All new clinical research NEALS-based initiatives will utilize the TREAT ALS platform. This platform adds efficiency, standardization and regulatory compliance to the conduct of ALS trials.

Acknowledgements: We thank The ALS Association for its continuing support.

C43 THE EFFECT OF INCLUSION CRITERIA ON OUTCOMES: INTERPRETING RESULTS IN HISTORICAL PLACEBO CONTROLLED TRIALS

KATZ JS, MILLER RG, MOORE DH

California Pacific Medical Center, San Francisco, CA, United States

E-mail address for correspondence: [email protected]

Keywords: trial design, historical controls, patient demographics

Background: Using historical placebo controls could reduce sample sizes and costs, and speed enrolment in ALS trials. The WALS Lithium study is the largest to use historical controls and was designed to test the large effect reported in an Italian study on Lithium. The large response would be easily refuted if the outcome appears only similar to historical controls, or confirmed if the outcome appears much superior. Detecting smaller effects, however, using this trial design is still challenging. Results from past trials have differed with respect to the observed rates of decline in placebo groups, and even small differences affect assumptions about what “true” rate of decline should be used for historical comparisons. Understanding what factors affect outcomes, including variations in enrollment criteria, recruitment methods, disease timing, phenotypes, and demographics, and creating mathematical models to control for differences would be a key step in the interpretation of results.

Objectives: To explain the differences in prior placebo outcomes from different ALS clinical trials; and to present novel methods for analyzing trial data in historically controlled trials.

Methods: Using our historical placebo database (containing 748 patients) we studied rates of decline among different trials, and created mathematical models to control differences in baseline patient characteristics that could have affected outcomes.

Results: Data from 4 past trials that used the ALSFRS-R slope decline as an outcome were used. We found differences in the mean distributions of slopes during the first six months of follow-up (range: −0.80 to −1.03). We found the differences had the closest correlations with predicted rate of decline, initial forced vital capacity (FVC), and the duration of symptoms since onset. Studies that set cutoffs with lower FVC or longer symptom durations found greater rates of decline than those using more restrictive criteria. In addition, individual patients with longer disease durations, higher baseline FVC, and slower pre-trial estimated decline tended to progress slower. The effect of timing was important because distributions of disease durations varied from trial to trial, perhaps due to definitions of “timing” that affected enrolment or due to differences in healthcare demographics. When corrections to slopes were made using a linear model, the differences narrowed and were no longer statistically significant.

Discussion and Conclusions: Differing enrolment criteria will affect mean rates of decline in trials, thus influencing observations of “true” slope declines in ALS. Subtle effects on timing could relate to regional differences in healthcare or study inclusion criteria. By using linear models that correct for the differences between the enrolled cohorts, the interpretation of outcomes in historically controlled trials may be improved.

SESSION 7A EMERGING DISEASE MODELS

C44 IPS CELLS AS IN VITRO MODELS OF ALS

CROFT GF¹, WEYGANDT MD¹, OAKLEY DH¹, BOULTING G², KISKINIS E², LAMAS NJ¹, ROYBON L¹, WICHTERLE H^1,3, EGGAN K², HENDERSON CE^1,3

¹Project A.L.S. Laboratory for Stem Cell Research, New York, NY, United States,²Harvard Stem Cell Institute, Boston, MA, United States,³Columbia University, Depts. of Pathology, Neurology and Neuroscience, Center for Motor Neuron Biology and Disease, New York, NY, United States

E-mail address for correspondence: [email protected]

Keywords: motor neuron differentiation, stem cells, drug testing

Research into ALS disease mechanisms, and the testing of candidate drugs, have been hindered by the inaccessibility of the cell types directly affected in human patients: motor neurons and surrounding glia. Since it is not feasible to sample these cells from human patients, we are exploring the potential of human stem cell-derived motor neurons. Our aim is to recreate in the culture dish the conditions that lead to ALS, using human cells with the same genotype as ALS patients. Several steps toward this goal have been accomplished. First, we have created multiple lines of induced pluripotent stem (iPS) cells from ALS patients and healthy controls. Second, we have shown that these iPS cells can be maintained as undifferentiated stem cells and then differentiated into spinal motor neurons. We are currently exploring means by which these iPS cells can be used to recreate the ALS disease process. Our results using human ES (embryonic stem) cells show that we can: (a) produce motor neurons of different subtypes by patterning with extrinsic factors; (b) generate enriched cultures of astrocytes; (c) detect toxicity of potential environmental factors for motor neurons; (d) validate survival assays for human motor neurons. Future research should determine whether iPS-MNs derived from ALS patients show different properties from control iPS-MNs in these or other respects.

Supported by Project A.L.S., the Harvard Stem Cell Institute, the Claire and Leonard Tow Foundation, MDA Wings Over Wall Street, and the New York Stem Cell Foundation.

C45 EVALUATION OF FUNCTIONAL MOTOR NEURONS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS (IPS) AND HUMAN EMBRYONIC STEM CELLS (HESC)

WIEDAU-PAZOS M¹, KARUMBAYARAM S¹, KORNBLUM H², NOVITCH B², UMBACH J³, LOWRY W²

¹Department of Neurology;²Broad Stem Cell Institute,³Department of Pharmacology, UCLA Los Angeles, CA, United States

E-mail address for correspondence: [email protected]

Keywords: stem cells, disease model, neurodegeneration

Background: Stem cells have the potential to treat neurodegenerative disorders. Their value may also lie in the opportunity to model human diseases in vitro, for which animal and other cell culture models have not yielded an understanding of underlying disease mechanisms, or new therapeutic approaches. The excitement surrounding pluripotent stem cell (IPS) technology is predicated upon the potential to treat disease or injury with derivatives of patient-specific stem cells. While the reprogramming of human somatic cells to an embryonic state is fast becoming commonplace, the potential for the differentiation of reprogrammed cells to prospectively defined post-mitotic neuronal phenotypes remains challenging.

Objectives: Here, we evaluated whether the methods shown to be effective at generating motor neurons from human embryonic stem cells (HESCs) were similarly efficient in differentiating motor neurons from iPS.

Methods: Fibroblasts from human skin samples were cultured and directed toward stem cell lineage by expressing the four transcription factors KLF4, OCT4, SOX2, and C-MYC.

Strains of IPS and hESC were differentiated in parallel using retinoic acid, sonic hedgehog and multiple growth factors. Maturation and lineage was followed using immunohistochemistry with neuronal and motor neuron specific markers, PCR of developmental and neuronal markers and patch clamping to assess single cell electrophysiology.

Motor neurons derived from ALS-subject IPS and hESC that express SOD1 mutations linked to familial ALS were analyzed for cell survival by manual cell counts and the distribution of intracellular SOD1 by immunocytochemical staining.

Results: The IPS and hESC appeared to follow a normal developmental progression when directed to motor neuron lineage expressing developmental stage-specific markers and neuronal markers such as TUJ1, ChAT, Islet1 and Hb9 at similar time lines. Importantly, the resulting motor neurons from both strains possessed cell-type specific electrophysiological properties.

When motor neurons from ALS subjects or from hESCs that express mutant A4V, G93A or I113T SOD1 mutants are evaluated for cell survival, all strains exhibit increased cell death when compared to controls, which was statistically significant on Student's t-test. In addition, when staining for intracellular SOD1 protein, IPS and hESC-derived motor neurons are positive for aggregated SOD1 inclusions.

Discussion: We demonstrate the feasibility of using IPS and transgenic hESC-derived motor neurons for in vitro modelling of motor neuron disease. Several typical features of motor neuron degeneration are present in our model system. Further evaluation will characterize the process of neurodegeneration in stem cell derived motor neurons in more detail to help optimize these cell model systems, which can aide in the study of disease pathways of ALS in sporadic and familial ALS and help screen for new therapies.

C46 A SOD1 ZEBRAFISH MODEL OF ALS SHOWS INDUCTION OF EARLY CELLULAR STRESS

RAMESH T¹, DILLER A², WANG C², BURGHES A², BEATTIE C²

¹University of Sheffield, United Kingdom,²Ohio State University, Columbus, Ohio, United States

E-mail address for correspondence: [email protected]

Keywords: animal models, zebrafish, HT screening

Background: Mutations in the SOD1 gene have been shown to cause classic ALS in numerous families. Transgenic mice over expressing mutant SOD1 suggest a dominant gain of function that is independent of SOD1 enzymatic activity but the mechanism of this toxicity is currently under debate. One excellent approach to uncover mechanism is to identify interacting genes. However the ability to perform suppressor screens is limiting in mouse models due to the low number of animals that can be tested and their high costs. Thus, additional animal models of ALS would benefit the field.

Objectives: Our goal is to generate another vertebrate model of SOD1 FALS that is amenable to genetic analysis.

Results: We have developed a transgenic SOD1 overexpression model of zebrafish that carries disease causing sod1 mutation in the zebrafish SOD1 gene (SOD1). The zebrafish and human SOD1 genes are very similar and 87% of the amino acids associated with causing ALS are conserved in the fish gene. A zebrafish BAC containing the genomic region of sod1 was used to clone a 20 kb fragment containing the SOD1 gene and flanking sequences for generating the transgenics. Founder (F1) fish were identified from F0 mating and the lines expanded. We show that like mice, the ageing adult transgenic fish show evidence of motor neuron loss, muscle atrophy and death. This demonstrates that zebrafish, like mice and humans, can mimic the ALS phenotype accurately, thus validating the use of zebrafish in studying ALS/MND.

In generating our transgenic lines, we incorporated the promoter for zebrafish heat shock protein 70 (hsp70) regulating the expression of the fluorescent molecule DsRed as a way to track our transgenic fish. We found that in SOD1 mutants, but not in SOD1 wildtypes, the hsp70 promoter was induced without heat exposure as indicated by DsRed expression. At this stage the mutant zebrafish embryos appear grossly normal and show no visible behavioural abnormalities as compared to wildtype SOD1 transgenics. The mutant sod1 specific heatshock response occurs as early as 24 hpf and occurs in the brain and spinal cord, the cell types affected in ALS.

Discussion: The sod1 zebrafish model of ALS mimics most aspects of motor neuron disease. The early embryonic readout of mutant SOD1 induced heatshock response could be very valuable for identification of chemical and genetic modifiers of mutant SOD1 activity and help in further understanding of ALS pathogenesis.

C47 FUNCTIONAL CHARACTERIZATION OF MUTANT TDP-43 IN ZEBRAFISH

KABASHI E^1,2, ROULEAU GA², DION P², DRAPEAU P¹

¹Department of Pathology and Cell Biology, Université de Montréal, Montréal, QC, Canada,²Centre of Excellence in Neuromics, Université de Montréal, Montréal, QC, Canada

E-mail address for correspondence: [email protected]

Keywords: TDP-43, transgenic zebrafish, pharmaceutical screening

Amyotrophic lateral sclerosis (ALS) is an adult-onset rapidly progressing neurological disorder characterized by loss of motor neurons, cells which control muscle movement. Approximately 10% of ALS cases are familial (FALS). Recently, we made the breakthrough discovery of a considerable number of TARDBP mutations in FALS and sporadic ALS (SALS) patients, the gene encoding TDP-43, the protein that aggregates in inclusion bodies from the spinal cord of ALS patients. For this project, we have developed a series of human TDP-43 cDNA constructs tagged at both the C-terminus and N-terminus which will be used to determine mutant and WT TDP-43 expression in cell lines, primary motor neurons and zebrafish embryos. Here we demonstrate that mutant TDP-43 causes a specific motor neuron disorder through a toxic gain of function since expression of mutant TDP-43, but not WT, causes selective motor neuron toxicity in primary spinal cord cultures. Further, expression of mutant causes a specific motor phenotype and motor neuron axonal defects in zebrafish embryos. On the other hand, decreased expression using anti-sense morpholino oligonucleotide (AMO) of TDP-43 yielded a similar phenotype in zebrafish; thus a specific loss of function may also be involved in the selective vulnerability of motor neurons to mutant TDP-43. A further characterization of these models will allow us to unravel molecular partners of mutant TDP-43 as well as to allow a better understanding of specific mechanisms of disease involved in mutant TDP-43 caused motor neuron degeneration. We are also generating TARDBP knock-outs in zebrafish as well as mutant TDP-43 transgenic fish lines. These lines will be functionally characterized to determine whether they properly model motor neuron disorders. These lines can then be tested for the screening of a large number of pharmaceutical compounds in transgenic zebrafish expressing mutant TDP-43, thus directly opening avenues for the development of therapies that could delay or prevent disease onset and progression in all ALS patients.

C48 TDP43 TOXICITY IN THE CHICK EMBRYO MODEL SYSTEM

TRIPATHI V¹, AL-CHALABI A¹, GUTHRIE S², SHAW C¹

¹Institute of Psychiatry, London, United Kingdom,²Department of Developmental Neurobiology, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: chick embryo, TDP43, apoptosis

Background: The TAR DNA binding protein (TDP-43) is the major protein in ubiquinated inclusions (UBIs) in FTLD-U and ALS. A phosphorylated 25-kD C-terminal fragment of TDP-43 and high-molecular weight ubiquitinated aggregates are enriched in detergent resistant fractions of FTLD-U and ALS brains. We aim to use the chick embryo model system to understand the function of TDP43 protein in vivo.

Methods: We electroporated TDP-43^WT, TDP-43^Q331K and TDP-43^M337V DNA into the spinal cords of HH stage 14 chick embryos using in ovo electroporation. All the 3 TDP 43 constructs were cloned into vectors carring different tags (Myc + HA or GFP). SOD1^G93A-dsRed, SOD1^WT-dsRed electroporation was used as a positive control and GFP electroporation as the negative control. After 24 hours the embryos showing normal development were processed for frozen sectioning and then apoptotic cell death was observed using TUNEL assay. Fluroescent microscopy was used to establish the cellular localisation of the wild type and the mutant TDP-43 protein.

Results: SOD1^G93A-dsRed transfected neurons showed cell death as demonstrated by TUNEL staining (number of embryos analysed = 36/36). We also observed a decrease in the population of motor neurons (stained for Islet1/2) on the transfected side of the embryo as compared to the non-transfected control side. TUNEL staining demonstrated that while GFP transfection results in some apoptotic cells, there is a significant increase in apoptotic nuclei in spinal cord sections of embryos electroporated with N-terminal HA and C-terminal Myc tagged TDP-43^WT, TDP-43^Q331K and TDP-43^M337V constructs. A two-tailed t-test of GFP vs HA-TDP-43^WT-Myc gave a p-value of 0.1252; GFP vs HA-TDP-43^Q331K-Myc gave a p-value of 0.0179 and GFP vs HA-TDP-43^M337V-Myc gave a p-value of 0.0079. In order to confirm that the Myc and HA tags were not causing the increased apoptosis, we carried out electroporation of GFP tagged TDP-43^WT, TDP-43^Q331K and TDP-43^M337V. Once again, TUNEL staining showed an increase in the number of apoptotic nuclei in embryos electroporated with either TDP-43^WT, TDP-43^Q331K or TDP-43^M337V as compared to those electroporated with pEGFPC1. A two-tailed t test of GFP vs GFP-TDP-43^WT gave a p-value of 0.0337; GFP vs GFP-TDP-43^Q331K gave a p-value of 0.0026 and GFP vs GFP-TDP-43^M337V gave a p-value of 0.0045. The mutant forms of TDP 43 showed pre-dominantly cytoplasmic localisation with nuclear inclusions while TDP 43^WT was pre-dominantly nuclear. A two-tailed t-test of of HA-TDP-43^WT-Myc vs HA-TDP-43^Q331K-Myc gave a p-value of 0.0650; HA-TDP-43^WT-Myc vs HA-TDP-43^M337V-Myc gave a p-value of 0.0263; GFP-TDP-43^WT vs GFP-TDP-43^Q331K gave a p-value of 0.0120 and GFP-TDP-43^WT vs GFP-TDP-43^M337V gave a p-value of 0.009

Conclusions: Over-expression of not only TDP-43^Q331K and TDP-43^M337V but also, TDP-43^WT causes neurotoxicity in the chick embryo spinal cord. The mutant forms of TPD-43 localise in the cytoplasm and show nuclear inclusions while TDP-43^WT localises in the nucleus.

SESSION 7B SPIRITUAL CARE AND MEANING IN LIFE

C49 MEANING IN LIFE AND PERSONAL VALUES IN ALS: EMPIRICAL DATA AND CLINICAL RELEVANCE

KOEGLER M, BRANDSTAETTER M, JOX R, WASNER M, HAARMAN-DOETKOTTE S, BORASIO GD, FEGG M

Interdisciplinary Center for Palliative Medicine, Munich University Hospital, Grosshadern, Munich, Germany

E-mail address for correspondence: [email protected]

Keywords: meaning in life, Schedule for Meaning in Life Evaluation (SMiLE), spirituality

Loss of meaning in life (MiL) is known to be one of the main determinants of requests for hastened death. Empirical data with respect to personal values and MiL in ALS patients and their clinical relevance will be discussed.

In a first study, the relationship between personal values and individual quality of life was evaluated in 28 ALS patients and 36 patients suffering from advanced cancer (1). The most important values were benevolence, self-direction and universalism, whereas power, achievement and stimulation were the least important. Self-transcendence values were higher than self-enhancement values in all patients. Compared with healthy adults, palliative care patients scored significantly higher in benevolence and lower in self-enhancement values. There were no significant differences between ALS and cancer patients. The observed shift towards self-transcendence values may be related to coping processes of terminally ill patients.

In another study (2), MiL was assessed with a newly developed instrument for the evaluation of individual MiL. In the Schedule for Meaning in Life Evaluation (SMiLE), the respondents list 3 to 7 areas which provide meaning to their life before rating the current level of satisfaction and importance of each area. Indices of total satisfaction (IoS), total weighting (IoW), and total weighted satisfaction (IoWS, range 0–100) are calculated. 49 ALS-patients were interviewed at the Interdisciplinary Center for Palliative Medicine, Munich University Hospital-Grosshadern. All open answers were assigned to 13 categories found in the representative survey (3). The results were compared to those of the representative sample (n = 1,004).

MiL was slightly lower in ALS-patients (SMiLE-Index = 76.6±20.2) compared to the representative sample (83.3±14.8; p = 0.03; d = 0.4). The areas of MiL mentioned most often were family (82%), partnership (61%), leisure activities (53%) and friends (39%). Partnership and leisure activities were listed significantly more often by ALS-patients compared to the representative sample, while work and health were mentioned significantly less often (p < 0.001 for each). This was shown by logistic regression analyses; age, marital status and education were included as additional predictor variables.

Overall MiL (SMiLE-Index) was slightly lower in ALS-patients compared to the representative sample. Health and work were less important for ALS-patients, relations to significant others, especially partnership and leisure time were listed more often. This could be due to “response shift” in the progress of the disease, i.e. a change of internal standards (4). Identifying ALS-patients who are at risk of losing their MiL and helping them to find alternative sources of MiL may be an important step towards preventing requests for hastened death.

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C50 EVIDENCE BASED SPIRITUAL CARE IN ALS: FACT OR FICTION?

ROSER T^1,2

¹Ludwig Maximilians University, Munich, Germany,²Evangelical-Lutheran Church of Bavaria, Munich, Germany

E-mail address for correspondence: [email protected]

Keywords: measuring spirituality, spiritual care training, pastoral care

Spiritual care is an essential part of holistic medicine, yet the methods of intervention in professional spiritual care seem to be somewhat obscure. What exactly does a spiritual care provider do in existential interventions for people facing a life threatening illness? Is there any evidence spiritual care is based on?

The talk starts with a wide and open understanding of spirituality where religion may or may not be part of a patient's spirituality: The realm of spirituality includes issues of meaning, transcending, becoming, and relating, depending on the individual. The spiritual relates to essential issues in palliative care, e.g. quality of life, hope, coping, anxiety, and awareness of dying. Tools measuring spirituality work with varying constructs of spirituality, depending on the expected outcome. Especially with ALS patients, constructs include an understanding of spirituality as a resource to the patient, as an area of conflict or suffering, as a search for meaning while nearing the end of life. These constructs affect the way, a multiprofessional team includes spiritual issues in care. Some studies show the importance of a patient's spirituality for treatment decisions at the end of life.

Spiritual Care, however, not only focuses on the spirituality of the patient, but also the needs and resources of relatives, and of care providers. Studies point out the importance of spirituality for care providers, raising the question whether training in spiritual care makes sense.

Since spiritual issues are an integral part of a whole person/whole care approach to medicine, the question remains to be answered, who (i.e. which profession) should provide spiritual care? Is spiritual care-performed by doctors-another tool that helps physicians to better connect with patients, to better understand them as people? Or should the provision of spiritual care be reserved to chaplains? In order to answer these questions, the talk will present findings from studies on pastoral care in palliative care settings in Germany, giving facts on actual spiritual care, and their inclusion in a multiprofessional approach to whole person care. Some cases with ALS patients will be discussed that put spiritual care into the frame of evidence based reality instead of fiction.

C51 SPIRITUALITY AND FAITH: MEANS FOR COPING WITH THE EFFECTS OF ALS/MND

O'BRIEN M¹, CLARK D²

¹Edge Hill University, Lancashire, United Kingdom,²Lancaster University, Lancaster, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: spirituality, faith, coping

Background: Spirituality and personal faith are recognised as important factors contributing to quality of life amongst those diagnosed with ALS/MND, assisting individuals to develop perspective and influencing their decisions as they adapt to living with the disease (1).

Objectives: To explore the personal experience of living with ALS/MND as documented in personal illness narratives, written by people diagnosed with the illness and examine the role of spirituality and faith as a means of coping with ALS/MND.

Methods: We previously reported the processes used for locating published and unpublished personal illness narratives about life with ALS/MND (2). The 161 narratives identified were subject to content and thematic analysis. Data management was aided by Nvivo 7 software.

Results: There is frequent reference throughout the narratives to the power of spirituality to impact positively on the illness experience. Authors refer to the strength acquired from seeking a spiritual understanding of their circumstances. There is a sense of being used for a ‘higher purpose’, being seen as a spiritual example to others. Religious convictions are frequently strengthened following the diagnosis, bound up in a feeling of not being burdened beyond their ability to cope. Faith is regarded by some as a cornerstone of the ability to cope. Belief in an afterlife sustains many authors as they recognise the present time as a small part of a much longer time span.

Conclusions: It is evident within the narratives that people with ALS/MND tolerate distress through maintaining hope by belief in a divine entity and through connection with a higher being (3). Spirituality should be regarded as an important resource for coping with ALS/MND.

Reference:

• Murphy et al. Neurology 2000; 55: 1581–1584
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C52 MANAGING DEATH IN ALS/MND: IDENTIFYING CAREGIVERS’ PERSPECTIVES OF THE FINAL DAYS OF LIFE

RAY R¹, BROWN J²

¹James Cook University, Queensland, Australia,²University of Southampton, Southampton, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: caregivers, dying, advanced planning

Background: Family caregivers usually remain central to the provision of care throughout the illness trajectory. Yet, little is known about how they manage or cope with the dying process and the death of their relative with ALS/MND.

Objective: This study sought to understand the experiences and support needs of family caregivers, during their relatives' final days of life.

Methods: Longitudinal studies of family caregiving in ALS/MND were undertaken in Australia and United Kingdom. Primary caregivers participated in in-depth interviews at four month intervals throughout the illness trajectory, including the bereavement phase. Five cases from each country were purposively sampled for analysis. A process of open and axial coding was used to categorise the data and identify themes as they emerged from each caregiver's story.

Results: In the broadly similar socio-cultural structure of Australia and the United Kingdom, family caregivers in both countries expressed similar experiences of their relative dying. However, their construction of the dying process varied, exhibiting delicately nuanced differences in their experiences and their responses. In eight out of ten cases, patients were cared for at home until their last days of life. All the United Kingdom cases died in hospital/hospice. Three of the Australian cases died at home and two died in hospital/hospice. Conversations between the caregiver and the dying person, about end of life, were only evident in six out of ten cases.

The most prominent themes across all cases were dignity, emotional labour, advanced care planning, spiritual sustenance, reflective guilt, and redefinition. Caregivers were often unprepared for the rapid deterioration and sudden death of their relative, prolonging their sense of loss and disconnection.

Discussion and Conclusion: The increasing trend towards dying at home is both challenging and rewarding for family caregivers. The quality of life focus, prominent during the earlier phases of living with ALS/MND, can be easily over looked in the medicalised processes of dying. When caregivers reflected on the dying experience and said “we got it right”, they lived with the positive affirmations of caregiving. However, if the dying experience was characterised by conflict about decision making, then negative memories are perpetuated.

This study demonstrated the need for an inclusive process of effective communication between the patient, the family and health and social care professionals, to prepare for dying. There is a need to develop communication skills to facilitate conversations that will support patient and family caregiver decision making and enhance their coping skills. While it is recognised that patients and caregivers have to be emotionally ready to have these conversations, the often unpredictable, degenerative nature of ALS/MND increases the need to facilitate preparation for dying in a timely manner.

SESSION 7C FUS MUTATIONS IN FAMILIAL ALS

C53 MUTATIONS IN THE RNA BINDING GENE FUS CAUSE FAMILIAL ALS

VANCE C¹, ROGELJ B¹, HORTOBAGCI T¹, DE VOS K¹, NISHIMURA AL¹, SREEDHARAN J¹, HU X¹, SMITH B¹, RUDDY D¹, WRIGHT P¹, WILLIAMS K², TRIPATHI V¹, AL-CHALABI A¹, LEIGH N¹, BLAIR I², NICHOLSON G², DE BELLEROCHE J³, GALLO J-M¹, MILLER C¹, SHAW C¹

¹Institute of Psychiatry, Kings College London, London, United Kingdom,²ANZAC Research Institute, University of Sydney, Sydney, Australia,³Imperial College, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: FALS, FUS, Mutations

Background: Familial ALS accounts for 5–10% of cases and recently mutations in the gene TDP-43 were described in both familial and sporadic ALS (1). Following this, genes with similar functions in the chromosome 16 linkage region (2) were prioritised for mutation screening.

Objectives: To identify the causative mutation in the chromosome 16 locus

Methods: In addition to the family (F1) who had previously been linked to chromosome 16, we identified 197 suitable FALS index cases from the UK and Australia, who had previously been screened and found to be negative for mutations in known ALS genes (SOD1, ANG, VAPB, Dynactin, CHMP2B and TARDBP). Exons of genes of interest were amplified and subsequently sequenced using BDT v1.1 on an ABI 3100.

Results: A point mutation was identified in exon 15 of the gene Fused in Sarcoma (FUS or TLS) in all affected members of the F1 family. This 1561 C- > T change in the gene was predicted to result in an arginine to cysteine change at position 521. In a survey of 197 FALS index cases we identified two further missense mutations in eight families. Post-mortem analysis of three cases with FUS mutations showed FUS-immunoreactive cytoplasmic inclusions and predominantly lower motor neuron degeneration. Cellular expression studies revealed aberrant localization of mutant FUS protein.

Conclusions: We have identified mutations in a new gene for familial ALS. FUS shares functional domains with TDP-43 and the identification of mutations in a second RNA binding protein indicates the importance of RNA metabolism to motor neurons.

Reference:

• Sreedharan et al. Science. 2008;319(5870):1668–72
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C54 FUS MUTATIONS IN A LARGE SERIES OF SPORADIC AND FAMILIAL ALS

Lai S-L¹, Abramzony Yg¹, Dunckley T², Stephan DA², Battistini S³, La Bella V⁴, Salvi F⁵, Mandrioli J⁶, Caponnetto C⁷, Siciliano G⁸, Monsurrò MR⁹, Mora G¹⁰, Sabatelli M¹¹, Brunetti M¹², Schymick JC¹, Traynor BJ¹, Restagno G¹², Chiò A¹³

¹Neuromuscular Diseases Research Group, Laboratory of Neurogenetics, NIA, Bethesda, MD, United States,²Neurogenomics Division, Translational Genomics Institute (TGEN), Phoenix, AZ, United States,³Department of Neuroscience, Neurology Section, University of Siena, Siena, Italy,⁴Department of Clinical Neurosciences, University of Palermo, Palermo, Italy,⁵Center for Diagnosis and Cure of Rare Diseases, Department of Neurology, Bellaria Hospital, Bologna, Italy,⁶Department of Neuroscience, S. Agostino- Estense Hospital, and University of Modena, Modena, Italy,⁷Department of Neuroscience, Ophthalmology and Genetics, University of Genova, Genova, Italy,⁸Department of Neuroscience, University of Pisa, Pisa, Italy,⁹Department of Neurological Sciences, Second University of Napoli, Napoli, Italy,¹⁰Salvatore Maugeri Foundation, IRCCS, Milano, Italy,¹¹Neurological Institute, Catholic University and I.CO.M.M. Association for ALS Reseach, Roma, Italy,¹²Molecular Genetics Unit, Department of Clinical Pathology, A.S.O. O.I.R.M.-S.Anna, Torino, Italy,¹³Department of Neuroscience, University of Torino, Torino, Italy

E-mail address for correspondence: [email protected]

Keywords: FUS gene, missense mutations, synonymous mutations

Background: Recently, the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene, located on chromosome 16p11.2, has been identified as a disease gene in familial amyotrophic lateral sclerosis (FALS).

Aim: The aim of this study was to further define the spectrum of FUS/TLS gene mutations in a large series of FALS and sporadic ALS (SALS) patients.

Methods: DNA samples from 52 index cases with FALS were collected from eight Italian ALS referral centres and from 1,749 SALS of American and Italian origin. The entire coding region was sequenced in all FALS and 276 SALS. As previous publications have implicated exon 15 as a hotspot for mutations within this gene, this exon was sequenced in an additional 1,473 SALS. Detected variants were evaluated in a control cohort consisting of 280 neurologically normal Italian subjects and 460 neurologically normal US subjects.

Results: Among the 1,749 SALS patients we found a total of 7 cases with missense mutations (0.4%). All mutations have been detected in exon 15: c.A1552G (leading to p.R518C) (1 case), cC1561T (p.R521C) (1 case), c.C1561G (p.R521G) (2 cases), c.G1562A (p.R521H) (1 case), c.C1574T (p.P525L) (1 case), andc.C1575T (p.P525R) (1 case). Moreover, in 5 cases we found the synonymous mutation c.G1566A (p.R522R). None of these variants were present in controls.

Among 52 index cases of FALS we identified a heterozygous c.G1542C missense mutation in a family of northern Italian origin, and a heterozygous c.C1574T missense mutation in a family of Sicilian origin. Both variants are located in exon 15 encoding the RNA-recognition motif, and result in a substitution of an arginine with a serine in position 514 (p.R514S) and substitution of a proline with a leucine at position 525 (p.P525L) respectively. Overall, the two mutations accounted for 3.8% of 52 non-SOD1 and non-TDP43 index cases of FALS. A synonymous mutation in exon 15 (c.G1566A, p.R522R) of another FALS index case was also detected. None of these variants were present in controls.

Discussion: FUS/TLS gene mutations have been found in 4% of non-SOD1 non-FTD43 FALS patients and in less than 0.5% of apparently SALS patients. Two novel heterozygous missense mutations in exon 15 have been found (c.A1552G and c.G1574T); also a synonymous mutation (c.G1566A) not detected in healthy controls has been found in 5 SALS cases and 1 FALS case.

The other members of the ITALSGEN Consortiumare: F Lombardo (Torino), I Ossola (Torino), A Calvo (Torino), C Moglia (Torino), S Cammarosano (Torino), K Maurinou (Milano), A Conte (Roma), M Luigetti (Roma), P Sola (Modena), I Bartolomei (Bologna), C Carlesi (Pisa), R Spataro (Palermo), P Paladino (Palermo), C Ricci (Siena), F Giannini (Siena), GL Mancardi (Genova), G Tedeschi (Napoli)

C55 IDENTIFICATION OF FUS/TLS GENE MUTATIONS IN A COHORT OF ITALIAN FALS PATIENTS

TICOZZI N^1,2, SILANI V¹, LECLERC AL², KEAGLE P², GELLERA C³, RATTI A¹, TARONI F³, KWIATKOWSKI TJ⁴, MCKENNA-YASEK DM², SAPP PC^2,5, BROWN RH², LANDERS JE²

¹Department of Neurology and Laboratory of Neuroscience, “Dino Ferrari” Center, University of Milan Medical School, IRCCS Istituto Auxologico Italiano, Milan, Italy,²Department of Neurology, University of Massachusetts Medical School, Worcester, MA, United States,³Unit of Genetics of Neurodegenerative and Metabolic Diseases, Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Milan, Italy,⁴Department of Neurology, Massachusetts General Hospital, Boston, MA, United States,⁵Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States

E-mail address for correspondence: [email protected]

Keywords: genetics, ALS6, FUS/TLS

Background: Approximately 10% of all ALS cases are familial (FALS). Mutations in SOD1 and TARDBP genes account for ~20% and ~5% of all FALS cases, while variants in other genes (ALS2, SETX, VAPB, DNCT1, and ANG) have been described in isolated pedigrees. Recently, mutations in the FUS/TLS gene have been identified in ALS families with linkage to chromosome 16 (ALS6).

Objectives: To perform a genetic screening of FUS/TLS to assess the mutational frequency among Italian FALS families.

Methods: We sequenced all 15 coding exons of FUS/TLS in 94 unrelated Italian patients with a diagnosis of probable or definite ALS according to the El Escorial revised criteria and a positive family history for motor neuron disease. All the participating individuals were negative for SOD1, TARDBP, and ANG mutations.

Results: We found four missense heterozygous mutations in five FALS patients. Two mutations (p.R521G and p.R521C) have been previously identified and were observed in one and two cases, respectively. Two novel mutations, p.G156E and p.R234L, were found in one patient each, and were absent in a panel of 376 age- and ethnically matched healthy controls.

Discussion: Arginine 521 is located in the Nuclear Localization Signal (NLS) of FUS/TLS protein, and its substitution with a glycine or cysteine has been shown to result in an aberrant subcellular distribution and apparent aggregation of the mutant protein in the cytoplasm. The p.G156E and p.R234L mutations are located in the SYQG-rich and RGG-rich domains of the protein, whose biological functions are still unknown. We hypothesize, however, that p.G156E and p.R234L are pathogenic since the residues at these positions are highly conserved throughout evolution and because these variants were not observed in our control panel. The clinical phenotype of our mutated patients was characterized by a predominance of lower motor neuron signs, and by a symmetrical, proximal and axial weakness at onset. Interestingly, a single patient also developed frontotemporal dementia (FTLD).

Conclusions: Our study provides evidence that FUS/TLS mutations account for ~3% of all Italian FALS patients, representing the third most important identified cause of FALS after SOD1 and TARDBP. It also suggests that patients carrying FUS/TLS mutations may have an uncommon clinical phenotype, possibly including FTLD.

C56 NOVEL FUS MUTATIONS IN A LARGE COHORT OF FALS PATIENTS

YAN J¹, DENG H-X¹, SIDDIQUE N¹, FECTO F¹, CHEN W¹, YANG Y¹, LIU E¹, DONKERVOORT S¹, ZHENG JG¹, SHI Y¹, AHMETI K¹, BROOKS BR², ENGEL WK³, SIDDIQUE T¹

¹Davee Department of Neurology and Clinical Neurosciences, Northwestern University Feinberg School of Medicine, Chicago, United States,²Department of Neurology, Neuroscience and Spine Institute Carolinas Medical Center, Charlotte, United States,³USC Neuromuscular Center, Good Samaritan Hospital, Los Angeles, United States

E-mail address for correspondence: [email protected]

Keywords: FUS, mutation, genetics

Background: Amyotrophic lateral sclerosis (ALS) is a fatal progressive paralytic disease caused by degeneration of motor neurons. Most of the ALS cases are sporadic (SALS), and approximately 5–10% of ALS cases are familial (FALS). Mutations in the Cu, Zn superoxide dismutase (SOD1) are responsible for about 20% of the FALS cases and mutations in the TDP43 cause ALS in about 3% of FALS cases. Mutations in the other identified genes are rare. Recently, 14 mutations in the FUS have been found in 26 FALS cases among a combined cohort of 491 FALS cases. Most of these cases are of Caucasian origin. The FUS mutations in the other populations have not been studied. The clinical features of the patients with the FUS mutations are largely unknown.

Objectives: To explore the spectrum of FUS mutations in FALS

Methods: Genetic variations of the FUS were determined by direct sequencing of all the 15 exons in 461 SOD1- and TDP43- negative FALS index cases, the largest single FALS cohort studied to date. Additionally, over 500 controls were analyzed for each genetic variant observed. Clinical data from the patients with the FUS mutations were compared to those from patients with known SOD1 mutations.

Results: We carried out comprehensive genetic and clinical analyses of the FUS mutations in a large cohort of FALS cases from different ethnic groups. We have analyzed all the coding exons of the FUS in 461 FALS cases, the largest data set analyzed to date. All the FALS cases were excluded for SOD1 and TDP43 mutations. We identified 13 FUS mutations in 22 FALS index cases or families, among which seven mutations are novel. Most of the mutations are missense, but mutations involving small deletions, splicing and truncations were also found. These mutations are aggregated in two clusters in FUS, thus providing potential functional relevance of the ALS-linked FUS mutations. We also found that the patients with the FUS mutations appeared to have higher rate of the bulbar onset and shorter duration of the disease when compared with the patients with the SOD1 mutations.

Discussion and Conclusions: Our data have demonstrated that the FUS mutations are a common genetic cause in FALS patients of different genetic backgrounds. The prevalence of the FUS mutations in the non-SOD1 and non-TDP43 FALS is about 5% (4.77%), or approximately 4% in all of FALS cases. Thus, the FUS mutations appear to be the second most frequent genetic cause of FALS after the SOD1 mutations. Patients with the FUS mutations manifest higher rate of bulbar onset and shorter duration of the disease.

C57 FUS MUTATIONS IN BELGIAN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS PATIENTS

VAN DAMME P^1,2, GORIS A¹, RACE V³, VAN DEN BOSCH L¹, MATHIJS G³, ROBBERECHT W^1,2

¹Experimental Neurology KU Leuven and Vesalius Research Center, VIB, Leuven, Belgium,²Neurology Department, Leuven University Hospital, Leuven, Belgium,³Center for Human Genetics, KU Leuven, Leuven, Belgium

E-mail address for correspondence: [email protected]

Keywords: FALS, FUS, dominant

Mutations in FUS were recently identified as a novel cause of familial amyotrophic lateral sclerosis (ALS). The frequency of occurrence of mutations in FUS in sets of familial ALS patients remains to be established. We therefore sequenced FUS in a Belgian set of familial SOD1-negative ALS patients.

The 15 exons and exon-intron boundaries of FUS were sequenced in 26 familial ALS patients from 19 different families. We identified a R521H mutation that segregated with disease in a kindred of dominantly inherited ALS. Mutations in FUS are responsible for 3% of pedigrees or 5% of familial ALS patients in our cohort of familial ALS.

These results show that mutations in FUS are a significant cause of familial ALS in Belgium.

C58 MUTATIONS OF FUS/TLS GENE IN A LARGE CHINESE PEDIGREE WITH AMYOTROPHIC LATERAL SCLEROSIS

ZHENG H, ZENG Y, FANG D, GUO X, SHANG H

Department of Neurology, West China Hospital, SiChuan University, Chengdu, SiChuan, China

E-mail address for correspondence: [email protected]

Keywords: SOD1 gene, liposarcoma (FUS/TLS) gene

Background: Amyotrophic lateral sclerosis (ALS) is the most common and fatal degenerative motor neuron disease in adults. Approximately 20% familial ALS (FALS) have been found Cu/Zn superoxide dismutase (SOD1) gene as the causative gene. Recently, 5% FALS was reported to be caused by liposarcoma (FUS/TLS) gene mutation.

Objectives: To screen for mutations of FUS/TLS gene in a big ALS Chinese family without mutation of SOD1 gene.

Methods: All living subjects who consented for this study were examined by at least two independent neurologists for the diagnosis of ALS (according to the El Escorial and revised El Escorial criteria). They also underwent electromyographic (EMG) examination to establish lower motor neuron (LMN) dysfunction in non-symptomatic body regions. FUS/TLS gene mutation screening was performed by directly sequencing after informed consent.

Results: This non consanguineous Chinese family consisted of 5 generations and had an autosomal dominant inheritance pattern. Twelve patients including 9 males and 3 females were found. Ten were dead. Mean onset age was 40 year-old. The duration of the disease is about 1.5 years. The cause of death is breathing difficulty. We found a mutation (R521C) of sarcoma/translated in liposarcoma (FUS/TLS) gene in the pedigree. However, two possible patients harbored two reported polymorphisms including rs741810 (p.G49G) in exon3 and rs1052352 (p.Y97Y) in exon 4 with absence of this mutation.

Conclusions: Our data supports the fact that SOD1 mutations are rare in Chinese familial ALS patients and indicates FUS/TLS mutations may be more common. However, variant intrafamilial phenotype in the family resulting from polymorphisms remains unknown.

C59 SCREENING FOR FUS MUTATIONS IN AMYOTROPHIC LATERAL SCLEROSIS

GROEN E¹, VAN ES M¹, VAN VUGHT P¹, VAN ROEKEL H², SPLIET W¹, VELDINK J¹, CUPPEN E², VAN DEN BERG L¹

¹Rudolf Magnus Institute of Neuroscience, Department of Neurology,²Department of Biomedical Genetics; University Medical Center Utrecht, Utrecht, Netherlands

E-mail address for correspondence: [email protected]

Keywords: FUS, mutations, familial ALS

Background: Recently, mutations in the FUS gene were shown to be associated with familial amyotrophic lateral sclerosis. Here we investigate these mutations in cases of hereditary motor neuron disease (including ALS, PMA and SMA).

Objectives: To investigate FUS mutations in familial forms of motor neuron disease.

Methods: We included 51 families with a family history of motor neuron disease. All patients were screened and were negative for mutations in SOD1, ANG, VAPB and TARDBP, as well as deletions of SMN1 and CAG repeats in the androgen receptor gene. We used capillary sequencing of whole-genome amplified DNA using BigDye 3.1 chemistry on all 15 exons. Mutations will be confirmed in a separate PCR and sequencing reaction on genomic DNA. For exons containing mutations, at least 900 neurologically normal controls are sequenced to confirm ALS specificity.

Results: Several mutations which were previously described are also found in our experiments in 5 out of 51 index cases (9.8%).

Conclusions: We are able to replicate previous findings on FUS mutations in our patient population. Work is as of yet ongoing and further research into clinical phenotype of these patients has yet to be carried out.

C60 MUTATION SCREENING IN TRANSLOCATED IN LIPOSARCOMA GENE IN PATIENTS WITH MOTOR NEURONE DISEASE

HEWITT C, KIRBY J, HIGHLEY R, HARTLEY J, HIBBERD R, HOLLINGER H, GOODALL E, INCE P, MCDERMOTT C, SHAW P

University of Sheffield, Sheffield, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Translocated in Liposarcoma gene, FUS, RNA splicing and transport

Background: Familial motor neurone disease (MND) accounts for 5–10% of all MND cases. Recently mutation in the Translocated in Liposarcoma (TLS) gene has been identified as a cause of MND. TLS, also known as FUS (fusion involved in t(12;16) in malignant liposarcoma), is a 53KDa widely expressed protein. TLS is a multifunctional protein, possessing distinct N-terminal transcriptional activating and C-terminal RNA binding domains and has been implicated in a number of cellular processes including transcriptional regulation, RNA splicing and transport, and maintainance of genomic stability. Previous neuropathological reports of TLS MND cases have demonstrated an absence of TDP-43 positive inclusions which is the major pathological change seen in sporadic and non-SOD1 familial MND.

Objectives: To identify the frequency, phenotype and molecular pathology of MND associated with TLS mutation.

Methods: DNA was extracted from CNS tissue and whole blood of 168 MND patients, including 49 familial cases. Intronic based primers were used to amplify the 15 exons of TLS/FUS. PCR products were bi-directionally sequenced and analysed using Sequencher software. Autopsy tissue from 4 cases with nucleotide substitutions was assessed for neuropathology.

Results: Two novel mutations (p.Arg524Trp and p.Gly507Asp), were identified in 2 familial and 1 sporadic MND patients. These changes were not identified in 295 control individuals. The phenotype in these individuals was of an upper limb onset with predominantly lower motor neurone involvement. Neuropathological study of two of these cases showed marked lower motor neurone loss and either absent or very few ubiquitinated inclusions and no TDP-43 positive inclusions.

Two further changes were identified (p.Gly228_Gly229insGly and pGly174ValfsX183) in 1 sporadic and 1 familial case. These changes were identified at low frequency in control cases (0.3%) raising the possibility that they may represent coincidental polymorphisms. Neuropathological study of these cases revealed lower motor neurone loss with TDP-43 positive inclusions. These neuropathological findings support the possibility that in these further cases, the MND phenotype was not due to the changes identified in TLS.

Discussion: Mutations in FUS/TLS have been found in a large cohort of MND cases, supporting this gene as a cause of MND. However, stringent screening of controls and demonstrable functional consequences of the “mutations” is necessary to ensure that the nucleotide changes identified are pathogenic for MND.

SESSION 8A PROTEIN REGULATION AND DEGRADATION

C61 MOLECULAR MECHANISMS FOR THE ATROPHY AND PROTEIN LOSS FROM SKELETAL MUSCLE UPON DENERVATION AND SYSTEMIC DISEASE

GOLDBERG A, ZHAO J, BRAULT J, COHEN SV

Dept of Cell Biology, Harvard Medical School, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: muscle atrophy, denervation, protein breakdown

Whether a muscle grows or atrophies depends on the balance between rates of protein synthesis and degradation. Much has been learned recently about the mechanisms for muscle wasting seen with nerve injury, disuse, and systemic diseases (e.g. cancer, cachexia, sepsis, fasting, excess glucocorticoids). In these diverse conditions, loss of muscle mass is due mainly to an acceleration of protein degradation, primarily by the ubiquitin proteasome pathway. In various types of atrophy, we have identified a common program of changes in the expression of about 100 atrophy-related genes which we've termed “atrogenes”. Amongst the genes most dramatically induced are the ubiquitin ligases, atrogin-1 and MuRF1 whose induction is necessary for rapid atrophy. During atrophy, certain proteins that stabilize the myofibril are selectively ubiquitylated by MuRF1 and are lost differentially, which appears to facilitate disassembly of the thick filaments. MuRF1, however, is not critical in the degradation of thin filament components which require different ubiquitination enzymes.

In atrophying muscles, signaling by the IGF-1-p13K-Akt-FoxO pathway decreases, and activation of the FoxO family of transcription factors play a critical role in muscle atrophy. Activation of FoxO3 is necessary for muscle wasting upon denervation or fasting and by itself causes rapid atrophy. FoxO3 causes induction of many atrogenes including atrogin-1 and MuRF1, and dominant negative FoxO3 mutations can block denervation atrophy.

The possible importance of lysosomes in muscle atrophy has received little attention. Using selective inhibitors of the lysosomal and proteasomal pathways, we were able to demonstrate that FoxO3 coordinately activates both processes. Activated FoxO3 stimulates lysosomal proteolysis in muscle (and other cell types) by activating autophagy and induces the expression of many autophagy-related genes. These autophagy-related genes are induced similarly in mouse muscles atrophying due to denervation or fasting and causes loss of mitochondria. These findings are the first evidence for coordinated regulation of the proteasomal and lysosomal systems which serve complementary roles in degrading different muscle components during atrophy, the myofibrillar apparatus by the ubiquitin-proteasome pathway and mitochondria by autophagy.

Muscle contractions can retard atrophy, in part by enhancing the production in muscle of IGF-1, which stimulates protein synthesis and blocks FoxO activation. Exercise also enhances production of the transcriptional coactivator, PGC-1a, which increases mitochondrial content, and in muscle directly inhibits the FoxO-dependent activation of the atrophy program. When overproduced, PGC-1a and its homolog PGC-1b were found to reduce muscle protein degradation and denervation atrophy. However, pharmacological enhancement of PGC-1a by AICAR treatment of mice maintained mitochondrial content but did not reduce fiber atrophy. By contrast, other approaches to block atrophy (e.g. by blocking the myostatin-Smad pathway) seem very promising.

C62 VPS54 MUTATION IN WOBBLER MOUSE MND CAUSES TDP-43 MISLOCALIZATION AND PATHOLOGY AS IN ALS

PIORO E, CHE K, KOSTENKO VR, MAHAJAN S

Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States

E-mail address for correspondence: [email protected]

Keywords: TARDBP, inclusions, ubiquitination

Background: The ubiquitously expressed nuclear TAR DNA-binding protein (TARDBP) 43 (TDP-43) has been implicated in the pathogenesis of ALS. Although TARDBP gene mutations occur in some patients with familial ALS, essentially all patients with sporadic and most with familial ALS contain TDP-43 which is insoluble, ubiquitinated, hyperphosphorylated, and mislocalized to the cytoplasm of motor neurons. Studying animal models with TDP-43 pathology not due to TARDBP mutations may provide insights into its pathogenic mechanisms in sporadic and non-TARDBP familial ALS. MND in the wobbler (wr) mouse results from a mutation in the vacuolar-vesicular sorting protein 54 (Vps54), which disrupts intracellular trafficking as occurs in human ALS. We have found TDP-43 pathology in the wr mouse.

Objectives: We will characterize the TDP-43 proteinopathy in wr MND and determine how closely it recapitulates this pathology in human ALS.

Methods: The presence of TDP-43 protein was examined by immunohistochemistry and immunoblotting in eight week-old wr cervical spinal cord (CSC), an age and location where pathology is prominent, in comparison to healthy littermates. One, two, and 20-week old mice were also examined immunohistochemically. Immunostaining of 30 µm-thick sections was performed for either TDP-43 alone or in relation to markers of pathology (e.g., ubiquitin) and cell type (e.g., ChAT). Relative amounts of TDP-43 immunoreactivity and its state of ubiquitination were examined in cytoplasmic and nuclear fractions of homogenized CSC. Soluble proteins in these samples were sequentially extracted to determine if TDP-43 was relatively insoluble, and ubiquitinated.

Results: TDP-43 immunoreactivity is mislocalized to the cytoplasm of primarily cholinergic CSC motor neurons as granular, globular, or filamentous inclusions where it becomes ubiquitinated, although it also appears increased in neuronal nuclei. Most prominent when motor neuron degeneration is advanced, intracytoplasmic TDP-43 reaction product increases just prior to onset of clinical disease (7–14 days old). Subcellular fractionation reveals a unique protein signature of increased TDP-43 immunopositivity in both the cytoplasmic and nuclear fractions of wr CSC, including ~25–30 kD bands not present in the wildtype. Wobbler TDP-43 protein is also ubiquitinated and insoluble as in ALS.

Discussion: TDP-43 proteinopathy occurs in the wr mouse in a way similar to human ALS, including cytoplasmic mislocalization, ubiquitination, insolubility, and the presence of ~25–30 kD bands, which in ALS represent pathologic cleaved C-terminal fragments. These abnormalities in the wr involve not only the cytoplasm but also the nucleus, suggesting pancellular TDP-43 pathology which may have implications to ALS.

Conclusions: The wr model of MND provides a framework for investigating the pathogenic mechanisms of TDP-43 toxicity in the absence of TARDBP gene mutations, which represents the majority of ALS. Identifying how the wr Vps54 mutation causes TDP-43 proteinopathy may reveal similar pathogenic mechanisms in human ALS.

C63 ABNORMAL INTRACELLULAR STORAGE IN LOSS OF FUNCTION OF FIG4

KATONA I¹, ZHANG X¹, BAI Y¹, HATFIELD J², SHY M¹, KUPSKY W³, LI J^1,2

¹Wayne State University, Department of Neurology, Detroit, MI, United States,²John D. Dingell VA Medical Center, Detroit, MI, United States,³Wayne State University, Department of Pathology, Detroit, MI, United States

E-mail address for correspondence: [email protected]

Keywords: Fig4, lysosomal storage, electron microscopy

Fig4, a phosphatase, regulates the level of PI(3,5)P2 and plays an important role in intracellular vesicular trafficking, particularly for the endosomes/lysosomes. Our previous studies have demonstrated that mutations in human Fig4 gene cause a recessive form of Charcot-Marie-Tooth disease type-4J (CMT4J) with a phenotype of rapidly progressive and asymmetric motor neuron degeneration, thus resembles motor neuron disease. A recent report also suggests heterozygous mutations of Fig4 are a genetic susceptibility factor for acquiring amyotrophic lateral sclerosis (ALS). Mice with homozygous loss of function mutation of the fig4 gene, called pale tremor (plt) mice, develop severe neuronopathy, and may serve as an animal model for investigating mechanisms of neurodegeneration. However, the pathological alterations in the plt CNS have not yet been well characterized. To investigate these issues, we performed an electron microscopic (EM) study on the mouse CNS. Cytoplasm of all cortical neurons and many glial cells in plt mice were filled with numerous high electron-dense granules that were reminiscent of lysosomes. Similar pathological changes were identifiable in about one third of the spinal anterior horn neurons. In contrast, this accumulation was not detectable in plt dorsal root ganglion (DRG) neurons and rarely visible in wild-type cells of CNS. Instead, plt DRG neurons contained excessive vacuoles that were single-membrane delimited and appeared electron transparent. Immunoreactivity against lysosomal proteins, NPC1 and LAMP2, was strong in many cells of plt brains, but hardly detectable in any wild-type cells. This was consistent with a robust increase of NPC1 and LAMP2 on Western blots, but not for mannose-6-phosphate receptor, an endosomal protein that is excluded from the lysosomes. In addition, cholesterol was also increased in plt cells. Taken together, our data suggest that Fig4 deficiency causes an abnormal lysosomal storage in the nervous system. We speculate that abnormal intracellular storage may be a pathogenic mechanism for neurodegeneration in the disease, and exert differential effect between spinal motor and sensory neurons. Supported by NINDS, MDA and Hiller ALS Foundation.

C64 DYSFUNCTION OF THE ENDOSOME PATHWAY INDUCED BY SOD1 MUTANTS

TURNER B, FARG M, HORNE M, ATKIN J

Howard Florey Institute, Melbourne, Australia

E-mail address for correspondence: [email protected]

Keywords: endosome, exosome, SOD1

Background: Secretion and extracellular action of SOD1 mutants is implicated in motor neuron degeneration in ALS, however the nature of the secretory pathway responsible remains unclear. ER-Golgi transport is proposed to mediate SOD1 secretion in culture, however evidence for SOD1 presence in the ER is conflicting at present. Endosomes represent one alternative export pathway and mediate secretion via exosomes originating from multivesicular bodies. The presence of SOD1 in the endosome compartment may therefore constitute another extracellular source.

Objectives: To investigate the contribution of the endosomal pathway to SOD1 secretion and relationship of endosomal function to cell pathology in familial ALS models.

Methods: NSC-34 cells stably expressing wild-type or mutant SOD1 were examined for expression of early and late endosomal markers using Western blotting and immunocytochemistry. Exosomes were purified by ultracentrifugation of cell conditioned medium and characterised by immunoblotting, sucrose density gradients and electron microscopy. Conditioned and exosome-depleted media were analysed as controls. Exosomes were also extracted and studied from cerebrospinal fluid from adult sheep as an animal model.

Results: SOD1 was identified in medium fractions enriched for the exosomal markers flotilin and TSG101, but not nuclear, mitochondrial or Golgi proteins. Isolated exosomes also conformed to predicted morphology, diameter and density. Exosomal SOD1 secretion was inducible by depolarisation and monensin treatment, while wortmannin blocked release in culture. In stable cell lines, wild-type SOD1 accumulated at high level in exosomes, while all mutants (dismutase active and inactive) were depleted from exosomes. Expression of late endosomal markers such as TSG101 was preserved in mutant cells, however early endosome proteins such as Rab5 were downregulated. Interestingly, analysis of exosome-depleted medium revealed presence of wild-type, but not mutant SOD1, suggesting a second pathway of secretion for normal SOD1. SOD1 was also identified in exosomes derived from ovine cerebrospinal fluid, confirming its exosomal secretion in vivo.

Conclusions: We demonstrate that SOD1 secretion is mediated by the endosome system which is disrupted by ALS-linked mutations, consistent with our previous findings of impaired mutant SOD1 secretion in a cellular model. The endosome pathway may therefore constitute a new possible therapeutic target for modulating extracellular SOD1 levels in ALS.

C65 LOSS OF RAC1-INDUCED MACROPINOSOMAL AND ENDOSOMAL LOCALIZATION OF ALS2 UNDERLIES THE PATHOGENESIS FOR MOTOR NEURON DISEASES CAUSED BY MISSENSE MUTATIONS IN THE ALS2 GENE.

OTOMO A, KUNITA R, SUZUKI-UTSUNOMIYA K, IKEDA J-E, HADANO S

Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan

E-mail address for correspondence: [email protected]

Keywords: ALS2, missense mutation, endosomal localization

Background: Mutations in the ALS2 gene account for a number of juvenile motor neuron diseases (MNDs), such as a juvenile recessive form of ALS (ALS2), a rare juvenile recessive form of primary lateral sclerosis (PLSJ), and an infantile-onset ascending hereditary spastic paralysis (IAHSP). Loss of function of its gene product ALS2/alsin accounts for neuronal dysfunction and degeneration. We and others have reported that ALS2 enhances endosome fusion by activating Rab5. Further, we have shown that ALS2 acts as a novel Rac1 effector, implicating a mode of endocytosis called macropinocytosis. Thus, ALS2 can mediate the Rac1-Rab5 signalling pathway, thereby regulating macropinocytosis and the following endosome trafficking and fusion. Recently, two missense mutations in the ALS2 gene were identified. These mutations result in mutants (ALS2^C157Y and ALS2^G540E) carrying a single amino-acid substitution in the regulator of chromosome condensation 1-like domain (RLD) located in the N-terminal region with the preservation of the C-terminal Rab5GEF domain. However, the molecular mechanisms by which these pathogenic mutants cause ALS2-linked MNDs are unclear.

Objectives: To elucidate the molecular mechanisms governing the functional loss of ALS2 due to a single amino-acid substitution in the RLD, we investigated molecular and cellular functions of these two pathogenic mutants.

Methods: Constructs expressing wild-type (ALS2^WT) and mutant ALS2 (ALS2^C157Y and ALS2^G540E) were generated. Interactions between ALS2 mutants and either Rac1 or Rab5 were examined by in vitro GST pulldown assays. Rab5GEF activities of the pathogenic mutants were measured by in vitro [³H]GDP dissociation assay on Rab5A. Homo-oligomerization of the mutant proteins was evaluated by co-immunoprecipitation experiments. Rac1-induced redistribution of the ALS2 mutants within the cells was assessed by immunocytochemical analysis.

Results: Similar to ALS2^WT, both ALS2^C157Y and ALS2 ^G540E interacted with Rac1 as well as with Rab5. Further, these mutants preserved the Rab5GEF activities in vitro, and the oligomerization capability, as does ALS2 ^WT. However, unlike ALS2^WT, these pathogenic mutants failed to localize to the Rac1-induced macropinosomes. These results indicate that while the ALS2 pathogenic mutants can still interact with Rac1, they lose their function as a Rac1 effector in cells. This could lead to the mislocalization of ALS2 within cells, and thus to loss of ALS2 function as a Rab5 activator on the macropinosome and endosome compartments.

Discussion and Conclusions: Our results indicate that the RLD of ALS2 is crucial to maintain the proper subcellular localization and function of ALS2. Thus, loss of Rac1-induced macropinosomal and the following endosomal localization of ALS2 might underlie the pathogenesis for MNDs caused by missense mutations in the ALS2 gene. Future studies will provide insights not only into the fundamental role of ALS2 in macropinocytosis and the endosomal maturation in neurons but also into the pathogenesis underlying the ALS2-linked MNDs.

SESSION 8B COGNITIVE CHANGE

C66 FRONTOTEMPORAL DEMENTIA AND MOTOR NEURONE DISEASE

NEARY D^1,2, SNOWDEN J^1,2, MANN D¹

¹University of Manchester, Manchester, United Kingdom,²Salford Royal Foundation Trust, Manchester, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: frontotemporal lobar degeneration, clinicopathological correlation, behaviour

An association between frontotemporal dementia (FTD) and motor neurone disease (MND) is now well recognised. Nevertheless, the precise nature of the relationship is not yet fully understood. It is not known whether people with FTD/MND are representative of the larger population of patients with forms of frontotemporal lobar degeneration (FTLD) or whether there are clinical, pathological and genetic differences. Similarly, it is unclear whether FTD/MND is a unique syndrome, clinically and aetiologically distinct from classical MND, traditionally thought to be a pure motor disorder, or whether there is a clinical continuum between the two.

In this talk I examine data from a cohort of more than 500 patients with clinical syndromes of frontotemporal lobar degeneration (FTLD) and compare the demographic, clinical and, where available, pathological characteristics of those individuals who develop MND and those who do not. The data reveal similarities between patients with and without MND in terms of age at onset and frequency of family history but highly significant differences in terms of gender ratio and illness duration. There are commonalities between patients with and without MND in cognitive/behavioural presentation, the majority showing the behavioural/executive disorder of FTD and a minority, features of semantic dementia or progressive nonfluent aphasia. However, in contrast to the pathological heterogeneity within FTLD, which encompasses tau and non-tau pathologies, and three sub-types of ubiquitin histopathology, the patients with MND show pathological homogeneity, sharing an identical tau-negative, ubiquitin positive histology. Molecular studies reveal no patient with MND to have mutations in the tau or progranulin gene.

Complementing these data, I describe the findings from a small-scale study of behaviour in MND. The findings point to a spectrum of behavioural change, with some patients exhibiting no behavioural alterations whereas others fulfil behavioural criteria for FTD.

The implications of these data for the “continuum” vs. “phenotypic variant” notion of the relationship between FTD and MND are discussed in the light of advances in molecular biology and genetics.

C67 EARLY VERBAL FLUENCY DEFICITS PREDICT COGNITIVE IMPAIRMENT IN ALS: A POPULATION-BASED LONGITUDINAL STUDY

PHUKAN J^1,2, GALLAGHER L³, JORDAN N³, PENDER NP³, HARDIMAN O^1,2

¹Department of Neurology, Beaumont Hospital, Dublin, Ireland,²Trinity College Institute of Neurosciences, Dublin, Ireland,³Department of Psychology, Beaumont Hospital, Dublin, Ireland

E-mail address for correspondence: [email protected]

Keywords: cognitive, population, longitudinal

Background: Up to 60% of people with ALS have mild cognitive decline; a smaller proportion develop frontotemporal dementia. The population-based frequency, clinical characteristics and natural history of cognitive decline in ALS are unknown.

Objective: To determine the frequency and natural history of cognitive decline in ALS in a defined population-based cohort, using the latest consensus criteria (1).

Methods: All incident patients with ALS are captured on the Irish ALS Register and are asked to participate in a longitudinal study of cognitive function. Each participant is assessed using an extensive neurological and neuropsychological battery at 3 time intervals over 18 months.

Results: A population-based cohort of 87 patients and matched controls were studied. Clinical characteristics reflect the overall demography of ALS in Ireland. Twenty two (25%) had bulbar-onset disease. A total of 46 (53%) had deficits in verbal fluency on first assessment. Based on consensus criteria of 2 standard deviations below the control mean on two or more cognitive tests, 32.2% of ALS patients had cognitive impairment (ALSci) at time of first assessment. 7 (8%) patients were diagnosed with behavioural impairment (ALSbi+ as per supportive Neary criteria); 20.7%of patients were however behaviourally impaired as measured by a 2-standard deviation (SD) change from premorbid levels on either a FrSBe subscale or FrSBe total score. Apathy was the most marked behavioural change. Total behavioural change in patients predicted caregiver burden and mood.

17 (19%) patients met Neary criteria for FTLD: 12 for behavioural variant FTD, 2 for non-fluent progressive aphasia, and 3 for semantic dementia. Of those who tested within the normal range on the first assessment, only 3 (7%) had evidence of cognitive impairment on subsequent testing. Conversely, those who had verbal fluency deficits on first testing had clear evidence of deteriorating function on subsequent testing.

Conclusions: ALS is associated with a high prevalence of cognitive and behavioural change. Early deficits in verbal fluency predict later cognitive impairment. However, those with normal cognitive function at baseline are unlikely to exhibit any cognitive or behavioural deterioration. These data suggest that ALS with cognitive impairment represent a distinct subpopulation of ALS and not a continuum.

1. Consensus criteria for the diagnosis of frontotemporal cognitive and behavioural syndromes in amyotrophic lateral sclerosis.

References:

• Strong et al. Amyotrophic Lateral Sclerosis, 2009; 10(3):131–46
   PubMed Web of Science ®Google Scholar

C68 VALIDITY OF A BRIEF COGNITIVE SCREENING EXAM FOR ALS PATIENTS

MURPHY J, AHMED F, LOMEN-HOERTH C

UCSF, San Francisco, CA, United States

E-mail address for correspondence: [email protected]

Keywords: ALS, FTD, screening

Background: Recent investigations suggest that 28–48% of ALS patients possess a spectrum of frontotemporal deficits on neuropsychological measures, and patients with these cognitive and behavioral changes have poorer compliance and reduced survival rates. In this study, a brief screening battery was compared with a thorough neuropsychological evaluation to determine whether a time-efficient, cost-effective screening exam can be a valid instrument in identifying ALS patients with cognitive and behavioral changes.

Objectives: This 30 minute cognitive screen is predicted to be a valid indicator of the cognitive and behavioral functioning of ALS patients. Specifically, diagnoses based on screening instruments are predicted to correlate with diagnoses based upon separate, standardized measures used in a full two hour neuropsychological battery. The diagnostic categories included ALS, ALS with Cognitive Impairment (ALSci), and ALS with Behavioral Impairment (ALSbi).

Methods: Based upon performance on a thirty minute screening battery, 14 study participants were classified into three diagnostic categories: ALS, ALSci, and ALSbi. Using a separate 2-hour neuropsychological evaluation, independent diagnoses were made. Comparisons were made between screening diagnoses and diagnoses made using standardized, age-matched norms. The screening diagnoses were based upon a cut off score of 12 or below on the ALS-Cognitive Behavioral Screen (ALS-CBS) and a score of 27 or above on the the Frontal Behavioral Inventory (FBI). Full battery diagnoses of ALSci were based on a 5^th percentile cut off on two or more measures of functioning. Full battery diagnoses of ALSbi were based on a T score of 65 or above on the Frontal Systems Behavioral Scale.

Results: In 12 of the 14 cases studied for cognitive functioning, matching diagnoses were made using the 30 minute battery. In two cases, the screening tests identified subjects as impaired but when given a full battery and requiring strict cut offs for pathology, they were diagnosed as normal. When measuring behavioral symptoms, 5 of the 7 subjects had matching diagnoses. In both cases, the screening test diagnosed the subject as normal but the full battery identified them as behaviorally impaired.

Discussion and Conclusions: These data suggest that the screening tool may be an effective instrument in identifying ALS patients with cognitive and behavioral changes, particularly when measuring cognitive changes. Behavioral changes may be more accurately measured with the full battery test (the FrSBe) as it was more sensitive than the Frontal Behavioral Inventory.

C69 VALIDATION OF THE PENN STATE BRIEF EXAM OF FRONTAL AND TEMPORAL DYSFUNCTION SYNDROMES IN AMYOTROPHIC LATERAL SCLEROSIS: APPLICATION OF GUILFORD'S STRUCTURE OF INTELLECT THEORY

FLAHERTY-CRAIG C, BROTHERS A, SIMMONS Z

Penn State College of Medicine, Hershey, PA, United States

E-mail address for correspondence: [email protected]

Keywords: Frontotemporal Dementia, assessment, treatment planning

Background: ALS-related cognitive behavioral impairments are estimated to occur in 30–50% of patients, primarily manifested as frontal and temporal dysfunction syndromes, with only a small percentage significant enough to meet the criteria for Frontotemporal Dementia. The Penn State Brief Exam of Frontal and Temporal Dysfunction Syndromes (PSFTS) is concise enough to be administered during the ALS multidisciplinary clinic visit, while sensitive enough to detect emerging declines in frontal and temporal cognitive behavioral capacities. Cognitive measures were chosen to detect declines in capacities recognized as important for decision making and problem solving. To account for the synergistic role of frontal and temporal cortical processing in decision making, we applied the Structure of Intellect Theory of Guilford during the selection of both brief exam measures and comprehensive neuropsychological measures to which we correlated brief exam findings. In Guilford's Structure of Intellect Theory, intelligence is viewed as comprising distinct Operations, Contents and Products. Operations include Evaluation, Convergent Production, Divergent Production, Memory and Cognition, the latter encompassing basic language skills. The Operation relevant to decision making is Evaluation, while Convergent and Divergent Production are Operations relevant to reasoning and problem solving.

Objectives: This study aimed to validate the PSFTS by correlation of findings to those obtained from a comprehensive neuropsychological battery of measures also constructed from Guilford's Operations, with an emphasis upon decision making and problem solving.

Methods: Thirty-five age, education and ALSFRS-R matched patients with an El Escorial diagnosis of probable ALS participated in the study. Non-parametric Spearman correlations were conducted between the brief exam and comprehensive exam measure findings classified by the Cognition, Evaluation, Convergent Production, and Divergent Production Operations.

Results: Statistically significant relationships were evidenced for all four Operations evaluated. Evaluation (decision making) relationships were evidenced for the brief exam task of Judgment and battery tasks of Consequences Obvious (p < 0.014) and Consequences Remote (p = 0.054). Problem Solving relationships included significance between 1) a brief exam task of Convergent Production (Similarities) and a battery task of Convergent Production (Missing Cartoons) (p < 0.007), as well as a battery task of Divergent Production (Alternate Uses), and 2) brief exam task of Divergent Production (letter fluency) and a battery task of Divergent Production (Alternate Uses) (p < 0.0001).

Discussion: Accounting for the synergistic role of frontal and temporal cortical processing in decision making by applying Guilford's Structure of Intellect Theory, we developed a brief exam applicable to the ALS multidisciplinary clinic, able to detect emerging declines in problem solving ability. Currently, we validated this brief exam by demonstrating the high degree of concordance between findings generated by 35 ALS subjects who completed both the brief exam and a comprehensive neuropsychological assessment, also selected by applying Guilford's Structure of Intellect Theory.

Conclusions: The PSFTS is a valid approach to early detection of frontal and temporal dysfunction syndromes by brief exam. The ability to identify emerging difficulties with decision making and problem solving in the ALS multidisciplinary clinic is of vital importance for optimal treatment planning.

C70 ANTI-SACCADE PARADIGM IN THE COGNITIVE FRONTAL ASSESSMENT OF AMYOTROPHIC LATERAL SCLEROSIS

POLETTI B¹, MARINGELLI F², LAFRONZA A¹, LOMBARDI C³, MERIGGI P⁴, SILANI V¹

¹Department of Neurology and Laboratory of Neuroscience, “Dino Ferrari” Center, University of Milan, IRCCS Istituto Auxologico Italiano, Milano, Italy,²Srlabs, MIlano, Italy,³Department of Clinical Medicine and Prevention, University of Milano, Bicocca, Department Cardiology, S. Luca Hospital, IRCCS Istituto Auxologico Italiano, MIlano, Italy,⁴Polo Tecnologico, Biomedical Technology Department, Fondazione Don Carlo Gnocchi Onlus, Milano, Italy

E-mail address for correspondence: [email protected]

Keywords: anti-saccade paradigm, neuropsychological assessment, eye-movements

Background: Eye movement abnormalities are sensitive markers of neurological diseases and have been studied in a variety of neurological conditions (1–3). The analysis of saccadic eye movements may provide a useful tool for investigating neurological or psychiatric disorders in which the frontal lobe is impaired. Frontal alterations in Amyotrophic Lateral Sclerosis (ALS) have been variously described and assessed in literature (4). Involvement of frontal function has recently been studied exploring ocular fixation with the aid of eye-tracking technology, thus revealing the importance of detecting the whole spectrum of frontal involvement characterizing motor neurone disease's cognitive pattern (5). Anti-saccade paradigm is ideal in exploring frontal cognitive functions (6). In the anti-saccade paradigm subjects are instructed not to make a reflexive saccade to an appearing lateral target but to make an intentional saccade to the opposite side. This ability depends on the integrity of the dorsolateral prefrontal cortex (DLPFC) (7).

Objectives: The purpose of this study was to analyze frontal cognitive functioning of ALS patients with cognitive computerized measures and the anti-saccade paradigm.

Methods: Fifteen patients fullfilling El Escorial Criteria (8) for ALS and fifteen controls underwent an extensive neuropsychological and psychodiagnostic assessment. Patients received the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRSr). Different cognitive domains were investigated with both traditional cognitive tools as well as computerized tests: reaction time, attention, eye-movements, executive functions, cognitive flexibility, language, problem solving. This battery included: computerized measures of reaction time and eye-movements (PVA test-Visuo-Attentional Performance Evaluation), Frontal Assessment Battery (FAB), Stroop Colour-Word Test, Symbol digit modalities test, Trail Making Test, Mini Mental State Examination (MMSE), Raven's Coloured Progressive Matrices (CPM). Eye-movements were measured using a 1750 Tobii eye-tracker. Clinical tools for assessing psychological and emotional status included: MOS 36-Item Short-Form Health Survey (SF-36), Beck Depression Inventory (BDI) and State-Trait Anxiety Inventory-Y (STAI-Y).

Results: Our data show quantitative and qualitative differences in cognitive performance between patients and controls, with higher difficulties in the anti-saccade task for the former and better scores in neuropsychological traditional tools for the latter. Significant differences and paradigmatic patterns of data emerge on different eye-movements/reaction times measures, while a general cognitive slowliness charcacterized patient's performances. Patients displayed lower performances on frontal measures of cognitive functioning. Subjects differed significantly for the presence of depressive/anxious symptoms.

Discussion and Conclusions: Neuropsychological assessment reveals specific cognitive and psychological patterns, as well as peculiar alterations of eye-movements patterns. Computerized neuropsychological assessment seem more sensitive in detecting small ‘frontal' cognitive changes frequently observed at the onset of the disease. Moreover this assessment seems to be an ideal tool in assessing ALS longitudinally. These data suggest that oculomotor assessment and the anti-saccade paradigm may be useful in this kind of diagnosis.

SESSION 8C INTERNATIONAL PERSPECTIVES ON CARE MANAGEMENT

C71 PREDICTORS OF SOCIAL SERVICES INPUT AMONGST PEOPLE WITH ALS/MND

O'BRIEN M¹, WHITEHEAD B^1,2, MITCHELL D², MURPHY P¹, CALLAGHER P², JACK B¹

¹Edge Hill University, Lancashire, United Kingdom,²Lancashire Teaching Hospitals NHS Foundation Trust, Preston, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: care, social services, predictive factors,

Background: A descriptive audit of the key characteristics of the MND population in a large UK specialist MND clinic was undertaken in 2008 as part of a larger study exploring the personal experience of living with MND. The audit revealed that a significant number of patients and carers were not accessing external caring support provided by local social services, despite increased levels of disability (1).

Objectives: The aims of this study were to document changes in the key characteristics over a 12 month period in an attempt to identify factors predictive of uptake of social services care.

Methods: A comprehensive case note review of patients was undertaken at 2 time points, T1 (March 2008) and T2 (March 2009). Statistical analysis was aided by SPSS version 16.0.

Results: The sample had a mean age of 64.4 years (SD = 11.7 years), with a mean onset age of 59.1 years (SD = 12.6 years). Age at T1 and duration of the illness were both significantly correlated with ALS Health Severity Scale (ALSHSS) ratings. Only 27.1% of the sample were in receipt of local authority care at T1, with this rising to 37.7% of the surviving sample at T2. The presence or absence of local authority care at T1 was taken as a dependent variable in a logistic regression analysis employing onset age, time from diagnosis to T1, and ALSFRS scores as a predictive model. The model was highly predictive of the dependent variable, with each unit rise in ALSFRS scores being related to an 8.1% decrease in the odds of receiving local authority care at T1.

A separate logistic regression showed that forced vital capacity (% of predicted) at T1, and the time from disease onset to T1, were both positively predictive of being alive at T2. In particular, each unit increase in vital capacity at T1 was related to a 4% increase in the odds of being alive at T2.

Conclusions: This study has provided detailed information regarding the level of uptake of care services over a one year period for the current MND population followed up by a specialist UK MND clinic. It confirms previous findings of relatively poor uptake of care services by people with MND which may be predicted by taking into account age at onset, disease duration and ALSFRS scores. Additionally, there is confirmation of the role of forced vital capacity as a predictor of survival.

References:

• O’Brien M., Whitehead B., Mitchell D. et al. ALS 2008, 9; Supp 1, 147
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C72 A CARING APPROACH TO ALS CARE

GOLDMAN B¹, HARTMANN S², WEBER M¹

¹Neuromuscular Disease Unit Kantonsspital, St. Gallen, Switzerland,²Palliative Care Unit Kantonsspital, St. Gallen, Switzerland

E-mail address for correspondence: [email protected]

Keywords: patient instruction, home care, quality of life

Background: Good patient care for ALS sufferers is a prerequisite for maintaining their quality of life. Knowledge about the disease and patient care is therefore essential for both patients and their caregivers. The patients' and caregivers' thirst for information was immense and could not be fulfilled in an adequate way during the regular outpatient consultations. To address this need, we designed a special training programme for ALS patients, caregivers and healthcare professionals (BG).

Objectives: The aim was to enhance patients' and caregivers' knowledge about the disease and patient care and to enable adequate care for a tetraplegic person in a home setting.

Methods: We therefore offered five theme-based afternoons, each was a module lasting five hours. These took place in comfortable surroundings and included presentations from experts, question and answer sessions, practical group exercises under supervision and ample time to exchange experiences. The themes of the modules were “mobility”, “breathing”, “nutrition”, “homecare” and “last phase”. The ALS Care Training Programme is based on the adult education concept consisting of information, practical experiences with supervised training and the exchange of experiences. The programme was mainly designed for patients (P) and caregivers (CG) but health care professionals (HC) could also participate. A booklet for everyone to take home was handed out at each module.

Questionnaires with a self-rating scale of 0 to 5 (0 = no knowledge, 5 = best knowledge) were given out before and after each training session addressing the major goals of each theme.

Results: A total of 42 patients, 52 Caregivers and 21 healthcare professionals participated during the years 2005, 2006 and 2008. A total of 532 questionnaires were completed. Improvement of knowledge was noted for all themes. Average scores increased for mobility from 2.3 to 3.7 (P); 1.9 to 3.2 (CG); 3.5 to 4.0 (HC), for breathing from 2.5 to 4.3 (P); 2.3 to 4.1 (CG); 3.7 to 4.7 (HC), for nutrition from 3.1 to 4.5 (P); 2.8 to 4.5 (CG) and homecare from 2.9 to 4.3 (P); 2.4 to 3.9 (CG) and “last phase” from 2.1 to 4.3 (P); 2.3 to 4.3 (CG); 3.4 to 4.7 (HC). All improvements were highly significant (p < 0.01; paired t-test)

Conclusion: To the best of our knowledge, this is the first systematic ALS Care Training Programme to have been reported. This project shows that systematic ALS Care Training is a particularly effective and appropriate way of transferring knowledge to improve confidence in care management of patients and their caregivers. It could be an inspiration for others.

We would like to thank for the support from Fresenius Kabi, Switzerland, Aventis Sanofi, Switzerland, patients' sponsoring and since 2008 by the Swiss Muscle Society.

C73 ALS OUTREACH SERVICES

CHAM E, POIRIER B

Vancouver Coastal Health, ALS Centre, Vancouver, British Columbia, Canada

E-mail address for correspondence: [email protected]

Keywords: client satisfaction, education, outreach

Background: The Vancouver Coastal Health ALS Centre is British Columbia (BC)'s trans-disciplinary team providing services to people with Amyotrophic Lateral Sclerosis (ALS) from time of referral to end of life. The team is composed of clinicians in Neurology, Dietetics, Nursing, Occupational Therapy, Physiotherapy, Social Work and Speech-Language Pathology and offers outpatient diagnostic, assessment, intervention, consultation, outreach and educational services in partnership with other community organizations throughout the province. With the support of the ALS Society of BC, the ALS Centre developed a Mobile Outreach Clinic to better serve ALS clients, families and local service providers. This started as a pilot project and is now a permanent service offered by the ALS Centre. Approximately four clinics per year are offered to different regions of the province. Education sessions to local service providers were later added to the mobile clinic schedule to meet the learning needs of the community.

Objectives: 1) To evaluate the effectiveness of the Mobile Outreach Clinic and Education sessions offered to local communities. 2) To evaluate satisfaction levels of clients, families and local health care providers with the Mobile Clinic and Education session outreach model.

Methods: Satisfaction questionnaires including 5 items on a 5-point Likert scale and open-ended questions were distributed to clients seen during Mobile Clinics and to health care providers who attended the education sessions. Complete data were obtained from 34 clients and 55 health care providers. Results were collated and analyzed with descriptive statistics (crosstab and bar graphs) using statistical software (SPSS V15).

Results: ALS clients, their family and the local health care providers all seem to be highly satisfied by the outreach services they received from ALS Centre.

Discussion: The high level of satisfaction of the ALS clients and their local health care providers illustrates the relevance of outreach services, in particular the mobile clinics and the education of local professionals about the management of ALS symptoms. As a result, those services have now been established as part of the permanent service provision offered by the ALS Centre.

Conclusions: The mobile outreach clinic project demonstrated high satisfaction from clients living in various regions of the province of British Columbia. The local health care providers involved with ALS clients expressed high satisfaction about education provided by the ALS Centre. However, this model of service provision does not serve some of the ALS clients living away from major urban centres: for example, clients living in facilities or home bound with limited transportation options. Future options to consider may include: home visits to facilities and client homes in rural areas, extending mobile clinic operating hours, and videoconference education sessions.

C74 OUTCOMES USING AN ON-LINE PRE-CLINIC ASSESSMENT TOOL

KITTRELL P, JACKSON C

University of Texas Health Science Center at San Antonio, San Antonio, TX, United States

E-mail address for correspondence: [email protected]

Keywords: pre-clinic assessment, communication, satisfaction

Background: Multidisciplinary ALS clinics use pre-clinic assessments to gather information before the patient is seen by each team member. Typically, these are sent by mail or completed over the telephone. Telephoning patients can be time consuming and, over time, disease progression can impact the patient's ability to communicate verbally.

Objectives: To develop an on-line survey that can be completed by the patient prior to their multidisciplinary visit. Secondary objectives were: to improve efficiency in obtaining information; to provide current information to the team members so that they can be prepared with equipment, teaching tools, information etc. to meet the needs of our patients; to prioritize the issues to be addressed by the team and to streamline the clinic process.

Method: Using Survey Monkey ®, a survey was developed by polling the team members for questions and concerns covered during the clinic visit. The survey included questions regarding change in medical status, medications, swallowing, nutrition, breathing, speech, mobility, medical equipment and family concerns (disability, wills, power of attorney). Patients were sent the survey link by E-mail one week before their visit. Patients without E-mail were called and the survey was completed by one of the team members. The survey link also served as a reminder of the upcoming visit. Patients with incomplete surveys were contacted to determine if a phone interview was needed. Surveys went to a secure server monitored by one of the clinic RNs. Identified concerns or questions needing immediate attention were addressed as indicated. Two days before clinic, responses were forwarded to all team members. Following the clinic visit, a questionnaire was sent to the patients who completed the on-line survey, asking for feedback on the process and suggestions for improvement. Revisions were incorporated into the survey.

Results: All patients but two (2) who attended the clinic between December 2008 and April 2009 completed the survey either by E-mail (78%) or with a team member via telephone (20%). Of the 67% of patients who completed the follow up survey, 78% perceived their quality of care at clinic was improved by completing the pre-clinic survey and 52% perceived their visit took less time and clinic flow was improved. A follow up survey with team members indicated they felt more prepared to answer questions and provide appropriate interventions. Loan Bank equipment utilization was improved with less need for home delivery, impacting time and travel costs. Time spent by team members on the telephone (pre and post clinic) was cut by more than 50%.

Conclusions: On-line pre-clinic assessment surveys can be a beneficial tool to help multidisciplinary teams prepare for and respond to patient needs.

C75 ENHANCING SPEECH DURING BIPAP USE

CHUA S, KRUCHTEN P, EISEN A

University of British Columbia, Vancouver, BC, Canada

E-mail address for correspondence: [email protected]

Keywords: BiPAP, speech, quality of life

Background: The efficacy and benefit of BiPAP use in amyotrophic lateral sclerosis (ALS) is well established with daily utilization varying from several hours to 24 hours depending on disease progression and stage. Problems associated with BiPAP use include, dry mouth, abrasion from the mask and claustrophobia. These can be dealt with relatively easily. A more major problem is its interference with speech caused by muffling of vocalization by the BiPAP mask and the associated airflow noise. For patients using BiPAP several hours a day this problem greatly limits their communication.

Objectives: To improve speech when using Bi-level Positive Airway Pressure (BiPAP)

Methods: To overcome muffled speech due to BiPAP use a small electret microphone was suspended near the top of the mask away from the direct path of the rushing air. The sound output was filtered and amplified by software on a digital signal processing board that computes an average of the noise during non-speech activity and then removes it from subsequent sound frames. Residual noise in a sound frame is then further attenuated based on information provided by an algorithm that examines adjacent sound frames.

Results: During preliminary testing, it was found that the airflow noise was removed almost entirely with very little residual noise from the sound signal and after amplification, allowing listeners to hear a BiPAP user's voice very clearly. Occasionally nasal bridge prominence caused the microphone to be flush against the skin and speech capture was impossible. Placing the microphone nearer the mouth overcame this problem but the airflow noise increased significantly affecting the performance of the noise filtering. Possible solutions involve finding an alternative optimal placement in the mask or designing a microphone housing that would help shield the microphone from noise. Sometimes reverberations in the mask resulted in a “radio-like” speech; a possible solution to this would be use of multiple microphones.

Discussion and conclusions: The prototype developed allows significant improvement in speech whilst using a BiPAP. We believe that this is the first attempt to solve the problem of audible speech whilst using a BiPAP. Several modifications are being tested to improve the aesthetics and other aspects of the present prototype and we are presently evaluating its use in a larger cohort of ALS patients.

C76 THE SURVEY REPORT OF THE ALS PHYSICIANS IN JAPAN

OGINO M¹, OGINO Y^1,2, HAMADA J¹

¹Kitasato University, Department of Neurology, Sagamihara, Kanagawa, Japan,²Toshiba Linkan Hospital, Sagamihara, Kanagawa, Japan

E-mail address for correspondence: [email protected]

Keywords: palliative care, withdrawal of permanent ventilation, questionnaire

In March 2009 we sent out a questionnaire to the 4,500 Board Certified Membership of the Japanese Society of Neurology to ask 30 questions about their clinical experiences, practice and thoughts in connection with palliative care at the terminal stage and patients’ rights, especially about the withdrawal of permanent ventilation. This is the very first of this kind of comprehensive survey in Japan to cover the exclusive population of ALS physicians. By the end of April, 1,470 anonymous responses (33%) had been returned. To date, 700 responses have been analyzed.

The purpose of the survey is to grasp what is happening to the physician's side in the Japanese ALS community where the high rate of the ventilated ALS patients and the low rate of morphine prescription for the ALS patients have been often pointed out.

As for the palliative care practice, 21% of the respondents prescribe morphine, which shows a drastic increase from the 14% in the 2007 survey. However, 72% of them had less than 5 patients. 50% of them studied and trained themselves when they began prescribing morphine. It illustrates that most of the neurologists are not well experienced with morphine, and that they are isolated in their practice. This may be explained by the fact that the Japanese national medical insurance does not pay for long acting morphine administered to ALS patients. However, 45% of the respondents answer that they would prescribe morphine whether or not the national insurance pays. This implies that the physicians' perspective about morphine prescription in the clinical setting is changing. On the other hand, approximately 30% of the respondents identify morphine prescription with euthanasia.

As for the withdrawal of the permanent ventilation which is not legal in Japan, 21% of the respondents were asked by their patients to turn off the ventilation, and 6% of them agreed and accommodated with the patient's wish. 77% of them declined and explained they were not allowed to do so. 18% of them declined and explained it is not supposed to do so. While 23% of the respondents believe that no discussion of whether the withdrawal right is permitted or not should be promoted, 46% believe that such rights should be granted if the decision made by the patient and/or his/her family members can explicitly be recognized. The results illustrate that the physicians are also divided.

It is worth noting that 20% of the respondents commented on their own, aside from answering the questionnaire, to share their thoughts. It may be the time to lay the foundation for the Japanese ALS physicians to discuss openly and candidly together to deal with the wants and wishes of their patients.

SESSION 9A INFLAMMATORY/IMMUNOLOGICAL AND GLIAL CONTRIBUTION TO MOTOR NEURON DEGENERATION

C77 NEUROINFLAMMATION – CAUSE AND CONSEQUENCE FOR DEGENERATIVE DISORDERS

HENEKA MT

Department of Neurology, Clinical Neurosciences, University of Bonn, Bonn, Germany

E-mail address for correspondence: [email protected]

Keywords: neuroinflammation, cytokine, microglia

Activation of the innate immune system either by external stimuli or by brain intrinsic mechanisms including aggregated, misfolded or post-translationally modified peptides and proteins has direct consequences for bystander cells including astrocytes and neurons. While generation of cytokines and chemokines in an acute phase may not necessarily be detrimental, the chronic exposure of glia and neurons to neuroinflammatory mediators is causing dysfunction and degeneration. Recent evidence indicates that all major neurodegenerative disorders, including Alzheimer‘s disease, Amytrophic Lateral Sclerosis and Parkinson‘s disease are characterized by an inflammatory component, in addition to their respective classical pathological changes. The presence of inflammatory mediators alone, is able to affect key neuronal functions such as hippocampal long-term potentiation, an integral mechanism of memory consolidation or axonal transport of synaptic vesicle precursors. Sustained exposure of glial cells in turn, reduces their trophic support for neighbouring neurons and the uptake of glutamate from the synaptic cleft. While it remains unclear whether inflammation is a primary cause, increasing evidence suggests that inflammatory mechanism interact with neurodegenerative pathways such as excitotoxicity, protein aggregation, APP processing and several others. Chronic neuroinflammation also compromises the neuronal antioxidative defense mechanisms thereby potentiating neurodegenerative events. Together this indicates that neuroinflammation, once stimulated may act as a motor for ongoing neuro- and glial dysfunction and death. New developments of PET tracers now allow for a direct assessment and monitoring of microglial activation in patients. Epidemiological data suggest a beneficial effect of anti-inflammatory treatment strategies at least for Alzheimer‘s and Parkinson‘s disease. Therefore, future therapies, when initiated within the time window of opportunity, should also successfully target the inflammatory component of neurodegenerative disease.

The presentation will give an overview of the mechanisms concerned and present results from cell culture experimentation, animal studies to human epidemiology and clinical studies, highlighting in particular the role in and for motor neuron disease.

C78 INNATE AND ADAPTIVE IMMUNITY IN AMYOTROPHIC LATERAL SCLEROSIS (ALS): EVIDENCE OF COMPLEMENT ACTIVATION.

STA M¹, SYLVA-STEENLAND RMR¹, CASULA M.², DE JONG JMBV³, TROOST D², ARONICA E², BAAS F^1,3

¹Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands;²Departments of Neuro-Pathology,³Neurology; Academic Medical Centre Amsterdam, University of Amsterdam, Amsterdam, Netherlands

E-mail address for correspondence: [email protected]

Keywords: innate immunity, complement, inflammatory cells

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by loss of motor neurons and gliosis in motor cortex (MCx) and spinal cord (SC). Although the etiology of ALS is still unclear, recent evidence suggests a role for the immune system in the disease process.

Objectives: To analyze the presence and distribution of inflammatory cells, such as microglia/macrophages and T lymphocytes as well as components of the complement system and their expression in sporadic and familial ALS cases.

Methods: We investigated the distribution of cellular components of innate and adaptive immunity using immunohistochemistry on SC and MCx sections of both sporadic (n = 16; sALS) and familial (n = 4; with frontotemporal dementia; FTD-ALS) ALS cases. We analyzed the expression and cellular distribution of complement components on both protein and mRNA levels as well. Quantification was performed for all stainings.

Results: In all ALS cases a prominent presence of microglial cells expressing class II-antigens (HLA-DR) and CD68-positive macrophages was found in both SC (ventral horn and corticospinal tracts) and MCx. We also observed perivascular and parenchymal T-lymphocytes (CD3⁺; with a predominance of CD8⁺ T-cytotoxic/suppressor cells) and the presence of dendritic cells (DCs; DC-SIGN⁺). Quantitative analysis showed a significantly higher number of HLA-DR⁺, CD68⁺, CD3⁺, CD8⁺ cells and DCs in ALS SC and MCx compared to control tissues. The number of microglia/macrophages and T-lymphocytes was higher in long term sALS patients as compared to sALS patients with rapid ALS progression. In contrast DCs were more prominently observed in patients who had a more rapid progression. Several components of the complement cascade (C1q, C3c, C3d and MAC) were observed in active microglia and reactive astrocytes in SC and MCx of ALS patients.

Conclusions: Our findings demonstrate a persistent activation of immune/inflammatory responses in ALS, including the activation of the complement system. Understanding the role of complement activation in motor neuron degeneration in ALS may be of great importance in the development of new therapeutic strategies.

C79 CD4 + CD25 + FOXP3+ REGULATORY T CELLS INFILTRATION INDUCES ALTERNATIVELY ACTIVATED M2 MICROGLIA AND A STABLE PHASE OF DISEASE IN THE MSOD1G93A MODEL OF ALS

HENKEL JS, BEERS DR, ZHAO W, LIAO B, WANG J, WEN S, APPEL SH

Methodist Neurological Institute, Houston, TX, United States

E-mail address for correspondence: [email protected]

Keywords: CD4 + CD25+ T-cells, microglia, alternatively activated

Background: Current evidence suggests that motoneuron injury in ALS and mSOD1 mice is non-cell autonomous and involves microglia and infiltrating immune cells. While previous data indicate that microglia play a pivotal role in rate of disease progression, recent evidence indicates that CD4+ T-cells also have a critical function modulating microglial activation and slowing motoneuron degeneration. A lack of functional CD4+ T-cells in mSOD1/RAG2^{−/ −} and mSOD1/CD4^{−/ −} mice resulted in attenuated microglial morphological activation, yet increased pro-inflammatory and cytotoxic factors, reduced anti-inflammatory and neurotrophic factors and glial glutamate transporters, and decreased survival due to the absence of a stable phase of disease. The stable phase of disease is not specific to the G93A mutation in SOD1; a similar phase has been described between onset and a rapid progression phase in several ALS mouse models with different SOD1 mutations.

Objectives: To investigate which sub-populations of CD4+ T cells could be responsible for inducing the stable phase of disease and examine whether alternatively activated (M2) vs. classically activated (M1) microglial phenotypes contribute to the stable and rapidly progressing phases of disease in mSOD1^G93A mice.

Methods: The sub-populations of T lymphocytes in the blood, lymph nodes, and spinal cord of mSOD1 mice were analyzed during critical time points based on disease progression by flow cytometry and QRT-PCR. Evaluations were also made of M2 markers, anti-inflammatory cytokines, neurotrophic factors and of M1 markers, reactive oxygen species generating enzymes, and inflammatory cytokines over time from spinal cord by QRT-PCR and flow cytometry.

Results: In mSOD1 blood, there were increased CD4 + CD25+, CD4 + FoxP3+, and CD25 + FoxP3 T lymphocytes at 11 and 14 weeks of age. End-stage mice contained less CD25 + FoxP3 T lymphocytes with no change in CD4 + CD25+ T cells. In lymph nodes, the increase in CD4 + CD25+, CD4 + FoxP3+, and CD25 + FoxP3+ T lymphocytes were not observed until 16 weeks of age. CD3+ and CD4+ were increased at end-stage disease in mSOD1 mice.

Evaluations of microglial phenotypes during the stable phase of disease reveal that microglia are converted to a neuroprotective M2 phenotype, expressing increased levels of Ym1, BDNF, and TNFa. However, during the rapid progression phase, the balance is shifted toward an M1 phenotype, expressing reduced levels of Ym1 and BDNF and increased levels of NOX2, IL-1β, and IFNγ. Without the presence of T cells, the balance is shifted more toward an M1 phenotype, expressing even more NOX2.

Discussion and Conclusions: These results reveal an expansion of CD4 + CD25 + FoxP3+ regulatory T cells which infiltrate the spinal cord and modulates the M1/M2 balance of microglia. These data provide further evidence that disease progression in ALS is non-cell-autonomous and is modulated by the innate and the adaptive immune system. These glial/T-cell interactions establish novel targets for therapeutic intervention and validate immunomodulatory therapies in ALS.

C80 ANTI-CD40L MONOCLONAL ANTIBODY THERAPY IMPROVES DISEASE PROGRESSION AND SURVIVAL IN A MURINE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

PERRIN S, LINCECUM J, THOMPSON K, VIEIRA F, WANG M, FERRARI A, SANCHEZ R, DEZUTTER J, GILL A

ALS Therapy Development Institute, Cambridge, MA, United States

E-mail address for correspondence: [email protected]

Keywords: SOD1_G93A, immunomodulation, biological

Amyotrophic lateral sclerosis (ALS) is a neurological disorder characterized by skeletal muscle atrophy, weakness, and ultimately total paralysis. The pathology results from an unremitting degeneration of motor neurons in the spinal column and brain. As of yet, there is no therapy that improves patient survival.

The precise mechanism of disease onset and progression remains unclear; however there is an humoral immune response prior to the onset of severe clinical symptoms. Microgliosis, astrocytosis and infiltrating inflammatory cells from the periphery into the central nervous system have been well described in ALS patients and in transgenic mouse models, such as SOD1-G93A mice. Recently, elegant murine genetic studies, crossing SOD1-G93A mice into either RAG2(−/ − ) or CD4(−/ − ) backgrounds, have provided evidence that infiltrating T cell populations are neuroprotective and not cytotoxic (1,2).

In an effort to extend on these findings, we have focused on modulating peripheral T cell activity using an antibody known to impact the T cell co-stimulatory pathway. A large body of work has established that blocking antibodies to CD40L ameliorate disease progression and survival in a variety of preclinical models of chronic inflammation including; experimental allergic encephalomyelitis (EAE) a model of multiple sclerosis, collagen induced arthritis, and systemic lupus erythematosus (3).

Here we report on a genome wide expression profiling study that has identified presymptomatic upregulation of the co-stimulatory pathway in the spinal cord, skeletal muscle, and sciatic nerve of SOD1-G93A mice. Based on these observations we hypothesized that systemic administration of anti-CD40L antibody would slow the onset and progression of disease in the SOD1-G93A mouse. Highly powered (n = 18 mice/group/gender), efficacy studies using optimized dosing protocols demonstrate that weekly administration of anti-CD40L antibody improves body weight and significantly slows the progression of paresis relative to age matched, gender balanced, litter mate controls. Furthermore, anti-CD40L antibody administration improves survival (median survival increase = 13±0.3 days in treated versus controls; 141d vs. 128 d; p < 0.007; hazard ratio: 0.35± 0.14) in this ALS preclinical murine model. Data will be presented showing that these therapeutic effects appear to be mediated by modulation of the immune response in the peripheral nervous system. Finally, this work raises the possibility that immunomodulatory biologics, with proven efficacy in other indications, could hold promise as a therapeutic strategy for ALS.

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C81 ASTROCYTIC MCT1 CONTRIBUTES TO MOTONEURON DEGENERATION IN ALS

KIERAN D, LENGACHER S, MAGISTRETTI P, AEBISCHER P

Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland

E-mail address for correspondence: [email protected]

Keywords: motoneuron, astrocyte, viral vector

In Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disease characterized by progressive loss of spinal and cortical motor neurons, astrocytes are proposed to play a significant role in disease pathogenesis. Astrocytes support neuronal function by secreting trophic factors, scavenging glutamate from the synapse, and providing metabolic support in the form of lactate. In this study, we were interested in examining the supply of lactate from astrocytes to motoneurons in ALS patients, and the mutant SOD1^G93A transgenic mouse model of ALS. We identified a selective loss in the expression of the astrocytic lactate transporter MCT1 in the spinal cord of both sporadic and familial ALS patients. Reduced astrocytic MCT1 expression was also evident in mutant SOD1^G93A transgenic mice. The effect of reduced astrocytic MCT1 expression on neuronal viability was examined both in vitro and in vivo. In vitro, reduced MCT1 expression in astrocyte-motoneuron co-cultures significantly reduced neuronal viability, while in vivo crossbreeding MCT1 heterozygous knockout mice with mutant SOD1^G93A transgenic mice resulted in an earlier onset of disease. The therapeutic effect of increasing MCT1 expression in mutant SOD1^G93A transgenic mice was examined using recombinant adeno-associated viral vectors, where intraspinal injection of AAV2/6:MCT1 had significant neuroprotective effects. Combined, these findings demonstrate that astrocytic MCT1 is required for neuronal survival and contributes to motoneuron degeneration in ALS.

SESSION 9B EXERCISE, METABOLISM AND NUTRITION

C82 IS EXERCISE A PREDISPOSING FACTOR IN ALS? THE CASE FOR

CHIÒ A

Department of Neuroscience, Torino, Italy

E-mail address for correspondence: [email protected]

Keywords: exercise, physical activity, ALS risk

ALS is a neurodegenerative disorder of the adult life. The cause of ALS in sporadic cases is still unknown. Among the possible causes, physical exercise, including sport activity, has been considered a predisposing factor. From the epidemiological point of view, several case control and cohort studies have evaluated the relationship between ALS and physical activity/sport participation. Most of these studies demonstrated an increased risk for heavy physical activity, with an odds ratio ranging from 1.5 to 2. While these data tend to support some effect of physical activity on ALS risk, these studies have several pitfalls, including the selection of cases and controls, the different operative definition of physical activity and the possible effect of confounding factors. According to one study (1) physical activity may be associated with an earlier age at onset of ALS. Cohort studies on professional athletes (soccer, bicycle riding, American football, basketball) did not demonstrate any specific effect of professional sport activity in ALS, since only professional soccer has been related with ALS (2,3). However, in this study the young age of onset of ALS in soccer players could indicate an effect of anticipation of ALS presentation related to strenuous physical activity. Physical activity could increase motor neuron degeneration because of stimulation of motor neurons in a pre-existent excitotoxic environment. Furthermore, physical activity may lead to increased motor neuron death through an unbalance between free radical formation and radical scavenger systems, with a consequent increased oxidative stress.

Data on the pre-clinical models of ALS are intriguing. In a study comparing SOD1^G93A mice under enriched vs. standard conditions better motor performances were observed in the preclinical phase, but an early age at onset of symptoms and a lower survival was observed in female mice (4). This finding has been explained by increased motor neuron firing rates and activity induced by environmental enrichment that may accelerate excitotoxicity. In another study, using the same animal model, male endurance-trained mice had an hastened death compared to sedentary mice (5). Conversely, a study (6) showed that regular exercise significantly increased the life span in SOD1^G93A male mice. Therefore, we have no unequivocal indication from the preclinical model of ALS on the effect of exercise on the onset and the course of neurodegeneration.

Data on physical activity and ALS are still too sparse and contradictory to be able to give a firm conclusion. Future epidemiological studies, looking at physical activity with a sound design, as well studies on the possible biological effect of physical activity on motor neurons are warranted.

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C83 IS EXERCISE A PREDISPOSING FACTOR FOR ALS? THE CASE AGAINST

WOKKE J, VELDINK J, VAN DEN BERG L

Department of Neurology, University Medical Centre, Utrecht, Netherlands

E-mail address for correspondence: [email protected]

Keywords: exercise, risk factor

The notion that exercise could be a risk factor for developing ALS may have been supported by the case of Lou Gehrig, the famous American baseball player with ALS who dramatically became manifest during a match when failing to hit the ball. A relationship between exercise and ALS was been suggested by Italian investigators who did a retrospective study of the occurrence of ALS in all Italian professional football players from the series A and B in the period between 1970 and 2001. Risk of developing ALS was higher (1). In a prospective extended study of the original cohort they identified 2 new cases. Mean onset was almost 20 years lower compared with population studies (2). Interestingly, they identified no ALS cases in large control cohorts of professional road cyclists and basketball players. Whether a relationship between exercise and ALS exists, cannot be concluded from these studies. We found no relationship between lifetime physical activity and sporadic ALS (3). However, increased leisure time activities were associated with earlier age at onset. This suggests that premorbid leisure time activities in a population at risk of developing ALS-for other reasons than physical activity-could accelerate onset of ALS.

Experimental studies in hSOD1 mice have not been very helpful to answer this question either (4). Exercise may even be beneficial in terms of survival (4,5). A small clinical trial of resistance exercise showed that patients who trained had higher ALSFRS and SF-36 scores (6). A beneficial effect of training has been demonstrated in other neuromuscular diseases, e.g. Guillain-Barré syndrome, myasthenia gravis and some muscular dystrophies (7). Psychological factor may play a role. Two Cochrane reviews failed to demonstrate a beneficial effect of exercise programmes on functional ability in patients with peripheral neuropathy or muscle disease (8,9). There were no adverse effects. Aerobic training was not well tolerated in patients with Kennedy disease (10).

ALS is considered a multifactorial disease with identified and unidentified risk factors. Identified risk factors include the male sex, smoking, and genetic factors. The next 5 years will reveal what other genetic factors are implicated and what functions these genes have. Excitotoxicity from various causes may induce neuronal death. Mitochondrial dysfunction may be a final common pathway. Disturbance of axonal transport can be a second mechanism. When reinnervation fails, skeletal muscle weakness in ALS results from a shift towards denervation of muscle fibres. The insulin-growth factor I could act at the level of the muscle fibre or on axonal outgrowth.

Prolonged neuronal excitation in epilepsy can lead to postictal paresis, but not to permanent deficit in itself. In conclusion, exercise cannot be hold responsible for development of ALS.

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C84 HYPERMETABOLISM IN ALS: IMPLICATIONS FOR NUTRITIONAL MANAGEMENT

DUPUIS L^1,2

¹INSERM U692, Strasbourg, France,²Université de Strasbourg, Strasbourg, France

E-mail address for correspondence: [email protected]

Keywords: animal models, energy homeostasis, dyslipidemia

ALS is increasingly recognized as a multi-system disorder. Beyond neurodegeneration, ALS patients display striking alterations of their energy homeostasis, including increased energy expenditure (hypermetabolism) and hyperlipidemia (1–5). Such abnormalities are also found in ALS animal models (6, 7), suggesting that they are intrinsic to the disease process. Most importantly, increasing energy intake of mutant SOD1 mice mitigated their symptoms (7) and increased lipemia positively correlated with survival in a cohort of 385 ALS patients (5). The importance of these findings for defining potential therapeutic strategies and improving nutritional management of ALS patients will be discussed, along with new results showing that muscle hypermetabolism is per se sufficient to drive motor neuron degeneration (8).

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C85 PROGNOSTIC SIGNIFICANCE OF NUTRITIONAL PARAMETERS IN ALS PATIENTS

OUADJIEMOM KAJEU P-J^1,2, MARIN B², DESPORT J-C^1,2, PREUX P-M², COURATIER P^2,3

¹CHU-Unité de Nutrition, Limoges, France,²EA3174-Faculté de Médecine, Limoges, France,³CHU-Centre SLA, Limoges, France

E-mail address for correspondence: [email protected]

Keywords: bioimpedance, phase angle, hydration

Background: Malnutrition has been shown to be associated with a poor survival of ALS patients. Malnutrition can be easily evaluated with% of weight loss or body mass index. Other more sophisticated methods have been used to assess the nutritional status of ALS patients. Of those, total body impedance analysis has been validated to assess lean mass and evaluate phase angle, a marker reflecting the nutritional status and the cellular membrane alteration.

Objective: Our aim was to assess the independently prognostic significance of nutritional parameters in survival of patients suffering from ALS.

Methods: Our study included all patients with available nutritional evaluation during their follow up in our referral center located in France. Evaluated variables were body mass index, weight, tricipital skin-fold thickness, midarm muscular circumference, phase angle, lean mass, hydration disorders (extracellular to intracellular water ratio (E/I)). We used the Cox proportional hazard model to perform a survival analysis from time to diagnosis until death or censoring time. We considered variables collected at first evaluation as fixed variables and during the entire follow up as time varying covariates. Our analyses were adjusted on sociodemographic characteristics and neurological clinical status at baseline.

Results: Among 175 patients with at least one nutritional evaluation, we identified that an increase in phase angle was significantly and independently associated with a better survival and that hydration disorders (extracellular to intracellular water ratio (E/I) increment) was significantly associated with a shorter survival.

Discussion: The identification of new prognostic factors of ALS may be useful for clinicians to monitor nutritional status of ALS patients and for researchers to emphasize new hypotheses on the physiopathology of the disease.

C86 DIET AT TIME OF PROCEDURE PREDICTS COMPLICATIONS AFTER PERCUTANEOUS ENDOSCOPIC GASTROSTOMY PLACEMENT IN ALS

LARSON S, TIRYAKI E

University of Minnesota, Minneapolis, MN, United States

E-mail address for correspondence: [email protected]

Keywords: percutaneous endoscopic gastrostomy, dysphagia, outcomes

Background: The current ALS practice parameters of the American Academy of Neurology state that PEG is indicated for patients with ALS who have symptomatic dysphagia and should be considered soon after symptom onset. These parameters state that PEG should be placed while the patient's vital capacity is still above 50% of predicted in order to ensure optimal safety and efficacy.

Objectives: The goal of this retrospective study was to assess predictors of outcome and survival after PEG placement at a single center.

Methods: Patients with probable or definite ALS seen between January 2006 and April 2009 were identified by chart review. Forced vital capacity (FVC), ALS Functional Rating Scale score, diet and BMI were captured at time of diagnosis and at time of procedure. The primary outcome measure considered was the presence of any complication (pulmonary, PEG-related, prolonged hospital stay, tracheostomy, and/or death).

Results: Fifty-eight patients (29 female, 29 male) had PEG tube placement. The mean age of patients was 56±13.3 years. Of these, 16 (27.6%) patients had bulbar onset of disease. Indication for PEG tube placement was weight loss in 39 (67%) patients, FVC < 50% of predicted in 11 (19%), and elective placement in 8 (14%). Diet at time of PEG tube placement was regular in 18 (33%) patients, dysphagia level 3 in 24 (44%), dysphagia level 2 in 6 (11%), dysphagia level 1 in 1 (2%), and NPO in 5 (9%). The rate of decline in BMI was 0.2±0.5 units per month (median 0.2, range 1.2 lost to 2.3 gained). The best predictor of complications from PEG placement in our patient sample was the consistency of diet at time of PEG. The more impaired the diet, the higher the likelihood of having at least one complication (p = 0.02), and pulmonary complications in particular (p = 0.005). The odds ratio for perioperative complications with significant modification in diet (dysphagia level 2, dysphagia level 1 or NPO) was 7.9 (CI: 1.9 to 33.4; p = 0.005) and increased after controlling for indication for PEG placement. FVC at time of diagnosis or time of PEG placement or impaired FVC (<50%) was not predictive of perioperative complications or survival (p > 0.40). Six month survival was 69% and was correlated with total weight loss between diagnosis of ALS and PEG placement (p = 0.03) and rate of weight loss (p = 0.02), as well as a slower rate of disease progression (p = 0.05) and the absence of any complications (p = 0.03).

Conclusions: In our sample, consistency of diet was a more reliable predictor of complications after PEG placement than reduced FVC and may be a good proxy for airway clearance. Careful monitoring of weight and placement of PEG prior to significant adjustment in diet may help prevent perioperative complications and improve 6 month survival.

SESSION 10A AXONAL TRANSPORT AND MAINTENANCE

C87 ANALYSIS OF AXONAL TRANSPORT: FROM IN VITRO SCREENS TO REAL TIME IN VIVO ASSAYS

BILSLAND LG¹, TERENZIO M¹, GREENSMITH L², SCHIAVO G¹

¹Cancer Research UK London Research Institute, London, United Kingdom,²Institute of Neurology, University College London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: axonal transport, siRNA screen, SOD1

Axonal transport is essential for the maintenance of neuronal function and deficits in transport are known to result in neuronal degeneration in several areas of the nervous systems. Evidence from animal models of Amyotrophic Lateral Sclerosis (ALS) suggests that axonal transport deficits may contribute to pathogenesis, although our understanding of how these relate to disease progression remains unclear.

A major focus of our laboratory is to understand the machinery controlling sorting and long-range axonal transport in motor and sensory neurons. To fulfil this task, we have extensively exploited the high affinity and specific traffic of the binding fragment of tetanus toxin (TeNT H_C) and more recently those of some neurotrophic viruses. TeNT H_C enters motor neurons at the neuromuscular junction and is targeted to the spinal cord in axonal carriers shared with neurotrophins and their receptors Trks and p75^NTR. This route requires a specific subset of small GTPases and relies on Rab7 activity for long-range axonal transport powered by cytoplasmic dynein.

To identify novel players in this transport mechanism, we have performed an siRNA screen in motor neurons derived from mouse embryonic stem (ES) cells using a library of siRNAs directed against genes involved in endocytosis and membrane traffic. A high-throughput transfection method was optimised and used to perform the screen using the endocytosis of TeNT H_C and an antibody directed against p75^NTR as a readout. Positive and negative regulators of TeNT H_C and p75^NTR trafficking were identified. Candidates were validated using independent siRNA pools and secondary screens implemented on selected regulators of cytoplasmic dynein. Hence, siRNA approaches using differentiated ES cells represent a powerful tool to investigate traffic events in neurons and for the discovery of novel players in this biological process.

To extend our understanding of the regulation of axonal transport in vivo and its alteration during disease, we have established in parallel a dynamic in vivo assay that allows us to monitor retrograde transport in the intact sciatic nerve in anaesthetised mice. This assay permits the quantitative analysis of physiological axonal transport in wild type (WT) mice in vivo and its changes in SOD1^G93A animals at different disease stages. Analysis of SOD1^G93A mice revealed significant alterations in retrograde transport of TeNT H_C from a presymptomatic stage. This presymptomatic stage appeared to represent the point of onset of retrograde transport deficits in this MND model.

The results of this study reinforce previous evidence that indicates altered axonal transport in ALS and provides a new method to closely examine and potentially dissect out the mechanisms underlying axonal transport defects in mouse models of ALS.

C88 AXONAL RNA TRANSPORT: ROLES IN MOTOR NEURON FUNCTION AND DEGENERATION

SENDTNER M, HERMANN T, DREPPER C, JABLONKA S

Institute of Clinical Neurobiology, University of Wuerzburg, Germany

E-mail address for correspondence: [email protected]

Keywords: axonal RNA transport, spinal muscular atrophy, active zones

Human motoneuron diseases are characterized by loss of motor endplates, axonal degeneration and cell death of motoneurons. The identification of responsible gene defects for familial ALS, spinal muscular atrophy (SMA) and spinal muscular atrophy with respiratory distress (SMARD) has pointed to distinct pathophysiological mechanisms responsible for the various forms of the disease. Evidence from mouse models suggests that enhanced vulnerability and sensitivity to proapoptotic stimuli is only responsible for some but not all forms of motoneuron disease (1–3). Reduced levels of the survival motoneuron (SMN) protein, which are responsible for SMA lead to disturbed RNA processing in motoneurons. A prominent phenotype of SMN deficient motoneurons is reduced axon elongation in the absence of defects that result in reduced motoneuron survival (4). In particular, the axonal transport of the mRNA for b-actin is severely reduced. The SMN protein is part of a complex in the cell body that assembles U snRNP particles. These U snRNP particles are central constituents of the spliceosome. In addition, the SMN protein is part of another complex in axons and axon terminals of motoneurons. This complex is distinct from the classical SMN complex, and it includes mRNA transport proteins, in particular the hnRNP-R protein (5). The hnRNP-R protein binds directly to b-actin mRNA, and both cell culture experiments in which hnRNP-R expression is reduced and in vivo studies point to an essential role of SMN/hnRNP-R interaction for axonal translocation of b-actin mRNA.

The consequence of reduced SMN levels in motoneurons is a severe depletion of b-actin-protein in axon terminals, resulting in disturbed axon elongation, reduced growth cone size and functional deficits in neurotransmission that are caused by disturbed integration and clustering of voltage-gated calcium channels in axon terminals (6). The deficit in clustering of voltage-gated calcium channel in growth cones of SMN-deficient motoneurons is accompanied by a significant reduction of spontaneous Ca^{2 +} transient frequency. Current research in our lab focuses on the development of imaging techniques to visualize local translation of mRNAs in axon terminals of cultured motoneurons, and to develop techniques that allow biochemical analysis and characterization of mRNA transport complexes in isolated motoneurons. In addition, new mouse models are generated that allow analysis of axon abnormalities and disturbed synaptic function at the neuromuscular endplate in vivo by multiphoton microscopy.

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C89 DOWNREGULATION OF GENES WITH A FUNCTION IN NEURITE OUTGROWTH AND SYNAPSE FORMATION IN MOTOR NEURONES OF THE VEGF^Δ/ΔMOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

BROCKINGTON A¹, HEATH P¹, HOLDEN H¹, KASHER P¹, AUTIERO M², CLAES F², LAMBRECHTS D², CARMELIET P², SHAW P¹

¹University of Sheffield, Sheffield, United Kingdom,²Vesalius Research Centre, Leuven, Belgium

E-mail address for correspondence: [email protected]

Keywords: Vascular endothelial growth factor, microarray analysis, neurite outgrowth

Background: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Understanding the molecular pathways to neurodegeneration in the VEGF^Δ/Δ mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease.

Objectives: The mechanism of neurodegeneration in VEGF^Δ/Δ mice is unknown. Chronic hypoxia has been proposed, as blood flow to neural structures is impaired. In addition to vascular development, however, VEGF plays a central role in the development of the nervous system, and may be required for survival of adult neurones. Disruption of these functions may cause neuronal degeneration in VEGF^Δ/Δ mice.This study aimed to determine the pathways to neurodegeneration in the VEGF^Δ/Δ mouse using transcriptional profiling of spinal motor neurones.

Methods: Spinal motor neurones were isolated by laser capture microdissection from 3 pairs of transgenic mice and wild-type littermates at 3 time points of disease. Extracted RNA was amplified, and gene expression profiles determined by microarray analysis, on the Affymetrix platform. Functionally important changes in gene expression were determined by analysis of gene ontology enrichment of significantly differentially expressed genes. A proportion of significant gene changes were confirmed by quantitative rtPCR.

Results: 324 genes were significantly differentially expressed in motor neurones of pre-symptomatic VEGF^Δ/Δ mice, 384 genes at disease onset, and 689 genes at late stage disease. In early disease, there was transcriptional up regulation, with a switch to major transcriptional down regulation at late stage disease, associated with down regulation of genes involved in RNA processing. At symptom onset, the reduction in neural expression of VEGF observed in VEGF^Δ/Δ mice was accompanied by down regulation of genes involved in the maintenance of neuronal processes and synapses: genes encoding cholesterol synthesis enzymes, synaptic proteins, cell adhesion molecules and proteins involved in neurite outgrowth and axonogenesis.

Discussion and Conclusions: The mechanisms involved in maintaining neuronal integrity once circuits are established, in the face of the demands of synaptic plasticity, is little understood despite their potential relevance to neurodegeneration. We have presented evidence that VEGF is required in the adult mouse for the maintenance of distal neuronal processes and synapses, such that a reduction in VEGF expression in VEGF^Δ/Δ mice leads to axonal retraction and cell death.

C90 A HUMAN P190RHOGEF HOMOLOGUE, RGNEF, INTERACTS WITH HUMAN LOW MOLECULAR WEIGHT NEUROFILAMENT (NFL) MRNA.

VOLKENING K¹, DROPPELMANN C¹, LEYSTRA-LANTZ C¹, STRONG M^1,2

¹Robarts Research Institute, London, Ontario, Canada,²Clinical Neurological Sciences, The University of Western Ontario, London, Ontario, Canada

E-mail address for correspondence: [email protected]

Keywords: RNA stability, neurofilament, aggregates

Background: p190RhoGEF is an important modulator of low molecular weight neurofilament (NFL) mRNA stability in the mouse, with alterations in expression of p190RhoGEF leading to neurofilament (NF) aggregate formation. A C-terminal region of p190RhoGEF interacts with a small region of NFL mRNA extending from the 3’ end of the coding region into the 3’ untranslated region which results transcript stabilization. Alterations in the expression of NFL protein subunits appear to contribute to NF aggregate formation in murine models of ALS. To date, no p190RhoGEF homologue has been described in humans.

Objectives: To determine if a p190RhoGEF homologue is present in human brain and spinal cord, and if it can interact with NFL mRNA in a manner similar to that of murine p190RhoGEF.

Methods: Sequence data mining revealed the existence of up to four predicted human homologues of p190RhoGEF. Sequence analysis revealed that human RGNEF had similar protein structure and domain organization as murine p190RhoGEF, including the presence of a RNA binding domain in the C-terminal region of the protein. This domain was expressed as a GST fused protein, then gel shift assays were used to confirm interaction with human NFL mRNA. RT-PCR was used to detect RGNEF expression in human spinal cord and brain from both control and sporadic ALS (sALS) cases. GST-fused RGNEF protein was then supplemented to lysates from control (3 cases) and sALS (3 cases) spinal cords, immunoprecipitated, and the presence of NFL mRNA determined by IP-RT-PCR.

Results: RGNEF was expressed throughout brain and spinal cord tissues from control and sALS cases. The C-terminal region of RGNEF interacted with in vitro transcribed NFL mRNA as predicted. Interestingly, we detected strong interaction of RGNEF with NFL mRNA in sALS spinal cord lysates but not in control lysates. NFL mRNA expression was confirmed in all samples and found to be present in the supernatant fraction in control lysates, showing that the absence of interaction was not due to absence of NFL mRNA expression.

Discussion: These data indicate that RGNEF can interact with human NFL mRNA in a manner similar to that of seen with p190RhoGEF and murine NFL mRNA. RGNEF is also expressed in the brain and spinal cord from both control and sALS patients. However, differences in interaction between control and sALS lysates with respect to NFL mRNA binding to RGNEF were seen.

Conclusions: While p190RhoGEF can directly contribute to the etiology of NF aggregates in mice, it also appears that the human homologue, RGNEF, is an important human NFL mRNA stability determinant. Differential NFL mRNA binding between control and sALS lysates suggests that RGNEF may play a similar role to that of murine p190RhoGEF and NF aggregate formation.

C91 CMT2E-CAUSING MUTATION OF NFL INTERFERES WITH MITOCHONDRIAL FUSION AND DISRUPTS A NORMAL FUNCTION OF NFL IN DETERMINING MITOCHONDRIAL MORPHOLOGY

GENTIL B, DURHAM H

MNI- McGill University, Montreal, Canada

E-mail address for correspondence: [email protected]

Keywords: sensorimotor neuropathies, neurofilament, mitochondrial fusion-fission

Background: Neurofilaments (NFs) are implicated in the pathogenesis of motor neuron disorders and in the preferential vulnerability of motor neurons to initiating factors. Abnormal accumulation of NFs or aggregation of NF proteins occurs in ALS and in the sensorimotor neuropathy, Charcot -Marie -Tooth disease, due to mutations in the NEFL gene (CMT2E). In CMT2E, mutations in the low molecular weight NF subunit (NFL) induce aggregation of NFs. However, we previously reported that shortening of mitochondria is observed prior to NF abnormalities in cultured motor neurons expressing NFL mutants. This mitochondrial rounding is reminiscent of another form of CMT2 caused by mutations in the gene encoding the pro-fusion protein, mitofusin 2 (MFN2). MFN2 mutants induce shortening and clustering of mitochondria, suggesting a common pathway between these forms of CMT2.

Objectives: To determine the normal role of NF proteins in the control of mitochondrial dynamics and how CMT2E-causing NFL mutants cause mitochondrial rounding

Methods: To test the effect of individual NF proteins on mitochondrial morphology and distribution in comparison to MFN2 proteins, NFL, NFH, NFLQ333P (a CMT2E-causing NFL mutant), MNF2 or MFN2R94Q (a CMT2A-causing mutant that inhibits mitochondrial fusion) was co-expressed with mitochondrially targeted dsRed in SW13- cells (an adrenocarcinoma devoid of intermediate filaments). Mitochondrial length and clustering were measured by confocal microscopy. To quantify effects of NF and MFN2 proteins on mitochondrial fusion, each was co-expressed with a mitochondrial-targeted, photoactivatable eGFP in motor neurons of dissociated spinal cord cultures. A small area was photoactivated and fusion was measured as decay in the intensity of fluorescence of the spot by time-lapse confocal live imaging.

Results: SW13- cells expressing NFLwt had longer and more reticulated mitochondria, but not when fusion was prevented by co-expressing MFN2R94Q. Unlike NFLwt, NFLQ333P caused rounding and shortening of mitochondria similar to the MFN2 mutant. NFH had no effect on mitochondrial length, but rather promoted clustering of mitochondria. In motor neurons, mitochondrial fission/fusion was unaffected by over-expression of NFLwt, but was profoundly inhibited by expression of NFLQ333P, similar to MFN2R94Q.

Discussion and Conclusion: The data reveal involvement of NF proteins in mitochondrial dynamics, NFH influencing mitochondrial distribution and NFL influencing mitochondrial morphology, possibly by affecting the balance of fission/fusion. The CMT-causing mutant, NFLQ333P, induces rounding of mitochondria prior to NF disruption by impairing fusion/fission, an effect similar to CMT2A-causing mutations in the mitochondrial protein MFN2 and linking CMT2A and CMT2E to a common cellular dysfunction.

C92 THE LEGS AT ODD ANGLES MUTATION (LOA) RESCUES MITOCHONDRIAL FUNCTION IN SOD1G93A MICE

MORSI EL-KADI A, MOORE AL, HAFEZPARAST M

Biochemistry and Biomedical Science, School of Life Sciences, University of Sussex, Brighton, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: SOD1, Loa, mitochondria

Background: We and others have previously demonstrated that the F580Y point mutation in cytoplasmic dynein heavy chain (DYNC1H1) results in the loss of sensory and motor neurons in the Loa mouse. In addition, we have shown that the Loa mutation delays disease onset and motor neuron loss in double mutant (Loa/SOD1^G93A) transgenic mice, leading to a significant increase in life span in these mice.

Objectives: To determine the underlying molecular mechanisms of the improved mitochondrial function by the Loa mutation and its link with the amelioration of the disease phenotype in Loa/SOD1^G93A double mutants.

Methods: We used gradient sedimentation techniques to isolate enriched mitochondrial fractions from SOD1^G93A and double mutant Loa/SOD1^G93A mouse brain and spinal cord homogentes. This was followed by a cohort of biochemical assays including polarography and spectrophotometry, blue native gel and 2D-gel electrophoresis to compare the integrity, composition, and functions of the mitochondrial respiratory chain complexes and their potential association with mutant SOD1 in the two mouse strains at the late stage of the disease.

Results: Our data from spectrophotometric and polarographic assays indicate that while succinate dehydrogenase (complex II) activity is normal, the activity of cytochrome c oxidase (complex IV) is compromised in mitochondria isolated from SOD1^G93A. Importantly, these assays also demonstrate that complex IV activity is partially restored in Loa/SOD1^G93A mice. Moreover, the blue-native gel electrophoresis assays that we used to separate the mitochondrial respiratory chain complexes suggest strong association of mutant SOD1 with ubiquinone:cytochrome c oxidoreductase (complex III). Of particular importance was the finding that the association of ATP synthase (complex V) with mutant SOD1 is significantly reduced in the mitochondria isolated from Loa/SOD1^G93A brain homogenates.

Discussion and Conclusions: The amelioration of the disease phenotype in SOD1^G93A by defective dynein has highlighted the dynein mediated retrograde axonal transport as a potential target for therapy. The significant reduction in the association of mutant SOD1 with the mitochondrial fractions indicates a role for the Loa mutation in protecting the mitochondria from SOD1^G93A toxicity. Importantly, the impaired cytochrome c oxidase function in the SOD1^G93A mitochondria and the partial rescue of this defect by the Loa mutation suggest that the amelioration of the disease phenotype by mutant dynein is at the mitochondrial level. Moreover, the reduced association of mutant SOD1 with complex V in the mitochondria from Loa/SOD1^G93A mice indicates that the function of this complex could also be compromised in the SOD1^G93A and that the Loa mutation, by depleting mutant SOD1 from these complexes, could restore their functions. We will present our findings on these and other components of the respiratory chain in Loa/SOD1^G93A versus SOD1^G93A mitochondria.

SESSION 10B RESPIRATORY MANAGEMENT

C93 RESPIRATORY EXERCISES IN AMYOTROPHIC LATERAL SCLEROSIS (REALS)

PINTO S¹, DE CARVALHO M^1,2

¹Institute of Molecular Medicine, Lisbon, Portugal,²Dept Neurosciences, Hospital de Santa Maria, Lisbon, Portugal

E-mail address for correspondence: [email protected]

Keywords: respiratory exercises, delayed-start design, neuroprotection

Background: Respiratory insufficiency is the main cause of death in ALS. Non-invasive ventilation and gastrostomy increase survival and quality of life in ALS. Without newer effective pharmacological interventions, it is fundamental to develop programs to improve respiratory function.

Objectives: The authors aimed to test a respiratory muscle-training program in ALS.

Methods: Inclusion criteria: age 18–75 years, disease duration <18mo, ALS-FRS 25–38, with definite or probable disease, written informed consent. Exclusion criteria: bicipital and flexor digiti muscular strength <4 MRC, weakness of lip sealing, forced vital capacity (FVC) <70%; maximal inspiratory (MIP) and/ or expiratory (MEP) pressures <50%, patients on NIV, gastrostomy, other concomitant diseases such as diabetes and pulmonary diseases, sternocleidomastoid (SCMAmpl) and diaphragmatic (PhrenAmpl) motor amplitudes <1mV or <0.3mV respectively. Patients performed a 8-month respiratory muscle strengthening programme using the “Threshold IMT®”. They were randomized in 2 groups: the efficient load group (G1) and non-efficient load group (G2). However, patients in G2 also performed the exercise with efficient load in the last four months (delayed start study design). Efficient load was individually calculated as 30–40% from MIP and in the first 4 months patients in G2 worked-out with the lowest possible load. Patients were evaluated 3 times, at entry and every 4 months, with ALS-FRS, FVC, MIP and MVV (maximal voluntary ventilation), sniff nasal inspiratory pressure (SNIP), PhrenAmpl and SCMAmpl, VAS for fatigue and dyspnoea, subjective respiratory control feeling, Fatigue Severity Scale (FSS), Epworth's scale, Functional Independence Measure (FIM), Euro-QoL 5D and Hamilton's scale.

Results: 19 patients (13 men, aged 57.7±8.8yrs, 3 bulbar-onset form, 2 definite disease, mean disease duration of 13.2±7.7 months) were included, 4 dropped-out due to rapid disease progression. There were no demographic differences between groups at entry. We observed a higher ALS-FRS and MVV decrease in G2 in the first four months. No other differences were found. All patients in both groups described a better voluntary control over respiratory dynamics.

Discussion and Conclusions: Exercise is controversial in ALS. Nonetheless, it seems that aerobic exercise at moderate load is related to longer survival and better quality of life. To our knowledge, there has been no specific respiratory exercise programme tested in ALS. In our study, patients in both groups referred a subjective improvement in the voluntary control of the ventilation, as well as a trend for a better outcome in G1 for ALS-FRS and MVV. However, these preliminary results show a trend towards a better outcome for patients included in G1. Although the completion of this investigation is essential before more definite conclusions, our findings suggest that these studies on respiratory exercise are relevant in ALS.

C94 HOME INITIATION OF NON-INVASIVE VENTILATION FOR MOTOR NEURONE DISEASE

JOHNSON O, RODGER J, ROBERTSON A

Mid Yorkshire NHS Trust, Pontefract, West Yorkshire, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: non-invasive ventilation, DGH, home initiation

Background: The local MND team has been in existence since 1995. Based in a local District General Hospital (DGH), the team provides patient-centred, key worker-led multidisciplinary team (MDT) care. As the team has developed so has the non-invasive ventilation (NIV) service. The service is unusual in that ventilation is usually initiated in the patients' homes. The team believes that home initiation of NIV by a DGH based team is at least as effective as initiation in a specialist secondary healthcare setting, and is popular with patients.

Objectives: To compare outcomes against published data from the Newcastle NIV study (1).

Methods: Retrospective caseload review of 42 patients who had died between 1996 and 31 March 2009 and who had had a trial of NIV was conducted. The case notes were analysed to include all patients who had NIV irrespective of type of presentation. The outcome was mean length of survival from initiation of ventilation to death. Data are also presented for survival of all patients.

Results: Bourke reported a survival of 219 days from initiation of ventilation. Our mean length of survival from initiation of NIV was 348 days. Our mean length of survival for all patients from diagnosis was 580 days. Over 90% of patients with MND within the service have NIV initiated at home. This avoids hospital admission for initiation of NIV. Our data indicate that it is safe and effective as mean length of survival is comparable to published data and patients prefer to have NIV initiated at home rather than in hospital. The level of patient satisfaction with the service is also very high.

Discussion: We have shown that home initiated NIV in MND is safe and effective over several years. The key factor in that success is the ability to monitor symptoms and detect the early onset of ventilatory failure in an MDT setting, using equipment such as transcutaneous monitoring of CO₂. The MND MDT is trained to recognize the early symptoms of respiratory failure. Early detection of symptoms is followed up by a team of specialist nursing staff with expertise in respiratory management. The team is based in a District General Hospital but outreach to the primary care setting. The respiratory team monitors patients regularly to optimize ventilatory settings and encourage early use of adjunctive therapies. This may include mechanical cough assistance and early antibiotic therapy.

Conclusion: Home initiation of NIV is safe and effective in MND.

References:

• Bourke SC, et al. Lancet Neurology 2006, 5:140–147
   PubMed Web of Science ®Google Scholar

C95 USUAL INDICATORS OF AMYOTROPHIC LATERAL SCLEROSIS (ALS) DISEASE SEVERITY DO NOT PREDICT OVERNIGHT EFFICACY OF SUBJECTIVELY PRESCRIBED NONINVASIVE POSITIVE PRESSURE VENTILATION

ATKESON A¹, ANDREWS J², GORDON P², MITSUMOTO HI², BASNER R¹

¹Department of Medicine,²Department of Neurology; Columbia University College of Physicians and Surgeons, New York, NY, United States

E-mail address for correspondence: [email protected]

Keywords: non-invasive ventilation, forced vital capacity

Background: Current guidelines for ALS recommend nocturnal non-invasive positive pressure ventilation (nNIV) for forced vital capacity (FVC) <50% predicted, to prolong survival, sustain respiratory muscle strength and improve sleep quality. nNIV is typically non-objectively prescribed and adjusted. The nocturnal physiologic efficacy of subjective nNIV use in ALS patients has not previously been assessed. Given the challenge of administering nNIV to such patients, we hypothesized that ALS patients commonly have failure of nocturnal oxygenation and ventilation with such nNIV, even when reporting adherence and subjective efficacy.

Objectives: To determine the efficacy of subjectively prescribed nNIV in ALS patients reporting adherence and benefit (improved dyspnea and/or sleep quality) with this treatment and ascertain if common measures of disease severity predict nNIV failure.

Methods: 20 consecutive ALS patients reporting successful use of nNIV (>4 h/night, >4 nights/wk and subjective benefit) were prospectively recruited from the Eleanor and Lou Gehrig ALS/MDA Center, and underwent home nocturnal polysomnography (PSG) using their current nNIV regimen. PSG included airflow, NIV pressure, thoracoabdominal effort, ECG and pulse oximetry (SpO₂). nNIV failure was defined as O₂ desaturation index >4% (ODI4%) ≥5/h recording time; and/or ineffective ventilation time (patient-ventilator asynchrony, central apnea, delivered inspiratory pressure (IPAP) >2cm H₂0 below set IPAP) >5% recording time. Data were analyzed with unpaired t-tests and Fisher's exact test.

Results: 20 patients were studied (6F/14M, mean age 57±10.8, FVC 42.1% predicted±16.4, DFS 0.62±0.48). 9 of 20 patients (45%) demonstrated nNIV failure, with mean±SD nadir SpO2 = 80±6%, ODI 4 = 6±4/hr, SpO₂<90% = 6±6% recording time, ineffective ventilation time = 18±16% recording time. nNIV failure and success groups were similar for age (failures 60.6±11.6 years vs 54.4±9.7 years, p = 0.2), FVC at the time of PSG (failures 48±18% predicted vs 38±14%, p = 0.2), ALSFRS-R score at the time of PSG (failures 25±7 vs 22±9, p = 0.5), rate of functional decline (DFS, failures 0.65±0.43 vs 0.60±0.54, p = 0.8), bulbar onset (failures 56% vs 64%, p = 0.9), presence of PEG (failures 33% vs 64%, p = 0.9), level of set IPAP (failures 13±4cm H₂O vs 12±4 cmH₂O, p = 0.6), and NIV duration prior to PSG (failures 9 ±5 months vs. 7±5 months, p = 0.4). 5 subjects who did not meet minimal criteria for nNIV failure evidenced nadir O₂ saturation of <85% or ventilator double triggering >10 events/hr.

Discussion and Conclusions: These data suggest that current nNIV practice is likely not meeting the goals of improving sleep disordered breathing (SDB) or sustaining respiratory muscle strength for ~50% of patients prescribed such therapy. The prevalence of nNIV failure among all patients subjectively prescribed nNIV is likely higher than that seen in this data. Neither degree of disease progression nor impairment appears to predict nNIV success in this setting.

C96 MULTICENTER STUDY RESULTS OF MOTOR POINT STIMULATION FOR CONDITIONING THE DIAPHRAGM OF PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS/ MOTOR NEURON DISEASE: PRELIMINARY TREND TOWARD SLOWED RESPIRATORY DECLINE AND IMPROVED SURVIVAL

ONDERS R¹, KATIRJI B¹, SO Y², CHO C², KATZ J³, MILLER R³, NEWMAN DS⁴, SIMPSON E⁵, APPEL S⁵, BOYLAN K⁶, MARAGAKIS N⁷, ROTHSTEIN J⁷, GONZALEZ-BERMEJO J⁸, SALACHAS F⁸, MORELOT-PANZINI C⁸, MEININGER V⁸, SIMILOWSKI T⁸

¹University Hospitals Case Medical Center, Cleveland, Ohio, United States,²Stanford University Medical Center, Palo Alto, California, United States,³California Pacific Medical Center, San Francisco, California, United States,⁴Henry Ford Hospital, Detroit, Michigan, United States,⁵Methodist Neurological Institute, Houston, Texas, United States,⁶Mayo Clinic, Jacksonville, Florida, United States,⁷John Hopkins Medical Center, Baltimore, Maryland, United States,⁸Pitie Salpetriere, Paris, France

E-mail address for correspondence: [email protected]

Keywords: diaphragm pacing, respiration, surgery

Background: The diaphragm pacing system (DPS) is a standardized minimally invasive laparoscopic technique intended to maintain and provide natural diaphragm ventilation. Respiratory insufficiency through diaphragm dysfunction is the major cause of mortality in ALS/MND and presently available therapies are inadequate to address this problem.

Objectives: Assess the safety and efficacy of DPS for diaphragm conditioning in ALS/MND.

Methods: Prospective, nonrandomized, controlled, multicenter, interventional trial with a lead-in design and 9 month post -implantation treatment period. Prospective efficacy and safety measures were obtained including the rate of decline of pulmonary function, quality of life measurements, adverse event rates and survival or full time tracheostomy with mechanical ventilation.

Results: 145 subjects enrolled with 106 implantations from March 2005 to January 2009 (most common reason for not implanting was a drop of FVC below 45% of inclusion criteria). Subject demographics from the first 88 implanted patients are: mean age 54.9±10.3, 71.6% male, 27.9% bulbar onset, ALSRS-r total score 27.8±7.2, riluzole use of 74%, symptom onset to implantation 41.5 months ±27.1, FVC (% predicted) 61.1±11.8, average pCO₂ 39mm Hg (max = 60), SF 36 average physical of 36, SF 36 average emotional of 53, and non-invasive ventilation use of 82% throughout the study. With a cumulative 1,346 months of device usage (average 1.1 years/patient), safety analysis showed no serious unanticipated adverse device effects with 3 (2.8%) serious adverse events related to surgical procedure. There were 17 reported respiratory events during the study period, and only one patient stopped pacing. Interim analysis shows a significant (p < 0.001) improvement (7%) in ratio of the ALSFRS-R respiratory subscore to the total ALSFRS-r score from implant to treatment. In the subset of subjects with at least 6 months of treatment data and a declining FVC during the lead-in period (n = 45), there was a significant reduction in FVC rate of decline (p = 0.01), with a paired (treatment to lead-in for each patient) FVC improvement of 1.1% change from 2.7% decline during lead-in to 1.6% decline post implantation. The 30 day survival was 100% (106/106), the 6 month survival was 92%( 90/98) and the 12 month survival was 78%( 56/72). Using Kaplan-Meir analysis the mean survival is 25.7 months post implant. When analyzing combined DPS and gastrostomy placement subset the 6 month survival was 89% (24/27) and the 12 month survival was 74% (17/23). Respiratory events accounted for 41% of the end events.

Conclusion: The DPS system can be safely implanted and utilized in ALS patients. DPS seems to have positive effects in maintaining respiratory function and decreasing respiratory decline in those declining prior to implant. Overall survival and survival with a gastrostomy seems higher than historical comparisons.

C97 DIAPHRAGM PACING IN ALS: PRELIMINARY RESULTS SUGGEST SIGNIFICANT SLEEP IMPROVEMENT

GONZALEZ-BERMEJO J¹, MORÉLOT-PANZINI C¹, STRAUS C¹, ARNULF I¹, MENEGAUX F¹, LACOMBLEZ L¹, BECQUEMIN M-H¹, SALACHAS F¹, ONDERS R², MEININGER V¹, SIMILOWSKI T¹

¹Groupe Hospitalier Pitié-Salpêtrière, Paris, France,²University Hospitals Case Medical Center, Cleveland, Ohio, United States

E-mail address for correspondence: [email protected]

Keywords: respiratory insufficiency, diaphragm, sleep

Background: During amyotrophic lateral sclerosis (ALS), respiratory insufficiency and diaphragmatic involvement cause major sleep disturbances. Their correction by non-invasive ventilation (NIV) largely contributes to the improvement in quality of life associated to this therapeutic. It has been suggested that diaphragm pacing (DP) using a laparoscopically implanted device (NeurRx, Synapse, Oberlin, OH, USA) could slow down decline in lung function. To test this hypothesis, a prospective, non randomized, multicenter interventional trial has been conducted among 106 patients at 8 centers. In the subset of patients implanted in Paris, we further tested the hypothesis that DP could maintain diaphragm strength and improve sleep.

Patients: 11 patients from the Paris cohort (n = 18) had completed 4 months of DP at the time of the present submission; 6 men, 5 women; median age 68 years, 95%CI 63–74; median ALSRS-r total score 34 (29, 40); riluzole 100%; NIV n = 5; bulbar onset n = 1; median interval between diagnostic and implantation 30 (19,42) months

Methods: In addition to the evaluation of the rate of decline of lung function, quality of life, safety and survival, the Paris patients had measurements of twitch esophageal and trans-diaphragmatic pressures (Pes,tw and Pdi,tw) in response to bilateral anterior magnetic stimulation (BAMPS) and sleep assessments including polysomnographic recordings (PSG) immediately before the implantation and after 4 months of diaphragm stimulation.

Results: During the conditioning period, forced vital capacity (FVC) significantly declined. There was no change in its rate of decline as compared to the lead-in period, although none of the patients had to start NIV. Pdi, tw and Pes,tw were markedly altered initially (median 6.7 (4.6, 15.0) cm H₂O and 2.3 (1.9, 7.0), respectively). In contrast to spirometric variables, they remained unchanged after 4 months. Median sleep efficiency increased from 67 (51, 71)% to 74 (70, 85)% (p = 0.02) with a significant reduction in sleep fragmentation (median arousal index from 28 (14, 30) to 13 (10, 21) events/h, (p = 0.01). The amount of non REM sleep stage N3, the amount of REM sleep, and sleep latency were unchanged, arguing against a first night effect. In coherence with these results, the patients reported lower Epworth scores after the 4 months pacing period (from 6 (3, 12) to 5 (3, 6), p = 0.05).

Conclusion: The quality of sleep significantly improved after 4 months of diaphragm pacing in a subset of the Paris cohort of the international DP trial in ALS. This was not associated with a significant reduction in the rate of decline of FVC, but the strength of the diaphragm did not decline during the conditioning period. The mechanisms of the improvement in sleep remain to be elucidated.

C98 SURVIVAL IN ALS PATIENTS AFTER TRACHEOSTOMY

CONTE A¹, MADIA F¹, LUIGETTI M¹, DEL GRANDE A¹, MELEO E¹, PENNISI MA², TONALI PA^1,3, SABATELLI M¹

¹Department of Neurosciences, Rome, Italy,²Institute of Anesthesia and Intensive Care,Catholic University Pol A Gemelli, Rome, Italy,³Don Carlo Gnocchi Foundation, Rome, Italy

E-mail address for correspondence: [email protected]

Keywords: tracheostomy, survival, prognosis

Background: The most common cause of death in patients with Amyotrophic Lateral Sclerosis (ALS) is respiratory failure due to progressive impairment of the respiratory muscles. The median survival is 36 months in ALS patients who do not receive invasive mechanical ventilation. Little information is reported in the literature about the survival of ALS patients who undergo to tracheostomy.

Objectives: The aim of our study is to analyze the survival of ALS patients who receive pressure positive ventilation by tracheostomy.

Methods: 95 out of 646 ALS patients followed in our Center decided to receive mechanical ventilation by tracheostomy. Long-term follow-up of 85/95 patients was available and was analyzed in the present study. Survival was calculated using Kaplan-Meyer analysis.

Results: There were 48 males and 37 females. The age of onset ranged from 12–82 years (media 54.81, median 57 years); in 18 patients (21.2%) the disease began before 40 years, in 67 patients (78.8%) after 40. The site of onset was bulbar in 21 patients (24.7%) and spinal in the remaining 64 patients (75.3%). 81 patients had a sporadic ALS, while in 4 patients a familiar history was present. Dividing sporadic ALS group by clinical phenotype, we observed 62 patients (76.5%) with classic ALS, 18 patients (22.3%) with predominant upper motor neuron phenotype (p-UMN) and 1 (1.2%) with Flail Arm variant. The time from onset of disease to tracheostomy ranged from 6 to 134 months (mean 35.71, median 28 months). Before tracheostomy 24 patients used a NIV, for a mean duration of treatment of 8.84 months (range 1–21 months); only 2 patients used NIV 24 hours/24 before receiving tracheostomy, for 5 and 10 months, respectively. The median survival after tracheostomy was 40 months (95% CI 25.93–54.06 months). Age of onset before 57 years and the time from onset to tracheostomy longer than 28 months were significantly associated with longer survival. After tracheostomy, 10 patients (12%) had a survival longer than 5 years, while only one patient was alive after 10 years (1.2%). 4 patients (4.7%) developed a totally locked-in state after a mean period of 27 months (range 15– 48 months) after tracheostomy and 45 months (range 27–69 months) from onset of disease.

Discussion and Conclusions:. In our series, the median survival of ALS patients receiving tracheostomy is 40 months. Age of onset of the disease and the period of time from onset to tracheostomy are significant prognostic factors. The knowledge of the evolution of ALS after tracheostomy may be helpful in the hard task of end-of-life decisions.

C99 A POPULATION-BASED STUDY OF TRACHEOSTOMY IN ALS

Calvo A¹, Mora G², Balma M¹, Moglia C¹, Mazzini L³, Meineri P⁴, Ghiglione P¹, Chiò A¹

¹Department of Neuroscience, University of Torino, Torino, Italy,²Fondazione Salvatore Maugeri, IRCCS, Milano, Italy,³Department of Neurology, University of Eastern Piedmont, Novara, Italy,⁴ASO Santa Croce, Cuneo, Italy

E-mail address for correspondence: [email protected]

Keywords: tracheostomy, incidence, outcome

Objectives: Respiratory failure is frequent in amyotrophic lateral sclerosis (ALS). Its management is based on mechanical ventilation with non-invasive positive pressure (NIPPV) or tracheostomy when NIPPV is no longer effective. However, information about the outcome of tracheostomy in ALS is scarce.

Methods: We evaluated the clinical characteristics and outcome of tracheostomy in ALS using data from the Piemonte and Valle d'Aosta Register for ALS (PARALS), a prospective epidemiological register collecting all ALS incident cases in two Italian regions.

Results: Among the 1260 patients incident in the period 1995–2004, 134 (10.6%) underwent tracheostomy. Young male patients were more likely to be tracheostomized. Site of onset (bulbar vs. spinal) and period of diagnosis (1995–1999 vs. 2000–2004) did not influence the likelihood of being tracheostomized. The mean duration of hospital stay was 52.0 days (SD 60.5). Overall, 27 patients died while still in hospital (20.1%); in-hospital mortality was lower in subjects followed at ALS multidisciplinary centers (13.1% vs. 28.8%; p = 0.02). Sixty-five patients (48.5%) were discharged to home, while 42 (31.3%) were admitted to long term care facilities. The median survival time after tracheostomy was 253 days. In the Cox multivariable model the factors independently related to a longer survival were enteral nutrition, age, marital status, and ALS centre follow up.

Conclusions: In an Italian epidemiological setting ALS survival after tracheostomy was less than one year. Socio-cultural factors may influence the probability to be tracheostomized, even in a highly socialized health system as the Italian one.

SESSION 11 JOINT CLOSING SESSION

C100 CLINICAL BIOMARKER USE IN THE CLINICAL INVESTIGATION OF NEURODEGENERATIVE DISEASE

O'NEILL GN

Biogen Idec Inc, Cambridge, MA, United States

E-mail address for correspondence: [email protected]

Keywords: biomarker, pharmacodynamic, biological activity

The ultimate goal of clinical science is to identify novel therapeutics to relieve human suffering. Therapies for neurodegenerative diseases, including ALS, aim to arrest or slow the progression of disease and the worsening of disability.

Rational drug development identifies biological targets that may be important to a disease's pathophysiological process and then creates interventions that impact these targets. The key challenge to drug development is the translation of these discoveries from the laboratory bench into the human patient. It is in this endeavour that clinical biomarkers can be used. In considering clinical biomarkers it is important to distinguish between pharmacodynamic markers that measure the biological effect of a therapeutic intervention and other biomarkers that reflect the pathophysiological processes of the targeted clinical disease. A pharmacodynamic biomarker allows the investigator to ascertain if the study drug is interacting with and affecting its desired target and helps to identify the dosage range and exposures required to affect this target. A pharmacodynamic marker will not necessarily predict a therapeutically meaningful effect in the studied disease and population, but it will allow the investigator to confirm that the biological hypothesis has been tested in clinical trials leading to a definitive “positive” or “negative” outcome. Such a clear binary outcome is eminently more desirable than a “failed” study where the clinical outcome in the disease population is negative but it is not known if the targeted biology was altered by the study drug. Examples of pharmacodynamic markers include assays of dystrophin in studies of therapeutic ribosomal read-through of premature termination codons in Duchenne muscular dystrophy and Ab clearance from the brain in Alzheimer's disease. Biomarkers that reflect the pathophysiological process of the target neurodegenerative disease are used to identify the optimal test population for a new therapeutic, to monitor disease progression and to measure slowing of disease progression. Examples of biomarkers of disease pathophysiology include MRI brain lesion number and volume in multiple sclerosis (MS), Pittsburgh B PET scanning to confirm the presence of Ab plaque in the brain and viral load in HIV/AIDs. In some instances, changes in these biomarkers (MS MRI, HIV viral load) are also highly predictive of a clinical effect.

Identifying biomarkers is a very high priority for the neurodeneration field, in general, and the ALS field, in particular. For timely delivery of such biomarkers, their development should occur in parallel with transition of a molecule from non-clinical to clinical development.