Platform Communications

SESSION 1 JOINT OPENING SESSION

C1 NEW PERSPECTIVE ON AMYOTROPHIC LATERAL SCLEROSIS AS TDP-43 PROTEINOPATHIES

LEE V M-Y

Center for Neurodegenerative Disease Research, University of Pennsylvania, Philadelphia, PA, United States

E-mail address for correspondence: [email protected]

Keywords: TDP-43, degeneration, neurons

The disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), now referred to as FTLD-TDP, and amyotrophic lateral sclerosis (ALS) was identified recently as the TAR DNA-binding protein (TDP-43) thereby providing a molecular link between these two disorders. Moreover, the discovery of mutations in the TDP-43 gene (TARDBP) in familial ALS and FTLD-U with similar TDP-43 pathology suggests that it is a primary cause of these disorders. Although TDP-43 is normally a nuclear protein, cytoplasmic accumulations of pathological TDP-43 as well as nuclear clearance of endogenous TDP-43 are major neuropathology found in TDP-43 proteinopathies including ALS and other motor neuron diseases. This disease feature was recapitulated in cell models that accumulate cytoplasmic TDP-43 inclusions with concomitant clearance of nuclear TDP-43, suggesting both gain and loss of functions are potential mechanisms of disease pathogenesis. We next generated transgenic (Tg) mice conditionally expressing human wild type TDP-43 (hTDP-43-WT) and hTDP-43 with a defective nuclear localization signal (hTDP-43-ΔNLS) directed by the CaMKIIα promoter to elucidate mechanisms of neurodegeneration in TDP-43 proteinopathies. Although expression of hTDP-43 WT or hTDP-43-ΔNLS led to the formation of rare phosphorylated and ubiquitinated TDP-43 inclusions, significant neuron loss in a time dependent manner was observed in selectively vulnerable forebrain regions. This was accompanied by corticospinal tract degeneration and motor spasticity which taken together, recapitulates key aspects of FTLD and primary lateral sclerosis. Remarkably, neurodegeneration was linked to a dramatic downregulation of endogenous mouse TDP-43 in nuclei of affected neurons associated with changes in gene expression, especially up regulation of genes involved in chromatin assembly. Our data suggest that perturbation of highly regulated endogenous nuclear TDP-43 results in loss of functions and changes in downstream gene regulatory pathways that trigger degeneration of selectively vulnerable neurons.

C2 AAN PRACTICE PARAMETERS: WHAT THEY TELL US AND WHAT THEY DON'T

MILLER RG¹, JACKSON CE², KASARSKIS EJ³, ENGLAND JD⁴, FORSHEW DA¹, JOHNSTON WS⁵, KALRA S⁵, KATZ JS¹, MITSUMOTO H⁶, ROSENFELD J⁷, SHOESMITH C⁸, STRONG MJ⁸, WOOLLEY SC¹, THE ALS PRACTICE PARAMETER TASK FORCE AND THE QUALITY STANDARDS SUBCOMMITTEE OF THE AMERICAN ACADEMY OF NEUROLOGY¹

¹California Pacific Medical Center, San Francisco, CA, United States, ²University of Texas Health Science Center, San Antonio, TX, United States, ³University of Kentucky and VA Medical Centers, Lexington, KY, United States, ⁴LSUHSC School of Medicine, New Orleans, LA, United States, ⁵University of Alberta, Edmonton, AB, Canada, ⁶Columbia University, New York, NY, United States, ⁷UCSF-Fresno Central California Neuroscience Institute, Fresno, CA, United States, ⁸University of Western Ontario, London, ON, Canada

E-mail address for correspondence: [email protected]

Keywords: patient management, practice parameters, guidelines

Background: The American Academy of Neurology (AAN) issued evidence-based practice parameters for managing patients with ALS/MND in 2009.

Objective: To critically review the value and limitations of the AAN guidelines.

Methods: The authors identified 10 Class I studies, 13 Class II studies, and 73 Class III studies for managing ALS.

Results: Evidence-based recommendations include the use of riluzole to slow disease progression, but cost-benefit analyses for this therapy are needed, and the literature failed to identify other effective disease-modifying agents. Noninvasive ventilation lengthens survival and improves quality of life, yet there was no evidence regarding the most sensitive tests of respiratory function in ALS or the optimal time to initiate noninvasive ventilation. The literature also lacked evidence regarding the most reliable measure of respiratory insufficiency in bulbar patients, and did not adequately address strategies for decision making regarding tracheostomy.

Percutaneous endoscopic gastrostomy (PEG) or radiologically inserted gastrostomy (RIG) can stabilize weight and prolong survival. While nutrition has relevance, questions remain regarding when to initiate PEG or RIG and how it impacts quality of life and survival. Additionally, topics including dietary management (eg high calorie or high fat diet, blood lipids and statins), the value of PEG/RIG in patients without dysphagia, and the effect of vitamins and supplements all deserve further study.

Multidisciplinary clinic referral can optimize health care delivery, and correlates with prolonged survival and enhanced quality of life. While this new recommendation is pertinent, cost effectiveness studies of multidisciplinary clinics are needed.

Refractory sialorrhea is improved by botulinum toxin B, and pseudobulbar affect responds to dextromethorphan with quinidine. However, optimal medical therapy for each intervention is not clear. Other symptoms have not been systematically studied (eg cramps, spasticity, fatigue, insomnia, laryngospasm, constipation, anxiety, depression, pain and terminal dyspnea). Nonetheless, clinical experience suggests symptomatic treatments that appear helpful until more evidence is available to guide management.

While cognitive and behavioral screening was recommended, the literature failed to clarify the impact of frontotemporal impairment on compliance and survival, and there were no trials evaluating pharmacologic intervention for cognitive impairment in ALS.

Comment: While we are waiting for the cure, there are treatments available for patients with ALS that alleviate suffering. Noninvasive ventilation, PEG/RIG, riluzole, and multidisciplinary clinics are the most important and have the best evidence. More high-quality, controlled studies are needed to guide management and assess outcomes in patients with ALS. These may address the domains reviewed in the practice parameters, as well as clinically important issues not addressed such as the role of gene testing, equipment for mobility, the role of exercise, and management of end of life issues.

SESSION 2A RNA BIOLOGY IN ALS

C3 USING EMBRYONIC STEM CELLS TO STUDY MOTOR NEURON/GLIA INTERACTIONS IN ALS

PHATNANI HP¹, FRIEDMAN BA¹, CARRASCO MA¹, PAULI F², REDDY T², MURATET M², MYERS R², MANIATIS T¹

¹Columbia University Medical Center, Department of Biochemistry and Molecular Biophysics, New York, NY, United States, ²HudsonAlpha Institute for Biotechnology, Huntsville, AL, United States

E-mail address for correspondence: [email protected]

Keywords: glia, motor neurons

Amyotrophic Lateral Sclerosis (ALS) is a result of the selective dysfunction and progressive degeneration of motor neurons. Although the underlying disease mechanisms remain unknown, recent in vivo and cell culture studies have implicated glial cells in motor neuron degeneration in ALS.

We have made use of the SOD1 mouse model of ALS to study the effect of glial cells bearing the mutant SOD1 transgene on motor neuron viability in cell culture. Specifically, we have studied the gene expression profiles of co-cultured mouse embryonic stem (ES) cell derived motor neurons and primary glia using the Illumina deep sequencing platform (RNAseq). In this study, we vary both the genotype of the motor neurons and glia, as well as time in culture as a means of examining both cell autonomous and non-cell autonomous effects of the mutant transgene. In addition, we carry out parallel studies with spinal cord samples from mutant and wildtype SOD1 mice, and compare both the in vivo and in vitro derived data sets with laser capture microdissection studies of both the ALS mouse model and human ALS patient samples.

We have detected significant cell autonomous and non-autonomous changes in gene expression in both motor neurons and glia, indicating that the two cell types profoundly affect each other's gene expression. In addition, we find a remarkable concordance between the different data sets mentioned above, thus validating the in vitro approach. We are currently analyzing these data sets to identify changes in the expression of specific genes and signalling pathways that may contribute to motor neuron degeneration in ALS.

C4 ROLE OF RNA PROCESSING IN THE PATHOGENESIS OF AMYOTROPHIC LATERAL SCLEROSIS

LAGIER-TOURENNE C^1,2, POLYMENIDOU M^1,2, HUTT KR², LIANG TY², HUELGA S², LING S^1,2, MAZUR C³, WANCEWICZ E³, BENNETT FC³, YEO GW², CLEVELAND DW^1,2

¹Ludwig Institute for Cancer Research, La Jolla, California, United States, ²Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States, ³Isis Pharmaceuticals, Carlsbad, California, United States

E-mail address for correspondence: [email protected]

Keywords: RNA processing, TDP-43, high-throughput sequencing

Background: The identification of Amyotrophic Lateral Sclerosis (ALS)-causing mutations in TDP-43 and FUS/TLS, two RNA/DNA binding proteins, combined with TDP-43 mislocalization in most incidences of sporadic ALS, suggests that altered RNA processing may play an important role in the pathogenesis of ALS. A fundamental issue is the precise role(s) of TDP-43 in RNA metabolism regulation and how alterations in its properties may underlie neurodegeneration. TDP-43 has been proposed to participate in several steps of RNA processing including alternative splicing regulation. Few RNA targets of TDP-43 have been identified and a comprehensive protein-RNA interaction map still needs to be defined.

Objectives: To identify in vivo RNA targets of TDP-43 and validate the roles of TDP-43 in the processing of these targets.

Methods: We have used cross-linking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP or CLIP-seq) to identify RNAs bound by TDP-43 in mouse brain. We have subsequently determined the effects of TDP-43 loss of function on RNA expression and splicing patterns by using high-throughput sequencing of cDNA (RNA-seq).

Results: Greater than 2 million uniquely mapped reads enabled the accurate generation of clusters using gene-specific thresholds to define TDP-43 binding sites. Consistent with a proposed role of TDP-43 as a splicing regulator, multiple binding sites proximal to alternatively spliced exons were identified. To validate the role of TDP-43 in the regulation of alternative splicing via these sites, downregulation of TDP-43 in vivo was achieved in an otherwise normal adult mouse brain using direct injection of antisense oligonucleotides against TDP-43. Transcriptome profiling from brains with TDP-43 knockdown to 80% of endogenous levels confirmed its roles in alternative splicing and gene expression regulation.

Discussion and conclusions: Genome-wide identification of validated RNA targets is a first step in the elucidation of the molecular mechanisms underlying death of motor neurons in ALS. This study reinforces the crucial role of splicing regulation for neuronal integrity and potentially identifies candidate genes whose altered processing is central to ALS pathogenesis.

C5 GENETIC AND BIOCHEMICAL ANALYSIS OF TDP-43 PROTEINOPATHY

KIM SH, HANSON K, TIBBETTS R

University of Wisconsin, Madison, WI, United States

E-mail address for correspondence: [email protected]

Keywords: TDP-43, FUS/TLS, motor neuron

It was recently found that dominant mutations in two related RNA-binding proteins, TDP-43 (43 kDa TAR DNA-binding domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a subset of ALS. The convergent ALS phenotypes associated TDP-43 and FUS/TLS mutations are suggestive of a functional relationship; however, whether or not TDP-43 and FUS/TLS operate in common pathways is not known. We have employed biochemical and genetic approaches to discover pathways controlled by TDP-43 and FUS/TLS in mammalian neurons and in the fruit fly, Drosophila melanogaster. We have found that TDP-43 and FUS/TLS directly interact to form a complex at endogenous expression levels in mammalian cells. Binding was mediated by an unstructured TDP-43 C-terminal domain and occurred within the context of a 300-400 kDa complex that also contained C-terminal cleavage products of TDP-43 linked to neuropathology. On the other hand, FUS/TLS and TDP-43 C-terminal fragments were excluded from large molecular mass TDP-43 ribonucleoprotein complexes that also contain the nuclear polyA-binding protein, PABP2. TDP-43 and FUS/TLS collaborate to regulate mRNA stability of HDAC6, and likely other mRNA targets, in mammalian cells, suggesting that these two ALS-associated proteins participate in common biochemical processes. We have further explored the mechanisms of TDP-43-dependent neurodegeneration in Drosophila, where expression of mutant or wild-type human TDP-43 proteins in motor neurons causes age-dependent paralysis and lethality. We are using the Drosophila ALS model to discover neuron-selective gene expression changes occurring before, during, and after onset of paralysis. The goal of these studies is to identify genes and small molecules that modify TDP-43-associated motor neuron death and to apply these findings to mammalian preclinical models. We will discuss our initial findings implicating two pathways as potentially key determinants in TDP-43-dependent neurodegeneration in vivo.

C6 CHARACTERIZING THE ROLE OF TDP-43 IN ALS

FREIBAUM B, SMITH R, ALAMI N, TAYLOR PJ

St. Jude Children's Research Hospital, Memphis, TN, United States

E-mail address for correspondence: [email protected]

Keywords: TDP-43, RNA metabolism, proteomics

Background: The TAR DNA-binding protein 43 (TDP-43) is the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. TDP-43 is a highly conserved nuclear heterogeneous ribonucleoprotein (hnRNP) that has been implicated in regulating the transcription, splicing and translation of target genes. In the brain, TDP-43 is normally found in the nuclei of neurons and some glial cells, although dynamic studies performed in vitro have shown TDP-43 to shuttle between the nucleus and cytoplasm. In TDP-43 proteinopathies, TDP-43 is frequently found redistributed from the nucleus to insoluble aggregates in the cytoplasm, although the significance of this finding is unknown. Mutations in the gene encoding TDP-43 have been identified in association with sporadic and familial forms of ALS, strongly implicating this protein as contributing to pathogenesis in these diseases.

Objectives: In this project, we seek to define the role of TDP-43 in disease pathogenesis using biochemical and molecular genetic approaches in both in vitro and in vivo model systems.

Methods: We characterized the TDP-43 interactome using proteomics in human tissue culture cells and developed transgenic Drosophila expressing wild type and numerous mutant forms of TDP-43. The mutant forms of TDP-43 we have evaluated are designed to interrogate the contribution of distinct TDP-43 domains to pathogenesis.

Results: Global proteomic analysis reveals that TDP-43 interacts with two distinct protein interaction networks, each involved in different aspects of RNA metabolism. The first is a group of predominantly nuclear proteins that regulate RNA splicing; the second is a group of predominantly cytoplasmic proteins involved in mRNA translation. Many of these interactions were found to be dependant on the RNA binding domain of TDP-43. Additionally, TDP-43 was found to interact with components of RNA granules and is recruited to RNA granules in an RNA dependant manner. To date, we have not identified any altered interactions in disease causing TDP-43 mutants.

In vivo studies using transgenic Drosophila demonstrate motor neuron degeneration in response to TDP-43 expression. This phenotype is enhanced by the M337V mutation associated with familial ALS, or by mutations designed to promote cytoplasmic localization of TDP-43.

Discussion and conclusion: Based on the results of these studies, we hypothesize that altered RNA metabolism plays a key role in the pathogenesis of ALS. Our proteomic analysis of TDP-43 interacting proteins reveals strong association with RNA binding proteins in support of this hypothesis. Ongoing work is being done to characterize the TDP-43 interactome in motor neurons as well as identifying additional genes that are required for ALS pathogenesis.

C7 RNA TARGETS OF TDP-43 IDENTIFIED USING UV-CLIP ARE DEREGULATED IN ALS

XIAO S, SANELLI T, ZINMAN L, ROBERTSON J

University of Toronto, Toronto, Canada

E-mail address for correspondence: [email protected]

Keywords: TDP-43, CLIP, RNA

Background: Tar DNA Binding protein-43 (TDP-43) is a major component of the pathological inclusions characteristic of Amyotrophic Lateral Sclerosis (ALS) and mutations in the TDP-43 gene are causative of about 4% of fALS cases and ∼1% of sALS cases. TDP-43 plays important roles in RNA processing and regulation, including transcriptional repression, mRNA stability and alternative splicing. Based on this, it is therefore likely that abnormalities of TDP-43 in ALS will be reflected in defects of RNA processing.

Objectives: Our first objective was to identify the RNA targets of TDP-43. Our second objective was to determine if any of the identified RNA targets are disrupted in ALS.

Methods: RNA targets of TDP-43 were identified using UV-CLIP (cross linking and immunoprecipitation) applied to the human neuroblastoma cell line, SHSY5Y.

In this method, UV-treatment of SHSY5Y cells covalently crosslinks TDP-43 to its target RNAs. The TDP-43-RNA complexes are then immunoprecipitated with polyclonal TDP-43 antibody, treated with RNAse, radiolabeled with γ-³²P and resolved by SDS-PAGE. The TDP-43-RNA complexes are excised from the gel and the bound RNA tags (CLIP-tags) released with proteinase k, cloned and sequenced. The sequences are then BLASTed to identify the corresponding genes.

Results: Conventional cloning and sequencing was used to validate the UV-CLIP technique, as high throughput sequencing generates an overwhelming abundance of data that can be difficult to process. After TA-cloning of the CLIP-tags, 250 clones were empirically selected for analyses. Of these, after numerous quality control steps, 101 genes were identified as RNA binding targets of TDP-43. These genes represented multiple biological pathways. The TDP-43 binding sites were predominantly within intronic sequences (77%), but also included 3'UTRs (4%), non-coding RNAs (5%) and intergenic regions (14%). Importantly the two most common recognition motifs were TG-rich (49%) and polypyrimide-rich (17.65%) sequences, as has been previously reported by others, validating our methodology for isolating bona fide TDP-43 RNA targets. RT-PCR of spinal cord mRNA was used determine if candidate genes were deregulated in ALS. The strategy for primer design was based on the location of TDP-43 binding and from web-based predictions as to whether or not the adjacent exons were alternatively spliced. Using this approach we show that 5 of the genes identified by TDP-43-CLIP are abnormally spliced in ALS versus controls.

Discussion: We have used UV-CLIP to identify RNA targets of TDP-43 and shown that 5 of the identified genes are deregulated in ALS versus controls. This supports the hypothesis that abnormalities of TDP-43 in ALS cause defects in RNA processing.

Conclusion: The genes we have identified not only provide candidates for genetic analyses, but will also provide insight into the biological pathways that lead to degeneration of motor neurons in ALS.

C8 INCREASING AUTOPHAGY RESCUES NEURODEGENERATION IN FLIES LACKING ADAR RNA EDITING

PARO S, LI X, MCGURK L, O'CONNELL M, KEEGAN L

MRC-Human Genetics Unit, Edinburgh, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: RNA editing, autophagy, Drosophila

Background: It has been shown that a reduction in RNA editing at the GluR2 Q/R site by ADAR2 is observed in motor neurons (MNs) of patients with sporadic ALS. This lack of RNA editing causes glutamate excitotoxicity and subsequently neuronal death. It has been shown that accumulation of altered mitochondria and intracellular aggregates within amyotrophic lateral sclerosis (ALS) MNs were associated with a defect in autophagy. One possibility is that a reduction in autophagy activation in MNs could contribute to ALS pathogenesis. Autophagy is a process involved in maintaining cellular homeostasis. It degrades long-lived proteins and organelles by enclosing them in a double-membrane vesicle (autophagosome) and delivering them to lysosomes for digestion. Increased numbers of autophagic vacuoles (AVs) are seen in a variety of stress and pathological states. The biological role of autophagy and its relationship to cell death in general and to excitotoxic neuron death in particular is still poorly understood.

Objectives: We have studied the effect of loss of ADAR RNA editing in neurons using Drosophila. ADAR2 has one homolog gene in Drosophila melanogaster. Immediately upon eclosion from the pupa, Adar^5G1 null flies show locomotion defects and later develop age-dependent neurodegeneration. EM analysis of both brains and retina of mutant flies reveals abnormal intracellular double-layer membrane vacuoles containing cytosolic material. This study aims to elucidate the pathways that cause neurodegeneration in Adar null flies using Drosophila genetics.

Methods: A genetic screen for dominant suppressors of reduced Adar^5G1 viability was performed. We crossed virgin females of y, Adar^5G1, w/FM7 with males from a series of DrosDel strains. We calculate Adar^5G1 male viability relative to FM7, Bar in the presence of the hemizygous DroDel deletion that might show suppressive and/or enhancing effects on viability. The mutant flies that had an increase of viability were aged for 30 days; locomotion activity was assayed using a two-minute open-field locomotion test and hematoxylin and eosin staining of the brain sections was used to investigate neurodegeneration.

Results: The screen revealed that decreasing Tor expression with either DrosDel deficiencies or specific P-element insertions in Tor suppresses Adar mutant phenotypes. The same effect was observed by inducing autophagy by overexpression of Atg genes using the Cha-Gal4 driver. Furthermore viral anti-apoptotic protein p35 expression did not rescue the phenotypes. Additionally, TUNEL-positive apoptotic brain cells were not detected in Adar^5G1 flies.

Conclusions: For the first time we provide in vivo evidence that Adar^5G1 mutant phenotypes are associated with the autophagy. We did not find evidence of neuron death and autophagy appears to be protective in flies. Autophagy may not be the cause of death in ALS motor neurons but may be protective.

C9 MICRORNA DYSREGULATION IN HUMAN SPORADIC ALS

LIOU L¹, KIM JM², RAVITS J², MÖLLER T¹

¹University of Washington, Seattle, WA, United States, ²Benaroya Research Institute at Virginia Mason, Seattle, WA, United States

E-mail address for correspondence: [email protected]

Keywords: microRNA, motor neuron biology, laser capture microdissection

Background: MicroRNAs are noncoding single-stranded RNA molecules about 22 nucleotides in length that function in the post-transcriptional regulation of gene expression by binding to mRNAs. Many genes contain predicted microRNA target sites in their 3’-untranslated regions (UTRs), and computational estimates suggest that about one-third of all genes that encode proteins may be regulated by microRNAs. MicroRNAs have been implicated in neurodegenerative diseases, but relatively little is known about the dysregulation of CNS-specific microRNAs. Differential expression of microRNAs has been seen in brain and CSF from Alzheimer's disease patients, and specific microRNAs have been found to be involved in spinocerebellar ataxia and Parkinson's disease. However, little is known about the role of microRNAs in ALS.

Objectives: To investigate if ALS is associated with a dysregulation of microRNAs.

Methods: Motor neurons and motor neuron-depleted anterior horns were collected from lumbar sections by Laser Capture Microdissection from 10 sporadic ALS patients and 10 controls. Total RNA was isolated using the RNAqueous Micro kit (Ambion). The microRNA expression was assessed using the microRNA TaqMan® qPCR Megaplex pools array (AB) with preamplification according to the manufacturer's protocol. Data analysis was performed with DataAssist V2.0 software (AB).

Results: We found that in motor neurons from control patients on average 104 microRNAs were detectable. In contrast in motor neurons from sporadic ALS patients only 86 miRNA were detectable. The expression level of still detectable miRNA was considerably reduced and no miRNA showed increased expression. In contrast, similar analysis on the surrounding anterior horn depleted of motor neurons, showed no statistically significant difference between ALS and control tissue.

Discussion: We have found a novel and seemingly general (all 10 tested sporadic ALS patients) dysregulation in microRNA levels in sporadic ALS. We are currently investigating the potential underlying mechanisms which might lead to a global reduction in microRNAs (eg expression, processing, localization).

Conclusions: Sporadic ALS is associated with a motor neuron-specific loss in miRNA expression. Our finding might provide novel avenues to investigate the motor neuron loss in ALS.

SESSION 2B AUTONOMY AND QUALITY OF LIFE

C10 QUALITY OF LIFE: INDIVIDUAL VALUES, STANDARDIZED ASSESSMENT?

COHEN SR

Lady Davis Research Institute, Jewish General Hospital, Quebec, Canada, McGill University, Quebec, Canada

E-mail address for correspondence: [email protected]

Keywords: quality of life, assessment, carer

Quantitative assessment of quality of life must begin by ensuring that instruments to measure it include all the relevant content and only relevant content. Otherwise one can measure something with precision, but it will not be quality of life that is measured. However, quality of life can be a nebulous concept. Who decides what is relevant? Is it even possible to have a standardized measure given differences among individuals in terms of what is important to their quality of life? How can there be a good quality of life if one has motor neuron disease, or is caring for someone with it?

Much of the current literature regarding quality of life for people with motor neuron disease does not distinguish between quality of life and health status, and the field may therefore be heading down the wrong path. The following will be explored in this talk. 1) The concept of quality of life, and its relationship to other important outcomes such as health status. 2) Critical questions that need to be answered before embarking on quality of life studies. 3) Different purposes for measuring quality of life and the implications of this for quality of life assessment. 4) The state of quality of life assessment for people with motor neuron disease and for their carers. The talk will conclude with a proposal for the way forward.

C11 THE OREGON DEATH WITH DIGNITY ACT: WHY DO PATIENTS REQUEST PHYSICIAN-ASSISTED DEATH?

GANZINI L

Oregon Health and Science University (OHSU), Portland, OR, United States

E-mail address for correspondence: [email protected]

Keywords: euthanasia, end of life care, terminal illness

The Oregon Death with Dignity Act, which legalized physician-assisted death (PAD) for terminally-ill patients, was enacted 13 years ago. This law allows a competent, requesting patient, with an estimated life expectancy of less than six months, to receive a prescription for a lethal medication from a physician for the purposes of self-administration. Support for the law varies among Oregon health professionals ranging from 40% of hospice chaplains and 51% of physicians, to 78% of psychologists. Only one third of Oregon physicians are willing to prescribe under the law. On the other hand, nine in ten Oregon hospice nurses who oppose the law would not actively oppose a client's choice for PAD.

Since enactment of the law, 460 patients have died by lethal medication, approximately 2/1000 Oregon deaths. Patients who die by PAD are more likely to have a college education, and a scant 2% lack medical insurance. Although only 35 ALS patients have died by PAD, ALS is the disease associated with the highest likelihood of PAD; ALS patients are over 30 times more likely to use PAD compared to other Oregon decedents. Over half of ALS patients in Oregon indicate they might consider legalized PAD, especially men, those who have high scores on measures of hopelessness, and those who are not religious. Studies of physicians and hospice workers who have cared for requesting patients; family members; and patients themselves, all point to the importance of staying in control, not being dependent on others, maintaining self-sufficiency and not burdening family as reasons patients pursue PAD. Among ALS patients negative views on the future predict persistent interest in PAD over time. Most requesting patients do not have major depressive disorder, however, a small number of patients with depression, who have not been evaluated by a mental health professional, access PAD. Over 80% of patients who have died by PAD in Oregon are hospice enrolled. Hospice referral and provision of non-judgemental support are the most effective interventions resulting in patients finding alternatives to PAD.

Among those whose family member requested PAD, whether or not the patient accessed a lethal prescription had no influence on subsequent depression, grief, or mental health services use; however, family members of Oregonians who received a lethal prescription were more likely to believe that their loved one's choices were honored and less likely to have regrets about how the loved one died. Family members of Oregonians who requested PAD felt more prepared and accepting of the death than comparison family members.

C12 DO RELIGIOSITY AND SPIRITUALITY CORRESPOND WITH ALS PATIENTS’ VIEWS ON END-OF-LIFE ISSUES? AN EXPLORATIVE STUDY WITH ALS PATIENTS AND THEIR PRIMARY CAREGIVERS

STUTZKI R¹, WEBER M², REITER-THEIL S¹

¹Department of Medical and Health Ethics, Medical Faculty, University of Basel, Basel, Switzerland, ²Neuromuscular Diseases Unit/ALS Clinic of the Cantonal Hospital, St. Gallen, Switzerland

E-mail address for correspondence: [email protected]

Keywords: religiosity, spirituality, suicide

Background: In the secular society of Switzerland self-determination, especially regarding end-of-life decisions, is highly esteemed. As suicide and also assistance to suicide are not legally punishable acts (unless in cases of ‘selfish motivation’), health care professionals as well as family members or friends of severely ill patients are facing ethical dilemmas, when the patient asks them to help terminating his/her life. This has been observed in groups of patients with malignancies and progressive diseases, including those suffering from ALS (Amyotrophic Lateral Sclerosis).

Objectives: To determine the correlation of personal faith, religious denomination and spirituality with ALS patients’ views on end-of-life issues.

Methods: Explorative interview study with 30 patients and their primary caregivers; semi-quantitative questionnaire and qualitative interview study (2008-2010). Measures: Demographics; Questions on end-of-life decisions; Hospital Anxiety and Depression Scale (HADS); Idler Index of Religiosity (IIR); Schedule for Meaning in Life Evaluation (SMiLE); The Neurobehavioural Rating Scale NRS on quality of life, feeling lonely, being a burden to others; semi-structured, tape-recorded interviews.

Results: Thirty patients and their caregivers were interviewed. The median age of the patients (10 female and 20 male) was 59 years. 15 patients were Roman-Catholic, 10 Protestant and 5 non-denominational. Median age of the care givers was 56 years (10 male, 20 female). 14 caregivers were Roman-Catholic, 7 Protestant and 9 non-denominational.

Significant differences between patients and caregivers were found in questions concerning quality of life (P=0.004), loneliness (P=0.005), religious self-assessment (P=0.001) and life-prolonging measures (PEG: P= 0.007).

Thirty seven per cent of the patients had already either thought about or discussed the option of ending his or her life with the help of a relative, close friends, pastor or medical doctor. At the same time, none of the interviewees showed any sign or interest to commit suicide or actively asked for assistance to terminate their life.

Discussion: The results suggest that patients affiliated to a religious denomination are less likely to consider suicide and also assistance to suicide as an option of action in comparison with patients who are not practising any faith.

Conclusion: Religious confessional faith does have an impact on a patient‘s view towards end-of-life issues. There is a significant difference, however, between Roman-Catholic and Protestant patients’ views.

C13 THE DIAGNOSTIC JOURNEY FROM SYMPTOM ONSET: EXPERIENCES OF PEOPLE WITH MND AND FAMILY CARERS

O'BRIEN M¹, WHITEHEAD B^1,2, MITCHELL D², JACK B¹

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

E-mail address for correspondence: [email protected]

Keywords: diagnostic journey, personal experiences, qualitative research

Background: Studies addressing the diagnosis in MND polarise upon diagnostic delays (1) or communication of the diagnosis (2). There has been little investigation of the journey from symptom onset through to diagnosis as a means of capturing and exploring the full diagnostic experience for patient and family.

Objectives: To use qualitative methodology to explore the personal experiences of patients and family carers to capture the diagnostic journey from initial symptom development through to delivery of the diagnosis. Additionally we sought to identify issues which may impact positively or negatively on these experiences.

Methods: Narrative interview data was collected from a purposively selected sample of people with MND (n=24), current (n=18) and former family carers (n=10). Thematic analysis was aided by NVIVO 7.

Results: A number of themes were apparent. Delays in arriving at a diagnosis arose from a failure to recognise the significance of intermittent and insidious symptoms which were frequently dismissed by patients, carers and health professionals. A lack of urgency within primary care resulted in protracted periods of time between initial GP consultation and onward referral for specialist investigations. Within secondary care there were extended waiting times for investigations. There was consensus amongst participants that these interrelated factors impacted on patients’ and carers’ well-being. Participants reported a mixture of experiences surrounding the communication of the diagnosis; for some the process was handled sensitively with appropriate explanation and empathy. However, many described interaction at this time as blunt and unsatisfactory revealing poor communication skills and a lack of consideration for the impact of the diagnosis on the patient and their family. Although the diagnosis was traumatic, it came as a relief for some to have their condition named so they could finally understand the cause of their symptoms. Comparisons were made with the assistance available for people with cancer; the impression being that support was routinely provided at the point of diagnosis in cancer, but not so in MND. Previous knowledge of MND did have an impact on the reaction to the diagnosis for a number of participants.

Conclusions: This study has provided insight into the experiences of people affected by MND during the time from symptom onset through to diagnosis. It highlights areas where delays have occurred and reveals the impact on patients and carers of these delays. The need for better and more sensitive healthcare interaction with patients and carers at this traumatic time is apparent.

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C14 COMMUNICATION STRATEGIES THAT ALS PATIENTS USE AT END OF LIFE: RESULTS OF A THREE YEAR SURVEY

BROWNLEE A^1,2, BRUENING L³

¹ALS Association, National Office, Calabasas Hills, CA, United States, ²ALS Center, University of Pennsylvania Health System, Philadelphia, PA, United States, ³ALS Association, Northern Ohio Chapter, Cleveland, OH, United States

E-mail address for correspondence: [email protected]

Keywords: communication, end of life, AAC

Background: Many people with ALS are not able to verbally communicate at end of life and rely on many different forms of communication systems to address medical and social needs, ask questions, and convey fears and concerns about the dying process. Many allied healthcare professionals have expectations that patients will utilize an electronic communication device until death. The results of this three year survey find that people with ALS are not using electronic communication systems at end of life but in fact are using multiple modalities of communication, the primary system being low technology like eye blinks, gestures, and letter boards. The speaker will present the results of this survey and recommendations to the field to address the communication issues of people with ALS at end of life.

Objectives: 1) Determine how many ALS families were aware of speech/communication issues related to ALS; 2) List specific communication related topics people with ALS are communicating at end of life; 3) Identify what communication strategies people with ALS are using at end of life; 4) Identify the number of people that were enrolled in hospice.

Methods: Surveys were sent to caregivers of ALS patients in various US States for a three year period from 2007-2010. Caregivers we asked specific questions as to how their loved one was communicating 3 months prior to death, 1 month prior to death, and the last few weeks and days of life. Surveys were sent via regular US mail.

Results: Almost 75% of those surveyed had signed on to hospice services; most people with ALS were communicating via speaking 2-6 months prior to death and 4 weeks prior to death. Days before death, most people with ALS were using gestures, eye blinks, letterboards, and electronic communication devices. In the last 2-6 months, less than 20% of people that had electronic devices used them. In the initial survey, sent to caregivers from 2007-2009, there were a total of 93 caregivers who reported their loved one had an electronic device in the home. 48 caregivers reported that their loved one had a device and chose not to use it – over 50%. Another 19 reported that their loved one sometimes chose not to use it for a total of 72% that had a device and chose not to use it. Most people with ALS used their electronic devices for less than one month.

Conclusions: More stress needs to be placed on low technology options and/or multi-modalities of communication systems. Caregivers and allied healthcare professionals need to be trained in various low technology options since it seems families use multiple systems for communication when people with ALS are near the end of life.

C15 AN ADVANCE-CARE PLANNING DECISION AID FOR PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS

BROTHERS A, GREEN M J, LEVI B, FARACE E, SIMMONS Z

Penn State College of Medicine/Hershey Medical Center, Hershey, PA, United States

E-mail address for correspondence: [email protected]

Keywords: advance care planning, computer-based decision aid, end-of-life

Background: Patients with ALS inevitably face decisions about what medical treatments they would and would not want in the future. Common methods of advance care planning have not been particularly effective at facilitating such decision-making. We anticipated that a computer-based decision aid would help ALS patients with the process of advance care planning.

Objectives: To determine whether: 1) a computer-based decision aid improves knowledge and decision-making among patients with ALS; and 2) the intervention increases the multi-disciplinary ALS Team's awareness of their patients’ healthcare wishes.

Methods: Patients meeting El Escorial criteria for ALS were enrolled in the study. Participants completed pre- and post- intervention questionnaires, using a computer-based decision aid to generate advance directives. Members of the ALS Clinic team (neurologist, nurse, counselor, chaplain, social worker, physical and occupational therapists, speech/language pathologist, dietitian) were surveyed about what end-of-life treatment decisions they thought they would make based on 3 hypothetical vignettes, and a concordance score was calculated to determine the extent to which patients agreed with the ALS team's decisions. Paired-sample t-tests were conducted for the pre-and post-intervention outcome measures and concordance ratings. Descriptive statistics are reported for participants’ decisional conflict and satisfaction scores.

Results: There were 18 participants (12 male). 88.9% were married, 94.4% had at least a high-school education, and 94.4% lived in their own home. Participants’ knowledge of advance care planning significantly improved after using the decision aid (51.4% correct responses pre-intervention compared to 67.9% post-intervention; P < 0.001). Mean (M) Self Determination scores (feeling of control about what medical treatments one will receive in the future) did not change pre- and post-intervention (M=35, maximum score=40). Anxiety also remained unchanged (P = 0.64). Post-intervention Decisional Conflict (maximum score =80) was relatively low (M=29, SD = 8.3), indicating that participants felt clear about their decisions for end-of-life care. Satisfaction with the computer program (maximum score=60) was high (mean=52.2, SD = 5.8); similarly, participants were satisfied with their decisions (M=9.9, SD = 3.1; where 6 = highest satisfaction and 30 = lowest satisfaction). Decisions for end-of-life care made by the patient and by the ALS team (n=11) showed significantly higher post-intervention concordance (maximum score = 100%; pre-intervention M=61.6, SD=31.4 compared to post-intervention M=88.4, SD=13.5, P = 0.017) suggesting that the ALS team's understanding of patients’ wishes significantly improved after the intervention.

Discussion and conclusions: Use of the computer-based decision aid effectively increased participants’ knowledge about advance care planning without an increase in anxiety levels. It was favourably received by ALS patients, who indicated high levels of satisfaction with the computer program, and with their decisions. Finally, the intervention was effective in improving the ALS Clinic team's understanding of patients’ wishes.

SESSION 3A LESSONS FROM OTHER DISEASES

C16 RNA PROBLEMS AND SOLUTIONS: LESSONS FROM MYOTONIC DYSTROPHY

THORNTON C

University of Rochester, Rochester, NY, United States

E-mail address for correspondence: [email protected]

No abstract available.

C17 THE ROLE OF RNA SPLICING IN SPINAL MUSCULAR ATROPHY

PELLIZZONI L

Department of Pathology and Cell Biology, Columbia University, New York, NY, United States, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, United States

E-mail address for correspondence: [email protected]

Keywords: spinal muscular atrophy, survival motor neuron, RNA splicing

Spinal muscular atrophy (SMA) is an inherited motor neuron disease and the leading genetic cause of infant mortality. SMA is caused by reduced levels of the survival motor neuron (SMN) protein, a core component of a macromolecular complex that is required for the efficient and specific assembly of small nuclear ribonucleoproteins (snRNPs)-the essential constituents of the pre-mRNA splicing machinery. Although SMN is ubiquitously expressed and snRNP biogenesis is required in all cells, we found that SMN activity in snRNP assembly is regulated in a tissue-specific and time-dependent manner, suggesting a differential requirement for this function in distinct cell types and during development. Analysis of the functional consequences of SMN deficiency in mouse models of SMA revealed that snRNP assembly is defective and the extent of its reduction correlates with disease severity. Surprisingly, rather then causing a uniform decrease in the levels of all snRNPs, SMN deficiency causes tissue-specific changes in the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. Furthermore, the levels of a subset of snRNPs that are responsible for the splicing of a rare class of introns (U12-type) representing less than 1% of all eukaryotic introns are particularly affected in SMA tissues. Based on this observation as well as the finding that restoration of a normal snRNP profile coincides with phenotypic correction in animal models of SMA, we investigated a possible link between SMN-dependent defects in the U12 splicing pathway and SMA etiology. Using cellular and animal models we found that SMN deficiency affects splicing of U12 introns in a significant proportion of genes with this type of introns. We took advantage of the very small number of U12 intron-containing genes to carry out a genome-wide functional analysis in Drosophila mutants of SMN. In this model of SMA, SMN deficiency causes synaptic dysfunction at the neuromuscular junction (NMJ), decreased muscle size and impaired locomotor activity. We have found that expression of approximately 40% of U12 intron-containing genes is decreased in Drosophila SMN mutants and studied the contribution to the neuromuscular phenotype of each of these genes. Using RNAi inhibition and overexpression in specific neuronal types, we have identified a novel transmembrane protein whose restored expression in Drosophila mutants of SMN fully rescues neurotransmitter release at the NMJ and significantly improves muscle size and locomotion. These findings reveal that SMN deficiency can elicit neuronal dysfunction at the NMJ by affecting splicing of genes critical for synaptic transmission and support the conclusion that SMA is an RNA splicing disease.

C18 MUTANT SMALL HEAT-SHOCK PROTEIN 27 (HSPB1) MICE RECAPITULATE AXONAL CHARCOT-MARIE-TOOTH DISEASE AND DISTAL HEREDITARY MOTOR NEUROPATHY PHENOTYPES

D'YDEWALLE C¹, KRISHNAN J¹, CHIHEB D¹, VAN DAMME P^1,2, IROBI J^3,4, VAN DEN BERGHE P⁵, TIMMERMAN V^3,4, ROBBERECHT W^1,2, VAN DEN BOSCH L¹

¹VIB and University of Leuven, Leuven, Belgium, ²University Hospital Leuven, Leuven, Belgium, ³VIB & University of Antwerp, Antwerp, Belgium, ⁴Institute Born Bunge, Antwerp, Belgium, ⁵University Leuven, Leuven, Belgium

E-mail address for correspondence: [email protected]

Keywords: Charcot-Marie-Tooth disease, peripheral neuropathy, mouse models

Background: Charcot-Marie-Tooth (CMT) disease is the most common inherited disorder of the peripheral nervous system. CMT patients show progressive distal muscle weakening and atrophy, foot deformities and sensory loss leading to steppage gait. CMT is classified into three groups: demyelinating (CMT1), axonal loss (CMT2) and mixed forms. When exclusively lower motor neurons are affected, CMT2 is referred to as distal hereditary motor neuropathy (distal HMN). Mutations in the gene encoding the small 27 kDa heat-shock protein (HSPB1) have been identified as the cause of CMT and/or distal HMN.

Objectives: To elucidate the pathological mechanism underlying mutant HSPB1-induced CMT2 and/or distal HMN in vivo, we aimed to develop and characterize transgenic mice expressing wild type (wt) or mutant HSPB1 selectively in neurons.

Methods: We created transgenic mice expressing wt and two different mutant (S135F and P182L) HSPB1 isoforms driven by a Thy1.2 expression cassette. Several phenotypical tests (including rotarod, grip strength, hotplate, gait analysis and nerve conduction studies) were used to characterize these transgenic mice. Real-time imaging of mitochondria was performed on DRG neurons isolated from symptomatic mice to assess axonal transport.

Results: S135F- and P182L-HSPB1 lines demonstrated limb-clasping behavior from the age of 6 months on. Both mutant HSPB1 lines showed reduced rotarod performance aggravating in function of age. Muscle force was severely affected in hind limbs from 6 months on, while forepaw muscle force decreased only later in disease in both mutant HSPB1 lines. Mutant HSPB1 mice demonstrated steppage gait. In contrast to P182L-HSPB1 mice, S135F-HSPB1 mice showed sensory loss as assessed by the hotplate test. Nerve conduction studies revealed signs of motor axonopathy in P182L-HSPB1 mice, and mixed sensorimotor axonopathy in S135F-HSPB1 mice. Histochemical analysis of muscle, neuromuscular junctions and peripheral nerves showed signs of peripheral neuropathy in both mutant HSPB1 lines. Mitochondrial transport was severely affected in DRG neurons isolated from symptomatic S135F-HSPB1 mice, but not from P182L- and wt-HSPB1 mice.

Discussion: The selective expression of mutant HSPB1 in neurons gives rise to CMT2 or distal HMN phenotypes in mice. Thus, mutant HSPB1 cell-autonomously leads to a peripheral neuropathy. S135F-HSPB1 mice display all key features of a mixed sensorimotor polyneuropathy while P182L-HSPB1 mice only demonstrate signs of a motor neuropathy. These findings are in line with the clinical symptoms in CMT2 and distal HMN patients, respectively. Disturbances of mitochondrial transport along peripheral axons might underlie the pathological mechanism of CMT2 and distal HMN.

Conclusion: This work describes new mouse models for CMT2 and distal HMN. These models accurately recapitulate all key symptoms of both human conditions, depending on the mutation in HSPB1. Moreover, these models provide powerful tools to elucidate the pathological mechanism underlying CMT2 and distal HMN, paving the way to develop therapeutical strategies.

SESSION 3B CARE EDUCATION AND PRACTICE

C19 PALLIATIVE CARE IN TERMINAL ILLNESS – THE IMPACT ON HEALTHCARE PROFESSIONALS

JACKSON V^1,2

¹Massachusetts General Hospital Department of Medicine, Palliative Care Unit, Boston, MA, United States, ²Harvard Medical School, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: end of life care, physician/patient communication, palliative care education

Caring for patients who are suffering and will eventually die is a particular challenge. Clinicians need to develop ways of coping with this stress that allows a continued empathic, therapeutic connection with the patient and family while promoting a sustainable model of clinical care. It is not uncommon for issues of conflict, grief, appropriate boundaries, and helplessness to complicate these relationships. In this session, the challenges in caring for these patients and methods for addressing this stress will be reviewed.

C20 DEVELOPMENT, IMPLEMENTATION AND TESTING OF AN EDUCATIONAL PROGRAM TO GUIDE PALLIATIVE CARE FOR PEOPLE WITH MOTOR NEURONE DISEASE

MCCONIGLEY R¹, AOUN S¹, KRISTJANSON L¹, CURROW D², O'CONNOR M³, COLYER S⁴, EDIS R⁵, HARRIS R⁶, ALLCROFT P⁷, YATES P⁸

¹Curtin University of Technology, Perth, Western Australia, Australia, ²Flinders University, South Australia, Australia, ³Monash University, Victoria, Australia, ⁴MND Association of Western Australia, Western Australia, Australia, ⁵Royal Perth Hospital, Western Australia, Australia, ⁶Motor Neurone Disease Association of Victoria, Victoria, Australia, ⁷Southern Adelaide Palliative Care Services, South Australia, Australia, ⁸Queensland University of Technology, Queensland, Australia

E-mail address for correspondence: [email protected]

Keywords: palliative care, education, evaluation

Background: The relative rarity of MND means that many palliative care professionals will not have sufficient knowledge of MND to provide a high standard of care to patients and families. A large Australian study of the needs of people with four neurodegenerative disorders found that this group of people and their carers had many unmet care needs. People with MND reported the lowest quality of life of the groups studied, as did their carers. Therefore, it is essential that education be provided to palliative care professionals about MND to ensure that care is appropriate.

Objectives: This project aimed to improve the quality of care for people with MND and their families through the development, testing and implementation of an educational program for service providers focused on the palliative care needs of this population.

Methods: This project used a three-phased approach to develop and implement an educational program about providing palliative care for people with MND. Phase 1 involved an extensive consumer consultation process, including conducting interviews and focus groups with patients, carers and health professionals to determine education needs. The second phase involved developing and implementing an educational program for people working in the palliative care area. The third phase involved testing the efficacy of the education program in improving the care of people with MND and their families.

Results: The results of the phase 1 consultation showed that the education program needed to provide information about what MND is and how to effectively manage practical problems experienced by patients and families using an experiential method of teaching. The resulting education package consisted of six one-hour modules that could be presented as stand-alone sessions or as part of a workshop or education series. Six one-day workshops were held to test the education materials developed and assess the effectiveness of the sessions at changing the knowledge and attitudes of participants. Results of the testing showed that following attendance at the workshops participants had increased knowledge of MND and were more likely to have positive attitudes to providing care for people with MND.

Discussion: The results of this study show that palliative care professionals are interested in learning more about MND and that a targeted education program can improve their knowledge and attitudes to providing care to this under-served group. An implementation plan has been developed to ensure widespread uptake of the educational program across Australia.

Conclusions: People living with MND and their families will benefit from increased access to palliative care services that are able to provide specialist care when required. A targeted education program for palliative care professionals will assist in improving the palliative care provided.

C21 PAIN IN ALS: FREQUENCY AND CHARACTERISTICS IN A POPULATION BASED SERIES

CALVO A, CANOSA A, GALLO S, MOGLIA C, BERSANO E, BALMA M, ILARDI A, CAMMAROSANO S, PAPURELLO D, CHIÒ A

Department of Neuroscience, University of Torino, Torino, Italy

E-mail address for correspondence: [email protected]

Keywords: pain, population-based, case control

Background: Pain is considered rare in early stages of amyotrophic lateral sclerosis (ALS), but it is more frequently reported during advanced stages. However, there are no studies devoted to the assessment of frequency and characteristics of pain in ALS, compared to a control population.

Aim: The aim of this survey was to determine the characteristics of pain in a consecutive series of ALS patients, comparing it to a control population.

Methods: A total of 120 ALS incident patients from the Piemonte Register for ALS (PARALS) were interviewed. Pain was evaluated with the Brief Pain Inventory (BPI) questionnaire. Patients’ physical status was evaluated with ALSFRS-R. The control population included people matched by age (±3 years) and gender, randomly selected from the patients’ general practitioners lists.

Results: Of the 120 ALS patients, 68 were men and 52 women. Their mean age at the time of the interview was 62.2 years, their mean disease duration was 44.4 months (median, 34 months), and their mean ALSFRS-R score was 30.0 (range 0-45). Controls were similar to patients for the main demographic characteristics. Pain was reported by 64 patients (53.3%) and 44 controls (36.7%) (P = 0.009). Maximum and mean pain intensity were similar in patients and controls. Forty-seven patients (73.4%) and 19 controls (43.2%) were treated for pain at the time of the interview (P = 0.0001). The efficacy of therapy for pain received similar rates in patients (63.3%) and controls (66.7%) (P = 0.88). The probability of being treated among patients was not related to the rating of severity pain, but was significantly higher in patients reporting negative effects of pain on social relationship and enjoyment of life. Among patients, the presence of pain was not related to age, gender, but significantly increased with the worsening of disability (ALSFRS-R score) (P = 0.04) and disease duration (P = 0.03). Patients reported that pain caused an interference with their mood and enjoyment of life and to a lesser extent with their social relationships and sleep.

Comments: In our series, pain was more frequent in ALS patients than in age and gender matched controls, although its intensity was similar. The presence was related to patients’ disability. More patients than controls received a treatment for their pain, but the control of pain was deemed not completely satisfactory both in patients and in controls.

C22 PATIENT CARE COORDINATION AT CAROLINAS NEUROMUSCULAR/ALS – MDA CENTER: NURSING MANAGEMENT OF E-PATIENTS AND T-PATIENTS

WILLIAMS NM¹, BLACK KJ¹, NICHOLS MS¹, BRAVVER E^1,2, BROOKS BR^1,2

¹Carolinas Medical Center, Department of Neurology Neuromuscular/ALS-MDA Center, Charlotte, NC, United States, ²University of North Carolina, Chapel Hill School of Medicine, Chapel Hill, NC, United States

E-mail address for correspondence: [email protected]

Keywords: tele-nursing, telephone advice, internet web patient portal

Background: Patient care coordination requires a communication infrastructure to provide contact between patients and caregivers with nurses, allied health personnel, physicians and others. Day to day contacts cover a number of patient care, caregiver, care provider needs but little information exists in the ALS literature concerning communication techniques and topics covered in this communications infrastructure.

Objective: To identify baseline communication interactions in a large ALS clinic situated in the third largest public healthcare system in the United States, Carolinas Healthcare System.

Methods: Email (E-patients)and telephone (T-patients) contacts are monitored for one week each quarter and contacts are categorized by subject and interaction type (1). Summary data is analyzed with descriptive statistics.

Results: Total weekly contacts (88.0±7.4 /wk (standard deviation)) by Email (2.3%) or telephone (97.7%) consisted of patient-nurse (71.7±7.5 /wk (80.7%)), nurse-nurse (16.0±1.5 /wk(18.8%)) and drug company representative-nurse (1.0±0.4 /wk(0.5%)) interactions. The bulk (50.0%) of weekly contacts concerned appointments for MD, allied health or test follow ups (44.0±5.6 /wk) while the second most common concern (17.0%) was medications (15.0±2.1 /wk) followed by equipment needs (9.1%) referred to physical, occupational and augmentative communication therapy (8.0±1.1 /wk) and requests (6.8%) for medical records (6.0±2.1 /wk). Changes in patient symptoms (6.0+1.3 /wk(6.8%)), disability forms-patient bills (5.0±0.7 /wk(5.7%)), patient laboratory results (4.0±0.4 /wk(4.5%)), home care issues (3.0±0.9 /wk(3.4%)) and patient transport issues (2.0±0.9 /wk(2.3%)) completed.

Conclusions: Nurse-managed average daily patient contacts by Email (0.5±0.2 /d(2.3%)) and telephone (17.6±1.5 /d(97.7%)) require periodic audit to justify allocation of resources. In this ALS Clinic communication contacts for follow-up appointments concerning care issues constituted 50.0% of daily nursing interactions similar to a specialty pediatric clinic (2). This analysis provides benchmark information concerning communication interactions among nurses, patients, other care providers relying primarily upon a telephone-based system prior to development of internet patient portals for more web-based communication with ALS patients.

Supported by: Carolinas ALS Endowment, Pinstripes Foundation, Carolinas Healthcare Foundation, Muscular Dystrophy Association.

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C23 A NATIONAL STUDY OF AMYOTROPHIC LATERAL SCLEROSIS MULTIDISCIPLINARY CLINIC UTILIZATION

YOUNG J¹, FELGOISE S¹, STEPHENS HE², WALSH S³, SIMMONS Z²

¹Philadelphia College of Osteopathic Medicine, Philadelphia, PA, United States, ²Pennylvania State University College of Medicine, Hershey, PA, United States, ³ALS Association, Greater Philadelphia Chapter, Philadelphia, PA, United States

E-mail address for correspondence: [email protected]

Keywords: multidisciplinary care, quality of life, social problem solving skills

Background: Some literature indicates that patients with ALS who attend multidisciplinary clinics (MDCs) have a higher quality of life (QOL) than those who do not. However, a previous study from our group demonstrated no QOL difference between attendees and nonattendees. Social problem solving (SPS) skills predict QOL in ALS caregivers. Therefore, SPS skills may affect choice of health care delivery or differentiate between those with higher and lower QOL.

Objectives: 1) To investigate differences between persons with ALS who access MDCs and those who do not with regard to: QOL, use of adjuvant therapies and support services, functionality, and SPS skills; 2) To obtain qualitative information to understand reasons why people choose to attend or not attend MDC.

Methods: Participants completed a web-based survey in response to announcements posted on Patientslikeme.com and the ALS Association website. The survey included the ALS Specific Quality of Life Questionnaire-Revised (ALSSQOL-R), the ALS Functional Rating Scale Revised (ALSFRS-R), the Social Problem Solving Inventory Revised (SPSI-R), and questions regarding MDC utilization. Multiple analysis of variance (MANOVA) was used to test for differences between attendees and nonattendees on the ALSSQOL-R and SPSI-R subscales and total scores. Univariate analysis was used to identify differences when the MANOVAs revealed significant differences. T-tests, analysis of variance, and descriptive statistics were used to test differences between the samples on personal characteristics, and to describe these characteristics.

Results: 403 ALS patients completed the survey –314 attendees and 89 nonattendees. Attendees had higher scores than non-attendees for average total ALSSQOL-R (mean 6.35 vs. 5.75, P=0.031). and for the subscales of Intimacy (P = 0.021), Physical Symptoms (P = 0.008), and Bulbar Function (P = 0.021). Both groups’ mean scores on SPSI-R and subscales were within the average range, and did not differ from one another (P = 0.147). Reasons for attending MDCs included: 1) Expertise of the specialist, 2) information and knowledge obtained, 3) convenience, travel and time, 4) support, and 5) research and clinical trials. Attendees reported more use of medications for depression, saliva, cramping, spasticity, pain and sleep. They also were more likely to have received services from other health professionals, complementary/alternative medicine, and in-home care.

Discussion: Individuals attending a MDC generally have a better QOL than those who do not. They can identify multiple reasons for attendance, and receive more treatments and services. Problem solving skills do not differ between attendees and non-attendees. It is possible that those who attend MDC are in need of more services and support than non-attendees to maintain QOL, or that those who attend a MDC perceive their QOL as better because they have more support.

SESSION 4A EMERGING DISEASE MODELS

C24 TDP-43 MUTANT TRANSGENIC MICE DEVELOP BIOCHEMICAL AND PATHOLOGICAL FEATURES OF AMYOTROPHIC LATERAL SCLEROSIS AND FRONTOTEMPORAL LOBAR DEMENTIA

SWARUP V, PHANEUF D, BAREIL C, JULIEN J-P

Centre de Recherche du CHUQ, Department of Neuroscience and Psychiatry, University Laval, Quebec, QC, Canada

E-mail address for correspondence: [email protected]

Keywords: TDP-43, mouse models, mutations

Objectives: Neuronal cytoplasmic and intranuclear aggregates of RNA-binding protein TDP-43 are a hallmark feature of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ALS and FTLD show a considerable clinical and pathological overlap and occur as both familial and sporadic forms. Mutations in TDP-43 are known to cause both the familial and the sporadic form of ALS in about 3% of patients. The goal of this study is to overexpress two such TDP-43 mutations – A315T and G348C and the wild-type human TDP-43 protein in transgenic mice, thereby modeling the human disease.

Materials and methods: TARDBP (NM_007375) was amplified by PCR from a human BAC clone (clone RPCI-11, clone number: 829B14) along with the endogenous promoter (∼5kB). Site-directed mutagenesis using the primer-based approach was used for the generation of mutants (TDP-43^A315T and TDP-43^G348C). The full-length TARDBP (wild-type, G348C and A315T mutants) were linearized by Swa-1 restriction enzyme and microinjected in mice embryos (having a background of C3H X C57BL/6). Later, they were transplanted in pseudo-pregnant mothers (having ICR CD1 background). Founders were bred with Ntg C57BL/6 mice to establish stable transgenic lines. Transgene expression was analyzed in brain and spinal cord by real-time PCR and in brain, spinal cord, muscle, liver by Western blot using monoclonal human TDP-43 antibody (Clone E2-D3, Abnova). Immunohistochemistry and immunofluorescence on spinal cord and brain sections of TDP-43 transgenic mice were done using monoclonal TDP-43 antibody, ubiquitin (Chemicon), Iba-1 (Wako), GFAP (Chemicon).

Results: Neurons in the affected spinal cord and brain regions showed accumulation of TDP-43 nuclear and cytoplasmic aggregates that were both ubiquitinated and phosphorylated as observed in ALS/FTLD patients. Moreover, the characteristic ∼25-kDa C-terminal fragments (CTFs) were also recovered from nuclear fractions and correlated with disease development and progression in TDP-43^A315T and TDP-43^G348C mutant mice as compared to TDP-43^wt mice. TDP-43^G348C mice showed increased ubiquitin positive TDP-43 aggregates in the cytoplasm. Also these mice had learning and memory deficits as evaluated by passive avoidance test compared to TDP-43^wt. TDP-43^G348C and TDP-43^A315T transgenic mice also had increased microgliosis and astrogliosis.

Conclusion: Our results suggest that mutation in TDP-43 (A315T and G348C) result in cytoplasmic aggregation of TDP-43 and its ubiquitination and phosphorylation. These mice have increased microgliosis and astrogliosis. These mice also have severe learning and memory deficits suggesting their usefulness in modeling ALS and FTLD-U.

C25 A NEW MOUSE MODEL OF ALS CARRYING A POINT MUTATION IN THE MOUSE SOD1 GENE

JOYCE P¹, FRATTA P², PHATAK V¹, MCGOLDRICK P², GREENSMITH L², FISHER EMC², ACEVEDO-AROZENA A¹

¹MRC Mammalian Genetics Unit, Harwell, United Kingdom, ²UCL Institute of Neurology, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: SOD1, mouse models, phenotype

Background: The array of SOD1 transgenic models created to study ALS has been extremely useful in furthering our understanding of the disease. However, a potential drawback of these models is that they express SOD1 at higher levels than in human ALS patients. Thus, some of the phenotypes described in SOD1 transgenic animals could be caused by the high level of expression as opposed to the actual effects of mutant SOD1.

Objective: To create a more physiologically relevant model of ALS and try and overcome the disadvantages that underlie the SOD1 transgenic models.

Methods: We screened the Harwell N-ethyl-N-nitrosourea (ENU) archive for mutations in the Sod1 gene. ENU is a potent mutagen that causes genome-wide point mutations. The MRC Harwell ENU archive consists of 10,000 matching DNA and sperm samples from first generation progeny of ENU mutagenised male mice. Following the identification of ENU-generated mutations, mutant mice can be re-created through in vitro fertilisation from stored sperm. We have identified an allelic series of ENU induced mutants carrying mutations in the mouse Sod1 gene: D83G, E109G and R115H. All three mutated residues are mutated in ALS cases, with the D83G mutation carrying the same amino acid change. As these are point mutations, animals express SOD1 mutant protein at endogenous levels. Using an array of behavioural, physiological and histological techniques, we have begun to characterise Sod1 D83G homozygote and heterozygote mutant animals.

Results: Phenotyping experiments are ongoing, however, initial behavioural phenotyping of homozygote Sod1 D83G mice has revealed ALS-like defects such as abnormal gait and decreased grip strength, body weight and startle response. Sod1 D83G homozygote mice generate less muscle force and fatigue less, similar to transgenic mice expressing the SOD1 G93A mutant protein. In addition, Sod1 D83G homozygote mice show a reduced number of motor neurons at 15 weeks. Heterozygote D83G mice appear similar to wildtype mice until approximately 7 months, at which time they develop behavioural and physiological abnormalities. Current ongoing work will establish how the Sod1 D83G homozygote and heterozygote animals deteriorate over time.

Discussion and conclusions: The identification of the Sod1 D83G ENU derived mutation provides the ALS community with a new model of disease. Early observations suggest that these animals display motor abnormalities and motor neuron cell death similar to that seen in ALS. Interestingly, the effect of the Sod1 D83G allele appears to be dosage dependent with homozygote animals displaying defects sooner than heterozygotes. This suggests that, even at physiological expression levels, the amount of mutant protein is critical for disease onset. In addition, wildtype Sod1 may be neuroprotective since homozygote Sod1 D83G mice, which do not express wildtype SOD1, develop ALS-like phenotypes sooner than heterozygotes. In conclusion, mice carrying the Sod1 D83G mutation could represent the next generation of Sod1-ALS mouse models and will be a powerful tool for analysing early stages of disease.

C26 DEVELOPMENT AND CHARACTERISATION OF A ZEBRAFISH MODEL OF MUTANT SOD1 MEDIATED MOTOR NEURON DISEASE

DA COSTA M, ALLEN C, HIGGINBOTTOM A, RAMESH T, MCDERMOTT C

University of Sheffield, Sheffield, South Yorkshire, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: zebrafish, SOD1, oxidative stress

Rationale and hypothesis: Mutations in the superoxide dismutase gene (SOD1) are known to cause familial Amyotrophic Lateral Sclerosis/Motor Neuron Disease (ALS/MND) in humans. SOD1 is a soluble cytoplasmic and mitochondrial protein, which converts superoxide radicals to oxygen and hydrogen peroxide. In order to test potential new therapies for MND, animal models of the disease are required. Zebrafish are an excellent model for human neurological diseases as they give plentiful transparent embryos, which are easy to manipulate and visualize. Furthermore, the human and the zebrafish SOD1 protein share 76% homology.

Methodology: TILLING (Targeting Induced Local Lesions IN Genomes) was carried out in collaboration with the Sanger Institute. DCF assays to detect oxidative stress were performed on NSC34 cells transfected with zfWT and zfT70I sod1. Oxidative stress assays were also carried out on 24hpf dechorionated embryos, using survival as a read out of chronic stress induced by H₂O₂, and DCF fluorescence as a read out of acute stress induced by CuSO₄. Neuromuscular junction (NMJ) staining using α-bungarotoxin (post-synaptic) and SV2 (pre-synaptic) was carried out on 11dpf larvae.

Results: Through TILLING, we identified a zebrafish carrying the T70I mutation in the sod1 gene. The zfT70I mutation occurs in the zinc-binding loop of the SOD1 protein and has been shown to significantly confer susceptibility to oxidative stress in both NSC34 cells (P < 0.001) and zebrafish embryos (P <0.001). The zfT70I sod1 mutation also causes a significant reduction in colocalisation of α-bungarotoxin and SV2 in NMJ staining in homozygous 11dpf larvae compared to WT (P <0.001) and T70I heterozygous (P <0.05) clutchmates. Preliminary data suggest that 14 dpf larvae show a motor phenotype, and further behavioural studies using 14 dpf larvae and adult homozygous T70I sod1 and WT zebrafish are currently ongoing.

Conclusions: In mammalian sod1 ALS models, sod1 mutation confers a susceptibility to oxidative stress and this mechanism has been identified as one of the key pathways in ALS pathogenesis. We have demonstrated that homozygous T70I sod1 embryos are also more susceptible oxidative stress induced by either hydrogen peroxide or copper sulphate. At 11 dpf, disruption of the neuromuscular junction is observed in T70I sod1 larvae. This is consistent with the current understanding of ALS pathogenesis in man, where the early pathology is observed in the neuromuscular junction. Our data therefore support the pathogenicity of the T70I sod1 mutation and that the zebrafish can be used as a model demonstrating key features of ALS pathogenesis. Our zebrafish model is also unique in that the TILLING missense mutation is more akin to MND patients with sod1 mutations than current transgenic murine models of MND which rely on high levels of over-expression of the hsod1 transgene and the mutant SOD1 protein.

C27 NOVEL ZEBRAFISH MODELS TO INVESTIGATE ALS DISEASE PATHOGENESIS

SAKOWSKI S, BUSTA A, PHILIP S, ROSENBERG A, DOWLING J, FELDMAN E

University of Michigan, Ann Arbor, MI, United States

E-mail address for correspondence: [email protected]

Keywords: zebrafish, neuroprotection, SOD1

Background: Amyotrophic lateral sclerosis (ALS) is a lethal multifaceted disease involving a complex interplay among many cell types and the unknown mechanisms of motor neuron (MN) degeneration in ALS make designing specific therapies challenging. In order to develop much needed therapies for this devastating disease, we have generated novel zebrafish models of ALS. Detailed observations of peripheral events occurring in MN axons and at neuromuscular junctions (NMJs) are possible in zebrafish, and they are permeable to small molecules and drugs, making them an ideal system for these studies.

Objectives: The objectives of this study are to generate novel in vivo models of ALS by expressing two forms of mutant SOD1 associated with familial ALS in zebrafish. G93A-SOD1, the most commonly studied SOD1 mutation, and A4V-SOD1, the most prevalent mutation in North America which is associated with a highly aggressive disease course, will be used to generate transient and stable transgenic zebrafish models.

Methods: Transient-transgenic zebrafish expressing G93A-SOD1 and A4V-SOD1 are generated by RNA microinjection following timed mating of wildtype AB zebrafish. Effects on early MN axonal development are examined by quantifying axon length following immunohistochemistry. Stable transgenic zebrafish expressing G93A-SOD1 and A4V-SOD1 are generated by Tol2-mediated transgenesis. Swimming ability is examined using the Noldus Larvae Activity Monitoring System to characterize symptom onset, and morphological characterization of NMJ and MN integrity is examined by immunostaining throughout the disease course in stable transgenic zebrafish.

Results: Mutant SOD1 expression results in a dose-dependant decrease in MN axon length in transient-transgenic zebrafish. Co-injection of RNA for insulin-like growth factor-I (IGF-I) upregulates neuroprotective pathways and rescues the phenotype. Stable transgenic zebrafish expressing G93A-SOD1 exhibit a progressive loss of swimming ability, indicative of symptom onset, around 15 weeks of age. Correlation of NMJ integrity and MN loss with symptom onset in stable transgenic zebrafish provide an established model for mechanistic and therapeutic discovery.

Discussion and conclusions: Rescuing the MN phenotype in zebrafish transiently expressing G93A-SOD1 and A4V-SOD1 using IGF-I validate the use of mutant SOD1 for the generation of stable transgenic ALS zebrafish models. Our successful development and characterization of transgenic ALS zebrafish enable a detailed look into the symptomatic and morphologic ALS disease course. Zebrafish can be obtained in large numbers, develop quickly, and are permeable to small molecules and drugs; therefore, transgenic ALS zebrafish provide a valuable vertebrate model to screen much-needed therapies. Knowledge obtained from these studies using transgenic ALS zebrafish will greatly advance our ability to treat ALS by identifying points for therapeutic intervention throughout the course of MN degeneration, and provides a novel in vivo system for therapeutic discovery.

Supported by the NIH (SAS: NS007222-26), the A. Alfred Taubman Medical Research Institute, and the Program for Neurology Research & Discovery.

C28 MUTANT HFE H63D PROTEIN IS ASSOCIATED WITH PROLONGED ER STRESS AND DECREASED NEURONAL VIABILITY IN ALS

LIU Y¹, LEE S¹, NEELY E¹, NANDAR W¹, SIMMONS Z², CONNOR J¹

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

E-mail address for correspondence: [email protected]

Keywords: HFE, ER stress, neuronal viability

Background: Endoplasmic reticulum (ER) stress appears to be present in patients with amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Accumulation of mis-folded proteins in the ER provokes the early ER stress response – unfolded protein response (UPR). UPR is a short-term and protective homeostatic mechanism. It initiates both apoptotic and adaptive pathways. It is not clear whether ER stress is largely neuroprotective or whether it directly contributes to the disease process in ALS. The HFE gene encodes for an iron regulating protein, and the H63D variant of this gene is found in increased frequency in patients with ALS and may increase the risk of ALS by four-fold.

Objectives: To determine the ER stress activation pattern associated with HFE H63D mutant protein and its contribution to neuronal viability, as the first step in understanding the mechanisms by which HFE H63D increases the risk for ALS.

Methods: We developed inducible neuronal cell lines expressing HFE wild type (WT) or H63D protein. This system allows us to faithfully capture the transit responses and follow the shift of those responses after the induction. We also expand our in vitro findings to a knock-in mouse model carrying the HFE H67D mutation, the mouse equivalent of human H63D.

Results: Our data demonstrated that the presence of HFE H63D mutant protein initially evoked UPR, as revealed by the elevated levels of the major UPR sensors. This response was followed by a persistent ER stress, as the signals of UPR sensors attenuated. At this time point, both Caspase-3 cleavage and activity were up-regulated. This ER stress pattern associated with HFE H63D was also seen in a HFE H67D knock-in mouse model, in which UPR was selectively activated in the lumbar spinal cord at 6-months then declined at 12-months, in association with increased Capase-3 cleavage. Based on the MTS assay, the number of cells in proliferation was decreased in the HFE H63D expressing cells, but no increased cytolysis was detected by measuring LDH activity in the medium. Thus, the data indicated that HFE H63D expressing cells had decreased cell proliferation compared to WT, but did not undergo extensive cell death. Additionally, in spite of increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status.

Discussion and conclusions: Our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and reduced cell proliferation, reflecting decreased neuronal viability. We speculate that in the case of HFE H63D, sustained ER stress attenuates the activation of the cytoprotective UPR and thus limits the ability to remove mis-folded proteins, creating an environment which is more conducive to the development of ALS.

C29 DYSREGULATION OF ER STRESS SIGNALING BY ALS-RELATED P56S VAPB MUTANT

KANEKURA K, SUZUKI H, LU S, KANEKURA M, MATSUOKA M, AISO S, URANO F

University of Massachusetts Medical School, Worcester, MA, United States

E-mail address for correspondence: [email protected]

Keywords: VAPB, ER stress, unfolded protein response

Background: P56S mutation in the VAPB gene causes autosomal-dominant ALS and spinal muscular atrophy. VAPB is a type II transmembrane protein localized to endoplasmic reticulum (ER) whose function remains elusive. We previously reported that VAPB is involved in the unfolded protein response (UPR), an adaptive response to ER stress, and ALS-related P56S VAPB acts as a dominant-negative mutant, resulting in motor neuron vulnerability to ER stress.

Objectives: 1) To investigate the precise mechanisms by which VAPB regulates the UPR. 2) To investigate how P56S VAPB mutant causes motor neuron vulnerability to ER stress.

Methods: To investigate how VAPB regulates the UPR, we developed a lentivirus-based inducible system of wild-type and P56S mutant VAPB in NSC34 cells. We monitored activation levels of the UPR by measuring expression of ER stress markers and activation of the UPR luciferase reporter in these cells. To identify genes and pathways required for VAPB to regulate the UPR, we established a genome-wide shRNA screening system.

Results: We found that VAPB plays a role in activating the IRE1-XBP-1 pathway and suppressing the PERK-ATF4 pathway of the UPR. In contrast, P56S VAPB mutant strongly activates the PERK-ATF4 pathway and causes upregulation of CHOP, a pro-apoptotic component of the UPR. Furthermore, we found that FFAT-motif containing oxysterol binding family proteins are required for VAPB to activate the IRE1-XBP-1 pathway. We are currently conducting a genome-wide shRNA screen to identify genes required for VAPB to regulate the UPR.

Discussion and conclusions: VAPB acts as an activator for the IRE1-XBP-1 pathway and a suppressor for the PERK-ATF4 pathway of the UPR. The ALS-related P56S VAPB mutant acts as a dominant-negative mutant, leading to the activation of the pro-apoptotic PERK-ATF4-CHOP pathway. Our results indicate that dysregulation of the UPR caused by P56S mutant VAPB plays a role in the pathogenesis of ALS.

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SESSION 4B SURROGATE MARKERS

C30 VALIDATION OF A NEW DEVICE TO MEASURE DISEASE PROGRESSION IN ALS

ANDRES P¹, SKERRY L¹, CUDKOWICZ M^1,2

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

E-mail address for correspondence: [email protected]

Keywords: outcomes measures, quantitative strength measures, disease progression

Background: Currently used outcome measures require large sample sizes and may be unable to detect small, but clinically important, treatment effects. Maximal voluntary isometric (MVIC) strength testing accurately reflects ALS disease progression. However, MVIC, using a strain gauge, is inconvenient and expensive. Hand held dynamometry is convenient but limited by the evaluator's strength.

To address the need for a convenient, accurate method to test MVIC, we developed a device called: Accurate Test of Limb Isometric Strength (ATLIS). This portable device consists of an adjustable tilting chair in a frame. A fixed wireless load cell measures MVIC of 12 limb muscle groups, tested in standard, gravity-eliminated positions. The testing protocol takes 15 minutes to complete.

Objectives: 1) To determine the test-retest and inter-rater reliability and the criterion-based validity of the ATLIS protocol; 2) To determine subject acceptance of this new testing protocol.

Methods: To determine test-retest reliability of the ATLIS protocol, 20 healthy adults and 10 subjects with ALS were tested twice by the same evaluator. Inter-rater reliability was determined by testing 20 healthy adults and 10 subjects with ALS twice: once by each of two evaluators. Concordance of each item was determined by calculating the mean absolute percent difference between the paired tests. To determine criterion-related validity, 20 healthy adults were tested using the ATLIS protocol and also using a well-validated protocol: Tufts Quantitative Neuromuscular Exam (TQNE).

Subjects were also asked to complete a five item survey using a Likert scale to assess their acceptance of the ATLIS protocol.

Results: The sample of healthy adults consisted of a 2/3 ratio of females to males with a mean age of 36 years. The sample of subjects with ALS was roughly equally distributed between males and females with an average age of 57 years. Mean absolute differences between tests ranged between 6 and 11% in all muscle groups. Comparisons between ATLIS and TQNE values of the 12 muscles groups demonstrated acceptable correlations (0.44 – 0.92), though TQNE values were higher than ATLIS values. Over 95% of ALS subjects reported agreement or strong agreement regarding the attributes of ATLIS.

Discussion and conclusions: Reliability studies indicate that reliability of ATLIS is equal to or better than the strain gauge or HHD testing. Test-retest reliability was only slightly better than inter-rater reliability indicating that perhaps subject performance accounted for the majority of variance rather than differences between the evaluators. Though the correlations of ATLIS and TQNE scores were acceptable, the higher scores seen with TQNE may be due to differences in testing positions. The survey of ATLIS attributes indicated overwhelming acceptance by subjects with ALS.

ATLIS shows great promise to become an efficient and precise outcomes measure for future clinical trials in ALS.

C31 MUSCLE ULTRASONOGRAPHY AS A DIAGNOSTIC TOOL FOR ALS: A DIAGNOSTIC STUDY ACCORDING TO THE STARD CRITERIA

ARTS I¹, PILLEN S¹, ZWARTS M¹, BOEKESTEIN W^1,2, KLEINE B¹, OVEREEM S¹, SCHELHAAS HJ¹

¹Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, ²Medical Spectrum Twente, Enschede, Netherlands

E-mail address for correspondence: [email protected]

Keywords: ultrasonography, diagnosis, STARD

Background: Ultrasonography can detect signs of lower motor neuron (LMN) loss, by visualizing fasciculations, diminished muscle thickness and increased echo intensity. Here, we evaluated the diagnostic potential of ultrasonography in ALS, in a prospective study according to the STARD criteria.

Objectives: To study the diagnostic value of ultrasonography in differentiating between ALS/SMA and mimics. Furthermore, to assess the ability of ultrasonography to detect LMN signs in clinical unaffected regions and thereby increasing the diagnostic certainty of ALS.

Methods: Fifty-nine patients, recruited from our MND clinic, received a diagnostic work up according to the standards of the revisited El Escorial criteria. In addition, bilateral transverse ultrasonography scans were made of the sternocleidomastoid, biceps, forearm flexors, rectus abdominis, quadriceps and tibialis anterior muscles. Muscle thickness and echo intensity were quantitatively measured. Each muscle was screened for fasciculations during 10 seconds. Ultrasonography was considered indicative for ALS/SMA when echo intensity was ≥ 1.5 SD above normal in at least 2 muscles and fasciculations were present in ≥ 4 muscles. In patients with ALS, we evaluated each region separately in order to quantify the number of affected regions. Requirements for ultrasonographic regional LMN involvement were: fasciculations in ≥ 1 one muscle for the bulbar and thoracic region, and fasciculations ≥ 2 muscles for the cervical and lumbosacral regions.

Results: Ultrasonography was able to differentiate between ALS/SMA and mimics with 96% sensitivity and 84% specificity. In the 24 ALS patients, ultrasonography detected 13 regions with LMN involvement that were negative using either clinical examination or needle EMG. In 10 of the 12 patients with clinical possible ALS, ultrasonography detected LMN involvement in ≥ 3 regions. The remaining two patients did not fulfil the EMG criteria for probable ALS laboratory-supported.

Discussion: Muscle ultrasound is able to differentiate between ALS/SMA and mimics with high sensitivity and specificity. In addition, non-invasive ultrasonography is superior as compared to needle EMG to detect additional affected regions in patients diagnosed with ALS. Especially the sensitivity to detect fasciculations is an important asset of ultrasonography.

Conclusion: Our findings warrant the incorporation of ultrasonography in the diagnostic work up of ALS.

C32 A NOVEL CMAP SCAN-BASED PROGRESSION SCORE FOR MOTOR NEURON DISEASE

MAATHUIS E, DRENTHEN J, VAN DOORN P, VISSER G, BLOK J

Erasmus Medical Center, Rotterdam, Netherlands

E-mail address for correspondence: [email protected]

Keywords: CMAP scan, progression, neurophysiology

Background: Motor neuron disease (MND) is marked by ongoing motor unit (MU) loss, reinnervation, and ultimately muscle atrophy. The effects of these three processes on existing markers of disease progression partly counteract each other (reinnervation masks MU loss). Hence, electrophysiological features of disease progression can only be monitored accurately if all three processes are taken into account. In the present study, we propose the first neurophysiological marker that combines quantitative information on the number of surviving MUs, reinnervation, and the extent of muscle atrophy: the CMAP scan progression score (CSPS). Each CSPS is derived from a CMAP scan, a novel electrophysiological assessment that can be performed easily and noninvasively in less than 10 minutes. The CMAP scan is basically a high-detail stimulus-response curve (normally smooth and sigmoid in shape), in which the results of reinnervation processes appear as so-called steps (‘gaps’ in the curve). Changes in the number and size of steps reflect the underlying process of loss of functioning MUs. The muscle fiber mass (reduced in atrophy) is expressed through the maximum CMAP amplitude.

Objective: To determine the value of the CSPS as a quantitative electrophysiological marker of disease progression in MND.

Methods: CMAP scans were recorded five times with a 2-3 week interval from the thenar muscles of 10 MND patients. After these 5 recordings at regular intervals, 8 patients had 1 to 8 additional recordings over a total follow up period that extended up to 85 weeks (median 34 weeks). Motor unit number estimation (MUNE) was performed in the same sessions for comparison.

The CSPS is the sum of 3 subscores: the reinnervation score (decreases with increase in step size, 1-5 points), the atrophy score (decreases with decrease in CMAP amplitude, 1-5 points) and the motor unit number score (based on average step size; decreases with increasing step size, 1-4 points). The total CSPS ranges from 14 (no change) to 3 (severe deterioration). To compare variables between days, paired nonparametric tests were used.

Results: Median CSPS on day 1 was 11.5 (range 9-14). Over the first 5 recordings, 9/10 (90%) of the patients showed a decrease in CSPS; median CSPS at the fifth recording was 11 (range 6-14) (P =0.03). MUNE and the maximum CMAP amplitude did not differ significantly between the first and fifth measurements. During long-term follow up, CMAP amplitude, MUNE, and CSPS decreased in all patients.

Discussion and conclusions: The CSPS is the first electrophysiological MND marker that combines the effects of MU loss, reinnervation and atrophy. It can be obtained with a simple, brief, and non-invasive test. Furthermore, the CSPS is objective, informative, easy to interpret and appears to represent disease progression more accurately than MUNE or CMAP amplitude.

C33 FURTHER EVIDENCE FOR MULTISYSTEM INVOLVEMENT IN ALS: MULTIPARAMETRIC MRI OF SENSORY STIMULATION

LULÉ D^1,2, DIEKMANN V¹, MÜLLER H-P¹, KASSUBEK J¹, BIRBAUMER N^2,3, LUDOLPH AC¹

¹Department of Neurology, University of Ulm, Ulm, Germany, ²Institute of Medical Psychology and Behavioral Neurobiology, University of Tübingen, Tübingen, Germany, ³Ospedale San Camillo, IRCCS, Instituto di Ricovero e Curo a Carattere Scientifico Venezia, Venice, Italy

E-mail address for correspondence: [email protected]

Keywords: sensory system, fMRI, DTI

Background: In recent years, evidence for cortical changes in amyotrophic lateral sclerosis (ALS) beyond the motor cortex has accumulated. Modern imaging techniques bear the potential to further investigate neurodegeneration and reorganisation in the cortex of ALS patients.

Objectives: Structural and functional imaging techniques were combined to investigate sensory system function in ALS.

Methods: Functional magnetic resonance imaging (fMRI) was used to investigate cortical activity during visual, auditory, and somato-sensory stimulation in fourteen ALS patients and eighteen control subjects. Changes in amplitude, latency and duration of the BOLD response were modelled. Furthermore, diffusion tensor imaging was used to investigate changes in white matter networks.

Results: During visual stimulation, fMRI demonstrated a decreased response in secondary visual areas in ALS possibly related to functional deficits of sensory nerve fibres. The stronger the decrease in physical functioning (measured with ALS-FRS) the higher was the brain activity in associative cortices. This might represent a compensatory process. Additionally, reduced functioning became evident for fibres projecting to extra-striate visual cortex.

For auditory stimulation, a delayed response in secondary auditory areas probably linked to prolonged conductance or synaptic transmission times and an altered cortical pattern in areas involved in target processing/detection became evident in ALS patients. Structural white matter differences in the primary and secondary auditory cortices were observed in ALS patients compared to controls. For somato-sensory stimulation a prolonged/reduced response in sensory integration areas of the parietal lobe was observed perhaps linked to the reduced visceral inflow due to immobility.

Discussions: Multiparametric MRI suggests a progressive functional deficit in secondary/higher order sensory processing areas in ALS. Accordingly, data provide evidence for a primary pathology of the sensory system in ALS highlighting the multisystematic character of the disease. Evidence for compensatory processes in multimodal associative cortices was found. This might be an expression of a general capacity for cortical plasticity in severe neurological disorders.

Conclusion: The present study provides evidence for structural and functional changes and reorganisation in the sensory cortex of ALS patients. Our data adds essential keynotes to the understanding of the multisystematic character of the disease ALS.

C34 BASELINE CORTICOSPINAL TRACT MRI REFLECTS CHANGE IN DISABILITY AT SIX MONTH FOLLOW-UP

ABRAHAM I, MENKE R, FILIPPINI N, TALBOT K, TURNER M

Oxford University, Oxford, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: diffusion tensor imaging, prognosis, biomarker

Background: The diagnostic pathway, prognostication and therapeutic trial monitoring could all be improved by the establishment of robust biomarkers in ALS. Diffusion tensor imaging (DTI) is a particularly promising application of MRI, with the ability to detect white matter (WM) damage. DTI has demonstrated consistent cerebral WM involvement in ALS within (though not limited to) the corticospinal tract (CST). There would be clear value in a non-invasive MRI marker of WM damage that could predict future disability, which might then be applicable to therapeutic trials.

Objectives: To identify whether regional baseline measurement of CST WM integrity, as assessed by DTI, reflects disease progression at six months.

Methods: Twelve ALS patients at various stages of disease underwent cerebral MRI at high-field (3T) with a six-month interval between the two studies. Fractional anisotropy (FA) maps were generated using tools from FMRIB Software Library (FSL). Tract-based spatial statistics (TBSS) were performed within the left and right CST in MNI space to assess voxel-wise correlations of DTI indices with the difference between follow-up and baseline ALSFRS-R scores. Results were considered significant at P <0.05 (corrected for multiple comparisons, FWE) using threshold-free cluster enhancement (TFCE).

Results: A highly significant (P = 0.002) negative correlation was noted between baseline FA values and change in ALSFRS-R score after six months, for a region spanning the internal capsules of the CST bilaterally.

Discussion: CST FA reduction is accepted to reflect the degree of WM involvement in ALS, and its baseline correlation with change in disability after six months in a heterogeneous group of patients suggests it may have prognostic potential. This must be explored further in larger studies with longer follow up and multivariate survival analysis.

Conclusions: DTI has clear potential to provide biomarkers that may be sensitive to both prognosis and therapeutic monitoring in ALS. Consideration should be given to cerebral DTI as a secondary outcome measure in future therapeutic trials to fully realise this potential.

C35 DIAGNOSTIC BIOMARKERS FOR ALS

GANESALINGAM J², SHAW C², SHAW G³, LACOMIS D¹, BOWSER R¹

¹University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, ²MRC Centre for Neurodegeneration Research, Kings College London, United Kingdom, ³University of Florida, Gainesville, FL, United States

E-mail address for correspondence: [email protected]

Keywords: biomarkers, mass spectrometry, cerebrospinal fluid

Background: Diagnostic biomarkers for ALS are desired to shorten the time between symptom onset and patient diagnosis. Diagnostic biomarkers may also be used to stratify the ALS patient population and potentially identify sub-populations that may best respond to specific treatments. Prior studies have identified a number of candidate biomarkers in the cerebrospinal fluid (CSF). We have further explored a specific set of proteins as diagnostic biomarkers for ALS within the CSF and plasma.

Objectives: To evaluate the diagnostic utility of neurofilament and complement c3 proteins as biomarkers for ALS.

Methods: CSF was obtained from 163 subjects and used for ELISA to measure levels of phosphorylated neurofilament heavy chain (p-NFH) and complement c3 proteins. The sample set was 71 ALS patients (median disease duration of 16 months from symptom onset), 40 healthy controls, and 52 disease controls that included 14 disease mimics. A training set of 106 and a test set of 57 CSF samples were generated from these samples. ROC curves of the training set were used to generate cut-off values for each protein that were subsequently used to determine diagnostic utility in the test set.

Results: The median level of pNFH was 1.77 ng/ml, 0.2 ng/ml and 0.165 ng/ml for ALS, disease control (DC) and healthy control (HC) groups, respectively. A p-NFH cut-off level of 0.635 ng/ml generated a sensitivity of 84% sensitivity and 93% specificity for ALS in the training set. Complement c3 was also significantly increased in ALS versus HC and DC versus HC groups, but not ALS versus DC. This was likely due to inflammation within the various members of the DC group. For complement c3, a threshold value of 3.62 µg/ml produced a sensitivity of 62% and specificity of 56% for ALS. However a ratio of p-NFH to complement c3 was found to provide the best overall diagnostic accuracy. A p-NFH/complement c3 ratio of greater than 0.000125 provided 91% sensitivity and 89% specificity for the training set. When this p-NFH to complement c3 ratio was applied to the test set, we obtained a sensitivity of 96% and specificity of 90%.

Conclusions: Levels of p-NFH and complement c3 are elevated in the CSF of ALS and other neurologic disorders. However we identified a specific ratio of the two proteins that provided over 90% diagnostic accuracy in CSF from 163 subjects. We also correlated CSF levels of p-NFH to plasma levels within the same subjects. Our results suggest that ELISA measurements for these two proteins within the CSF may provide a diagnostic predictor of ALS. Further studies are required to validate these results.

SESSION 6A TRANSLATIONAL STRATEGIES

C36 PLASTIC VIRUSES: ENGINEERING NANOPARTICLES FOR TARGETING THE CENTRAL NERVOUS SYSTEM

BATTAGLIA G

The Krebs Institute, Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: bionanotechnology, synthetic technology, delivery system

Provided the right hydrophilic/hydrophobic balance can be achieved, amphiphilic block copolymers are able to assemble in water into membranes. These membranes can enclose forming spheres with an aqueous core. Such structures, known as polymer vesicles or polymersomes (from the Greek -some = body of), have sizes that vary from tens to thousands of nanometers. The wholly synthetic nature of block copolymers affords control over parameters such as the molar mass and composition, which ultimately determine the structure and properties of the species in solution. By varying the copolymer molecular mass it is possible to adjust the mechanical properties and permeability of the polymersomes, while the synthetic nature of copolymers allows the design of interfaces containing various biochemically-active functional groups (1). In particular, non-fouling and non-antigenic polymers have been combined with hydrophobic polymers in the design of biocompatible nano-carriers that are expected to exhibit very long circulation times. Stimulus-responsive block copolymers have also been used to exploit the possibility to trigger the disassembly of polymersomes in response to specific external stimuli such as pH, oxidative species, and enzyme degradation (1). Such bio-inspired bottom-up supramolecular design principles offer outstanding advantages in engineering structures at a molecular level, using the same long-studied principles of biological molecules. Thanks to their unique properties, polymersomes have already been reported and studied as delivery systems for both drugs, genes, and image contrast agents as well as nanometer-sized reactors (1).

In the recent years we have studied and optimized the molecular parameters that control polymersomes assembly as well as indentified several formulation of their ad hoc disassembly (2–5). We are able to control the shape and the surface morphology of the polymersomes at the nanoscale (6). We are now using these molecular engineering tools to design nanoscopic vectors for the effective and targeted delivery of therapeutic and image contrast agents. We have particularly demonstrated that by formulating pH sensitive polymersomes we can achieve high cytosolic delivery of small molecules as well as large macromolecules like nucleic acids and proteins (7–9).

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C37 APPLYING IPSC TECHNOLOGY FOR MOTOR NEURON DISEASE: A PATIENT FIRST DRUG DISCOVERY PLATFORM

STRULOVICI B, JAVAHERIAN A

iPierian, San Francisco, CA, United States

E-mail address for correspondence: [email protected]

Keywords: iPSC, neurons, pharmaceuticals

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with complex genetic and environmental contributors. The current standard drug discovery platform for human disease typically relies on some knowledge about the molecular mechanism of the disease and a target in mind. However, for complex diseases such as ALS where the molecular mechanism underlying disease pathology is not well understood, target-based biochemical assays are, at best, speculative or not feasible. Due to the lack of predictive in vivo and in vitro models that represent sporadic as well as familial ALS, traditional drug discovery strategies using surrogate cell lines and animal models have not been successful for ALS. We have challenged the traditional drug discovery approaches to this disease by putting the patient at the forefront of drug discovery. Induced pluripotent stem cell (iPSC) technology allow us, for the first time in the history of drug discovery, to study neurologic disorders in patient-derived neural cells. We are using iPS cell lines from both familial and sporadic ALS patient fibroblasts differentiated into motor neurons and glia for identification of ALS disease phenotypes. We have industrialized the process of reprogramming patient cells to produce large numbers of neurons and glia, the cell types implicated in ALS, in a highly robust and reproducible manner. We are applying disease phenotypes discovered in patient motor neurons and glia in high throughput assays to screen libraries of compounds to identify therapeutic agents for ALS. We are executing this project in a scale large enough to enable testing of compounds across a large number of patient lines that represent different forms of ALS in a novel and innovative ‘in vitro clinical trial’ approach. The discovery of a novel therapeutic compound for ALS could have a major impact on over 25,000 patients in the United States. More importantly, if successful, our new drug discovery platform could be applied to a variety of complex diseases where animal and simple cell models are not adequate to address the complexities of the human population.

SESSION 6B EPIDEMIOLOGY

C38 CAN WE IDENTIFY RISK FACTORS FOR ALS/MND?

BEGHI E

Istituto Mario Negri, Milano, Italy

E-mail address for correspondence: [email protected]

Keywords: epidemiology, risk factors

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder with worldwide distribution. The geographical distribution of ALS is fairly homogeneous except for the Western Pacific variant of the disease (the ALS/parkinsonism dementia complex) formerly thought to occur at a 100^th fold rate. This significant increase in the risk of ALS in the local populations has been attributed, among others, to the cycad hypothesis, according to which ALS may be caused by a neurotoxic aminoacid, beta-methylaminoalanine (BMAA) present in a palm seed (the Cycad micronesica). Recent discoveries found that BMAA is produced by symbiotic cyanobacteria within the cycad roots and that animals forage on the palm's seeds, bringing the toxin into the food chain. This brings the attention back to the role of environmental factors as possible causes of ALS. In the western countries several occupational, alimentary and other environmental factors have been investigated with disappointing results. In fact, the assessment of the environmental risk factors in ALS is based on a high number of observations of uncertain etiological significance. These include conjugal ALS, correlation with antecedent poliomyelitis or concurrent neoplasms, exposure to (heavy) metals, to solvents or to electrical or electromagnetic fields, mechanical trauma, heavy physical activity, and living in rural areas or using chemical substances in agriculture. The inconsistent findings can be explained in light of the poor methodology of most published reports. The inclusion of non-representative study samples, the lack of standard definitions for exposures, and the small sample size are among the commonest methodological defects. The use of prevalent rather than incident cases is a source of selection bias. Differing definitions of putative risk factors prevent meaningful comparisons across studies to verify the consistency of the results. As the disease is rare and also several exposures are uncommon in the general population, most case-control studies have been insufficiently powered to detect significant differences in the rate of exposure among ALS patients and matched controls. In this regard, only large population-based case-control studies done in incident ALS patients and using standard definitions for exposures are proper tools to investigate risk factors for ALS.

C39 EPIDEMIOLOGY OF ALS IN THE NETHERLANDS, 2006–2009

HUISMAN MHB¹, DE JONG SW¹, VAN DOORMAAL PTC¹, WEINREICH S², SCHELHAAS HJ³, VAN DER KOOI AJ⁴, DE VISSER M⁴, VELDINK JH¹, VAN DEN BERG LH¹

¹Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, Netherlands, ²Dutch Neuromuscular Patient Association, Baarn, Netherlands, ³Department of Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, ⁴Department of Neurology, Amsterdam Medical Center, Amsterdam, Netherlands

E-mail address for correspondence: [email protected]

Keywords: epidemiology, population-based, incidence

Background: Prior population-based registries of ALS are limited by small population size and no adjustment for differential coverage of patients in different age and sex classes by making an estimate of the number of unobserved patients. Due to these limitations it is not clear whether the reported incidence rate decline in the very elderly and the decreased male to female ratio in postmenopausal age classes are real.

Shared exposure to an environmental risk factor might result in geographical clustering of ALS. Identification of these clusters could be a starting point to discover new exogenous risk factors.

Further clues for environmental and genetic risk factors could be retrieved by investigating the family history of ALS patients, since relatives share genetic and environmental risk factors.

Objectives: 1) To provide reliable data on ALS epidemiology; 2) To identify geographical clusters of ALS; 3) To determine whether the occurrence of ALS, Parkinson's Diseases (PD), dementia and vascular diseases in relatives of patients with ALS is different from the occurrence in relatives of controls.

Methods: A population based study has been performed in the Netherlands between January 2006 and December 2009 (mean population 16,426,273; area 41,528 km²). Patients were ascertained from five sources. Diagnosis was made according to the El Escorial criteria. Capture-recapture analysis in each 5 year age group for both genders apart was performed to correct incidence and prevalence rates for the estimated number of missing patients. Spatial scanning software was applied to test for geographical clustering by using residential zip code at diagnosis and 5 years before diagnosis. 635 patients and 1616 age- and sex-matched controls filled in questionnaires concerning the diagnosis of PD, dementia, myocardial infarction (MI) or stroke in first-, second- and third-degree relatives.

Results: The incidence rate of ALS was 2.77 per 100,000 person-years (95% CI 2.76-2.79). Prevalence rate at 31 December 2008 was 10.32 per 100,000 people (95% CI 10.27-10.37). Both incidence and prevalence peaked in the 70-74 years age group. Although the male: female incidence rate ratio in the premenopausal age group was higher than in the postmenopausal age group, 1.89 and 1.50 respectively, this difference was not statistically significant.

Two geographical clusters of ALS were identified; one with a relative risk of 3.1 (radius 15 kilometers, P-value 0.015) and one with a relative risk of 1.8 (radius 27 kilometers, P-value 0.034).

Relatives of patients have an elevated risk of ALS compared to controls (λ 2.22; 95% CI 2.16-2.26). Dementia (λ 1.14; 95% CI 1.09-1.18) and PD (λ 1.13; 95% CI 1.03-1.23) are more common among relatives of ALS patients compared with relatives of controls. The occurrence of stroke (λ 0.93; 95% CI 0.88-0.97) and MI (λ 0.84; 95% CI 0.80-0.88) in relatives of ALS patients is lowered.

Discussion and conclusions: We report the epidemiology of ALS in the largest individual population based register until now. Application of the capture recapture method confirmed that incidence rates of ALS decrease after age 74 years. Further, it showed that the previously found postmenopausal decrease in the male to female ratio is possibly a result of bias. Two significant clusters were detected which need further investigation and may provide clues to the etiology of MND. The results on the family history of ALS support an association between neurodegenerative diseases and ALS, indicating that these diseases share genetic and environmental risk factors. The lowered risk of vascular diseases in relatives of ALS patients may suggest that a beneficial vascular risk profile increases the susceptibility for ALS.

C40 ALS PHENOTYPIC HETEROGENEITY: EVIDENCE FROM A POPULATION-BASED STUDY

CHIO A¹, CALVO A¹, MAZZINI L², MORA G³, BOTTACCHI E⁴, BALMA M¹, GALLO S¹, CANOSA A¹, MUTANI R¹

¹Department of Neuroscience, University of Torino, Torino, Italy, ²Department of Neurology, University of Eastern Piedmont, Novara, Italy, ³Department of Neurorehabilitation, IRCSS Salvatore Maugeri, Milano, Italy, ⁴Regional Hospital, Aosta, Italy

E-mail address for correspondence: [email protected]

Keywords: phenotypes, incidence, outcome

Aim: The epidemiological and clinical characteristics of ALS phenotypes in the patients included in an Italian prospective epidemiological register have been assessed.

Background: The characteristics of ALS clinical phenotypes (bulbar, classical (Charcot), pyramidal, flail leg, flail arm and respiratory) remain poorly understood. Moreover, no studies have been performed on these phenotypes in an epidemiological setting.

Methods: The patients prospectively diagnosed and followed-up between 1995 and 2004 in Piemonte and Valle d'Aosta have been classified according to their clinical phenotype. The effect of the phenotypes on ALS prognosis has been also analyzed.

Results: Of the 1260 incident patients, 1241 (98.5%) had complete phenotypic data. The most common phenotype was bulbar ALS (mean incidence rate 1.1/100,000/year, with no difference between genders) and the second was classical ALS (incidence rate, 1.2 men and 0.9 women; men to women rate ratio 1.7:1). Flail leg syndrome and pyramidal phenotype had a similar frequency in both genders (incidence rate, 0.4 and 0.3, respectively). Flail arm syndrome and respiratory phenotype were largely more represented among male (men to women rate ratio 4.0:1 and 9.0:1, respectively). The oldest age at onset was found in the bulbar phenotype (68.8 years) and the lowest in the pyramidal phenotype (58.3 years). Frontotemporal dementia was more frequent in bulbar phenotype (9.0%) and very rare in the flail arm syndrome (1.4%). Significantly different outcomes were found: pyramidal and flail arm phenotypes had the better prognosis (median survival, 6.3 and 4.0 years, respectively), while bulbar and respiratory phenotypes had the worst prognosis (2.0 and 1.4 years, respectively).

Conclusions: ALS phenotypes are largely related to a complex interplay between gender and age. The reasons for the strong influence of these factors on ALS biology remains largely unknown. In turn, ALS phenotypes are the amongst the main factors determining the clinical outcome.

SESSION 8A GENETICS AND GENOMICS

C41 CLINICAL, PATHOLOGICAL AND GENETIC FEATURES OF AMYOTROPHIC LATERAL SCLEROSIS ASSOCIATED WITH OPTINEURIN MUTATIONS

KAMADA M¹, IZUMI Y¹, ITO H², MARUYAMA H³, KUSAKA H², MORINO H³, KAWAKAMI H³, KAJI R¹

¹Tokushima University, Tokushima, Japan, ²Kansai Medical University, Osaka, Japan, ³Hiroshima University, Hiroshima, Japan

E-mail address for correspondence: [email protected]

Keywords: Optineurin, ALS, NFkB

We have recently reported mutation of the gene of Optineurin (OPTN), an inhibitor of NFkB, in ALS. Although the clinical features fulfilled the El Escorial criteria (laboratory-supported probable), 2 families (heterozygous E478 mutation) out of 5 exhibited slow clinical progression (∼ 8 years without ventilation) with clinically symmetric onset. Another family with homozygous exon 5 deletion showed early and asymmetric onset and more rapid progression (intubated 4 years after onset). Two families with homozgygous Q398X mutation showed intermediate course between these. The phenotypic difference might be correlated with the site of mutation within OPTN. Because abnormal accumulation of OPTN in motor neurons was found not only in those with OPTN mutation but also SOD1 positive and sporadic ALS patients, NFkB pathway may have a pivotal role in ALS. The phenotypic difference among OPTN positive patients should give insights into the specificity and efficacy of NFkB inhibitors for the development of possible therapeutic agents for ALS in general.

C42 A LARGE CNV ASSOCIATION STUDY IN SPORADIC ALS

BLAUW H¹, AL-CHALABI A³, ANDERSEN P^4,5, VAN VUGHT P¹, VAN ES M¹, WAIBEL S⁵, MEYER T⁸, HARDIMAN O⁹, TOMIK B¹¹, SLOWIK A¹¹, BROWN R¹⁰, LANDERS JE¹⁰, ROBBERECHT W¹⁴, LUDOLPH AC⁵, OPHOFF R^2,17, VELDINK JH¹, VAN DEN BERG LH¹

¹Department of Neurology, UMC Utrecht, Utrecht, Netherlands, ²Department of Medical Genetics and Rudolf Magnus Institute, Utrecht, Netherlands, ³Department of Clinical Neuroscience, King's College, London, United Kingdom, ⁴Department of Clinical Neuroscience, Umeå University, Umeå, Sweden, ⁵Department of Neurology, University of Ulm, Ulm, Germany, ⁶Department of Internal Medicine, Erasmus University Medical Centre Rotterdam, Rotterdam, Netherlands, ⁷Department of Epidemiology, Biostatistics and HTA, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, ⁸Department of Neurology, Charité University Hospital, Humboldt-University, Berlin, Germany, ⁹Department of Neurology, Beaumont Hospital, Dublin, Ireland, ¹⁰Department of Neurology, Massachusetts General Hospital, Charlestown, United States, ¹¹Department of Neurology, Jagiellonian University, Krakow, Poland, ¹²Experimental Neurology, University of Leuven, Leuven, Belgium, ¹³Vesalius Research Centre, Flanders Institute for Biotechnology (VIB), Leuven, Belgium, ¹⁴Department of Neurology, University Hospital Leuven, University of Leuven, Leuven, Belgium, ¹⁵Division of Molecular and Clinical Neurobiology, Department of Psychiatry, Ludwig-Maximilians-University, Munich, Germany, ¹⁶Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, and German Center for Neurodegenerative Diseases, Tuebingen, Germany, ¹⁷UCLA Center for Neurobehavioral Genetics, Los Angeles, United States

E-mail address for correspondence: [email protected]

Keywords: genome-wide association study, CNV, rare variant

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease selectively affecting motor neurons in brain and spinal cord. Recent genome-wide association studies have identified several common variants (SNPs) that increase disease susceptibility. In contrast, rare copy-number variants (CNVs), which have been associated with several neuropsychiatric traits, have not been studied for ALS in well-powered study populations.

Objectives: To identify rare CNVs that increase disease susceptibilty in sporadic ALS.

Methods: We conducted a two-staged association study focused on rare CNVs, including over 19,000 individuals. We analysed genome-wide Illumina data and DNA samples using stringent quality control criteria. We used PennCNV software for CNV detection and applied extra quality control filters to reduce the number of false-positive CNV calls. We tested each gene for association by comparing the number of CNVs affecting the gene in cases versus controls. Loci (genes) with a nominal Fisher Exact P value < 0.01 and a frequency of <1% in controls were selected for follow-up after careful validation with TaqMan qPCR.

Results: In the discovery cohort of 1,875 ALS patients and 8,731 controls we identified two loci that met our criteria for follow-up: the DPP6 locus (CNVs in 10 of 1,875 ALS patients versus 13 of 8,731 controls, OR=3.59, P=6.6 3 102³), and the 15q11.2 locus, containing NIPA1, the gene causing hereditary spastic paraparesis (HSP) type 6 (CNVs in 8 of 1,875 ALS patients versus 3 of 8,731 controls, OR=12.46, P=9.3310²⁵). Validation experiments confirmed the presence of all (n=25) tested CNVs.

In our replication population of 2,559 ALS patients and 5,887 controls, the genes of interest again contained more CNVs in patients compared to controls, but did not meet our criteria for independent replication: DPP6 locus: 10 CNVs in 2,559 ALS patients versus 12 in 5,887 controls, OR=1.92, P=0.097, pooled results: OR=2.64, P=1.4310²³; NIPA1: 7 CNVs in 2,559 ALS patients versus 5 in 5,887 controls, OR=3.23, P=0.041, pooled results: OR=6.20, P=2.2310²⁵).

Discussion: We identified two genes that show suggestive evidence for association with ALS disease status. DPP6 has been suggested previously as a candidate gene for ALS, while NIPA1 has not been associated with ALS before. Mutations in NIPA1 cause HSP type 6, a disease characterised by the selective death of (central) motor neurons, suggesting an important role in motor neuron biology. Statistical power remains a problem in studies aimed at rare variants.

Conclusions: CNV analyses provide suggestive evidence for DPP6 and NIPA1 as candidate susceptibility genes for ALS.

C43 RARE MUTATIONS IN ANGIOGENIN CONFER LARGE RISK FOR SPORADIC ALS

VAN ES M¹, VAN VUGHT P¹, SCHELHAAS H², FUMOTO K¹, ANDERSEN P³, GROEN E¹, BLAUW H¹, DIEKSTRA F¹, CUPPEN E¹, LEMMENS R⁴, VAN DAMME P⁴, ROBBERECHT W⁴, DAHLBERG C³, DE VISSER M⁵, VAN DER KOOI A⁵, LUDOLPH AC¹, VELDINK JH¹, PASTERKAMP JR¹, OPHOFF R¹, VAN DEN BERG LH¹

¹UMC Utrecht, Utrecht, Netherlands, ²Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, ³Umeå University Hospital, Umeå, Sweden, ⁴University Hospital Leuven, Leuven, Belgium, ⁵Amsterdam Medical Center, Amsterdam, Netherlands, ⁶University of Ulm, Ulm, Germany

E-mail address for correspondence: [email protected]

Keywords: angiogenin, rare variation, association

Background: A large candidate gene study demonstrated an association between a SNP (rs11701) in ANG and sporadic ALS. Mutations in ANG were also reported in SALS patients. However, subsequent follow-up studies have unfortunately produced conflicting results. The association for the SNP could not be replicated and mutations were also identified in healthy controls.

Objectives: The aim of this study was to provide a definitive answer on the role of ANG in ALS.

Methods: The association for rs11701 was tested in a population of over 20,000 individuals. We performed a meta-analysis on all previous studies in ANG in ALS and performed additional sequencing experiments, allowing us to analyze sequence data from over 6,000 cases and 6,000 controls.

Results: Analysis of all data revealed a total of 16 rare non-synonymous mutations in 25 patients out of a total 6,006 sporadic ALS patients (0.42%). By comparison these variants were not observed in any of the 6,296 control subjects. Statistical analysis showed that this aggregation of rare variation in cases is significant with P = 3.28 x 10^-7. Results were consistent across all populations and a subsequent Woolf test did not detect evidence for heterogeneity between the strata with P = 0.97. These rare variants indeed confer a greater effect on disease risk than the common polymorphisms identified by GWAS and thus partially explain the missing heritability. We found that the odds ratio (OR) for rare mutations in ANG is 27.37 with 95% confidence interval (95% CI) of 6.31 – 449.68.

Discussion: We show a significant excess of rare mutations in ANG in sporadic ALS patients compared to controls. These mutations confer a very significant risk for ALS with OR > 25.0.

Conclusions: Mutations in ANG confer large risk for ALS.

C44 PARAOXONASE GENE MUTATIONS IN AMYOTROPHIC LATERAL SCLEROSIS

TICOZZI N^1,2, LECLERC AL², KEAGLE P², GLASS JD³, WILLS A-M⁴, VAN BLITTERSWIJK M², BOSCO D², RODRIGUEZ-LEYVA I⁵, GELLERA C⁶, RATTI A¹, TARONI F⁶, MCKENNA-YASEK DM², SAPP PC^2,7, SILANI V¹, FURLONG CE⁸, BROWN RH², LANDERS JE²

¹Department of Neurology, Università degli Studi di Milano, IRCCS Istituto Auxologico Italiano, Milan, Italy, ²Department of Neurology, University of Massachusetts Medical School, Worcester, MA, United States, ³Department of Neurology, Emory University, Atlanta, GA, United States, ⁴Department of Neurology, Massachusetts General Hospital, Charlestown, MA, United States, ⁵Faculty of Medicine, Universidad Autonoma de San Luis Potosi, San Luis Potosi, Mexico, ⁶Fondazione IRCCS Istituto Neurologico ‘Carlo Besta’, Milan, Italy, ⁷Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States, ⁸Department of Medicine, University of Washington, Seattle, WA, United States

E-mail address for correspondence: [email protected]

Keywords: genetics, paraoxonase, mutation

Background: The paraoxonase gene (PON) cluster on chromosome 7q21.3-q22.1 encodes for three homologous proteins (PON1, PON2 and PON3) involved in preventing lipid oxidation and detoxifying organophosphate compounds. Several reports have described an association between common SNPs in these three genes and sporadic amyotrophic lateral sclerosis (SALS) susceptibility.

Objectives: To assess whether mutations in the PON cluster are also involved in familial ALS (FALS) pathogenesis.

Methods: We screened for mutations in the PON1, PON2 and PON3 genes a cohort of 260 unrelated 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 patients were negative for SOD1, TARDBP and FUS mutations. Novel variants were genotyped in two panels composed of 1184 SALS and 1159 control samples.

Results: We identified 8 ALS-associated novel variants in the PON cluster in 12 samples (9 FALS and 3 SALS). All were heterozygous missense or splicing mutations, with the exception of a single homozygous mutation in PON2, identified in a patient whose parents were asymptomatic first cousins. The identified variants are predicted to disrupt protein function.

Discussion: Mutations in the PON cluster may contribute to ALS pathogenesis by impairing detoxification of exogenous toxins or by compromising the anti-oxidative capacity of the paraoxonase enzymes.

Conclusions: Our study suggests that mutations in the PON cluster may be responsible of ∼2.5% of all FALS cases.

C45 JUVENILE ALS WITH BASOPHILIC INCLUSIONS IS A FUS PROTEINOPATHY WITH FUS MUTATIONS

BÄUMER D¹, BIBA A¹, HILTON D², PAINE S³, LOWE J³, TURNER M¹, ANSORGE O¹, TALBOT K¹

¹University of Oxford, Oxford, United Kingdom, ²Derriford Hospital, Plymouth, United Kingdom, ³Nottingham University Medical School, Nottingham, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: FUS, basophilic inclusions, juvenile ALS

Background: The majority of cases of amyotrophic lateral sclerosis (ALS) start in late adult life and are characterised by TDP-43 positive ubiquitinated inclusions. Juvenile ALS is a rare but important exception with regard to age of onset and underlying neuropathology, which is TDP-43 negative, but characterised by basophilic inclusions on H+E stain.

Methods: We identified four patients with juvenile ALS with basophilic inclusions, analysed clinical features, characterised the neuropathology by immunohistochemistry and performed genetic and in vitro functional analyses, expressing FUS mutants in cell culture.

Results: Motor symptoms began between age 17 and 22, with rapid disease progression without dementia. No family history was identified. Basophilic inclusions were present in all cases and were immunoreactive for various RNA binding proteins, most prominently FUS (fused in sarcoma), but negative for TDP-43. A wide range of granular and compact FUS deposits was identified in glia and neuronal cytoplasm and nuclei. This was accompanied by disintegration of Nissl substance, ultrastructurally in keeping with aggregation of fragmented rough endoplasmic reticulum. Although no patient had a family history of motor neuron disease, genetic analysis revealed the presence of FUS mutations in all three cases with available DNA. Two patients carried apparent de novo P525L mutations. Post-mortem analysis and functional studies in cell culture indicate that these mutants may act by disrupting RNA binding protein complexes in the cytoplasm.

Conclusion: Juvenile ALS with basophilic inclusions is a FUS proteinopathy with features of endoplasmic reticulum disintegration. Most if not all cases are caused by mutations in FUS, suggesting that altered RNA metabolism is pivotal in disease pathogenesis.

SESSION 8B COGNITIVE AND PSYCHOLOGICAL ASSESSMENT AND SUPPORT

C46 SOCIAL COGNITION IN ALS: IMPAIRED COGNITIVE AND AFFECTIVE THEORY OF MIND

VAN DER HULST E-J, BAK TH, ABRAHAMS S

University of Edinburgh, Edinburgh, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: social cognition, cognitive change, prefrontal dysfunction

Background: A key component of Social Cognition is Theory of Mind (ToM) conceptualized as the ability to ascribe mental states to others. The Eye Gaze Test (EGT) is a classical ToM test and assesses an individual's ability to judge the preference of another by using the direction of their eye gaze as a cue. This process is fundamental for appropriate social interaction. Deficits on this test have been found in both ALS and FTD. ToM has been fractionated into cognitive and affective components, involving the recognition of the thoughts and feelings of another respectively. Disruption of affective ToM has been related to damage to the ventromedial prefrontal cortex, a region implicated early in FTD but which has not been as yet identified as typically affected in ALS.

Objectives: This study examined whether ALS patients display changes in both cognitive and affective ToM. Furthermore, the influence of attentional factors were explored.

Methods: Sixteen patients with ALS (non-demented) and 16 healthy participants, matched for age, sex and years of education, completed the cognitive and affective EGT. The EGT comprised of three features: type of judgement (cognitive, affective or physical control), attention (presence or absence of a distracter) and complexity of judgement (first or second order ToM). In addition, each participant was administered two tests of visual processing.

Results: The analyses revealed that ALS patients were significantly impaired in both cognitive and affective ToM trials. ALS patients displayed significantly lower scores for first order cognitive (P <0.01) and affective judgements (P <0.005) as well as second order cognitive judgements (P <0.05) with a trend for second order affective judgements (P=0.067). The patients were not impaired on physical control judgements or visual processing. Moreover there was no effect of having a distracter present.

Discussion and conclusions: Cognitive and affective components of ToM appear to be affected in some non-demented ALS patients who show difficulties in recognizing the thoughts and feelings of another. This deficit indicates involvement of the ventromedial prefrontal cortex and suggests that the prefrontal involvement in ALS may be extensive in those who do not show overt dementia. This selective deficit was found in patients with intact visual processing abilities. Moreover there was no effect of distracter present indicating that this was not the result of an attentional dysfunction. Hence this deficit reflects a basic social cognition impairment. These results could account for some of the behavioural abnormalities observed in ALS and implies that some ALS patients may have difficulties in primary interactions with carers.

C47 THE NATURE OF LANGUAGE DEFICITS IN ALS: SEMANTIC IMPAIRMENTS IN ACTION SEQUENCING

VAN DER HULST E-J, ABRAHAMS S, BAK TH

University of Edinburgh, Human Cognitive Neuroscience, Edinburgh, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: neuropsychology, language, embodied cognition

Background: A subset of patients with ALS show subtle difficulties in a range of cognitive functions, including language. In particular, recent research has uncovered deficits in action processing in ALS-patients both with and without dementia, although the exact nature of these deficits remains to be elucidated. Findings from these studies have been interpreted as support in favour of the influential theory of embodied cognition, which states that both motor functions and knowledge of actions are dependent on the same underlying neural networks.

Objectives: This study investigated the nature of linguistic and conceptual deficits in patients with classical ALS. In particular, it explored the distinction between two types of relationship between actions: parallel (actions of a similar character, eg typing – writing) and sequential (actions, which usually follow each other, eg peeling – cutting).

Methods: Twenty-one ALS-patients and 17 healthy controls (HCs), matched for age, education and sex, were compared on a neuropsychological battery of tests. The patients had to choose the correct answer from an array of two different choices. The battery comprised of language tests: semantic association for Objects and Actions (O and A) and Action Sequencing (AS). Furthermore, a relatively difficult test of Audio Visual Information Processing (AVIP) was added as a measure of complex non-semantic information processing. Both reaction times (RTs) and error rates were recorded.

Results: Analyses revealed that ALS-patients made significantly more errors than HCs on AS (P = 0.021), while both groups displayed an equal amount of errors on O and A (Ps>0.067). In terms of the reaction times, taking out the possible influence of motor slowing, basic motor speeds were subtracted in all three conditions, yielding cognitive decision times. Results showed a main effect of group (P = 0.016). Although ALS-patients exhibited longer cognitive decision times than HCs overall, patients were not slowed on any of the individual tests in particular (P = 0.158). There were no differences in reaction times and errors between patients and controls on AVIP (Ps>0.233).

Discussions and conclusions: Our study detected a specific deficit in the task requiring the comprehension of action sequencing in a group of non-demented ALS-patients. In contrast, no deficits were observed on action and object association tests as well as in audiovisual information processing. The deficits in action sequencing were confined to the accuracy of responses only and did not appear to influence cognitive decision times. Our findings suggest that action deficits observed in ALS patients might in fact be due to a sequencing deficit. Moreover, they raise the question whether sequencing deficits are specific to language or possibly extend to other neuropsychological domains, constituting one of the basic cognitive deficits in ALS.

C48 PREFRONTAL LOBAR DETECTING: A MORE SENSITIVE BATTERY FOR SCREENING COGNITIVE IMPAIRMENT IN EARLY-PHASE ALS?

JI Y¹, WEI L², DENG M¹, WANG K², FAN D¹

¹Peking University Third Hospital, Beijing, China, ²Anhui Medical University First Hospital, Hefei, China

E-mail address for correspondence: [email protected]

Keywords: prefrontal lobar dysfunction, prospective memory, early phase

Background: ALS is now considered as a multisystem disease with co-occurrence of frontotemporal syndrome. Imaging studies show the prefrontal lobe is the most involved area in ALS patients with behavioral and cognitive impairment.

Objective: The aim of this study is to find a screening battery to detect subtle cognitive deficits in ALS patients at early stage.

Methods: Eighty consecutive patients diagnosed as ALS according to the El Escorial criteria and 59 normal controls were matched for sex, age and education. Both groups were assessed with a series of neuropsychological tests state as follows: the Mini-Mental State Examination (MMSE), Neuropsychiatric Inventory (NPI), Frontal Behavior Inventory (FBI), verbal fluency test (VFT), Stroop Color Word Interference Test (CWT), and prospective memory (PM) including event-based prospective memory (EBPM) and time-based prospective memory (TBPM). To further explore the topic a subset of patients underwent further examination involving picture and music emotional perception, theory of mind or decision making (Iowa Gambling Task and risk-taking task).

Results: As previous studies reported, our patients also did not differ from normal controls on MMSE, but significantly differed on behavioral inventories including NPI (4.60±4.78 vs 0.16±0.59, P<0.001) and FBI (3.19±3.59 vs 0.18±0.54, P<0.001). However, we found our patients did not differ from normal controls in traditional neuropsychological tests on executive function including VFT and CWT. On the other hand, statistically significant differences were found between ALS patients and the normal controls on the prospective memory (EBPM: 5.55±2.20 vs 6.37±1.80, P=0.043; TBPM: 4.85±1.51 vs 5.65±1.10, P<0.001), faux pas task (23.13±10.09 vs 28.91±6.20, P=0.002) and the arousal of negative pictures (6.38±1.32 vs 7.18±1.12, P=0.008). More interestingly, we found TBPM is more sensitive than EBPM in early phase patients (possible and probable-laboratory supported ALS). We did not find differences between ALS patients and normal controls on the valence and recognition of pictures, music emotional perception, Iowa Gambling Task and risk-taking task.

Conclusions: Prefrontal lobar dysfunction does exist among ALS patients, and may be spread from medial to lateral part. Behavioral test, PM and theory of mind used in this study is more sensitive in detecting behavioral and cognitive impairment in ALS patients at early stage than the classical cognitive measures.

C49 DIAGNOSTIC VALIDITY OF THE ALS COGNITIVE BEHAVIORAL SCREEN

RUSH B¹, WOOLLEY SC², BOYLAN K¹

¹Mayo Clinic, Jacksonville, FL, United States, ²California Pacific Medical Center, San Francisco, CA, United States

E-mail address for correspondence: [email protected]

Keywords: cognitive screen, cognitive impairment, diagnostic validity

Background: Cognitive and behavioral symptoms of amyotrophic lateral sclerosis (ALS) are increasingly recognized. The ALS Cognitive Behavioral Screen (ALS-CBS) (1–2) is a 20 point screening measure with unclear diagnostic validity.

Objectives: To compare diagnostic classifications of cognitive impairment from the ALS-CBS to those from gold standard neuropsychological evaluation.

Methods: Retrospective review of 24 cases of ALS referred for evaluation of cognitive and behavioral symptoms between January 1, 2008 and March 31, 2010. Each participant was administered the ALS-CBS and a standard of care neuropsychological evaluation and received two diagnoses; one from the ALS-CBS and one from neuropsychological evaluation. The following diagnoses were considered: ALS, ALS with cognitive impairment/no dementia (ALS-ci), ALS with behavioral impairment/no dementia (ALS-bi), or ALS with frontal-temporal dementia (ALS-FTD). A score of ≤ 11 on the cognitive form of the ALS-CBS suggested cognitive impairment and a score < 5 suggested ALS-FTD. Diagnostic classifications from the ALS-CBS and the neuropsychological evaluation were compared and diagnostic validity was determined.

Results: Ten out of the 24 cases were female (42%), with a median age of 68 years (range: 43 – 83 years), median education of 14 years (range: 9 – 20 years), and median of 18 months (range: 6 – 55 months) duration between motor neuron disease symptom onset to cognitive evaluation. Fifty percent of cases had bulbar-onset disease. Neuropsychological evaluation confirmed cognitive impairment in 16 out of 24 cases (67% base rate; 9 ALS-FTD and 7 ALS-ci). There were 50% bulbar cases in the cognitively impaired and unimpaired groups. Using a cut-off score of ≤ 11 on the ALS-CBS, 46% cases (11/24) had cognitive impairment (8 ALS-FTD, 3 ALS-ci). This cut-off score yielded a sensitivity of 69% and a specificity of 100%. Of the 5 cases for which the ALS-CBS yielded a false negative diagnosis of cognitive impairment, 4 of the cases had a diagnosis of ALS-ci and 1 case had a diagnosis of ALS-FTD on neuropsychological evaluation.

Conclusion: The ALS-CBS demonstrates excellent specificity using a cut-off score of ≤ 11 for determining cognitive impairment. The ALS-CBS may have limited sensitivity to ALS-ci or cognitive impairment without dementia.

Discussion: Data support the validity of the ALS-CBS as a screen for cognitive impairment in ALS. The cut-off score of ≤11 on the cognitive form of the ALS-CBS is empirically justified particularly when ALS-FTD is present. Alternate cut-off scores or language screening items could improve the sensitivity of the ALS-CBS to ALS-ci when it is present.

References:

• Woolley SC. Third International Research Workshop on Fron-totemporal Dementia in ALS. London, Ontario, Canada, 2009.
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C50 LONGITUDINAL COGNITIVE SCREENING USING THE ALS COGNITIVE BEHAVIORAL SCREEN (ALS-CBS™)

WOOLLEY SC¹, MOORE DH¹, VALAN M¹, SPITALNY M², KUSHNER G¹, FORSHEW DA¹, MILLER R¹, KATZ J¹

¹California Pacific Medical Center, San Francisco, CA, United States, ²Research Computing, Mill Valley, CA, United States

E-mail address for correspondence: [email protected]

Keywords: cognition, screening, longitudinal

Background: The ALS Cognitive Behavioral Screen (ALS-CBS™) has been validated as a clinical measure but its utility in research is unknown. Its ease of administration is ideal for longitudinal assessment of cognition in large cohorts.

Objectives: To evaluate cognitive screening data obtained from the WALS multi-center trial of lithium carbonate and to characterize cognitive status of this research cohort over time.

Methods: 109 patients with ALS were enrolled. The maximum cognitive score on the ALS-CBS™ is 20; scores at or below 10 are suggestive of frontotemporal dementia (1). The mean score for a clinical cohort (n=112) was 14.6 (1). Longitudinal cognitive change was calculated using the difference in scores over 12 months. Age of onset, duration of symptoms, forced vital capacity (FVC), ALSFRS-R scores, and fatigue were measured in the trial.

Results: The mean age of subjects was 56 years, mean ALS-FRS-R at study initiation was 37.5, and mean FVC was 94.8%. Ninety-eight subjects completed the ALS-CBS™ longitudinally. The average cognitive score was 16.68 (2.67) at baseline and the cohort remained stable over 1 year (16.69). Only 7% of subjects revealed significant cognitive declines (>5 points) but 4% revealed significant increases. Declines in cognition were not associated with age, symptom duration, low FVC, low ALSFRS-R scores, fatigue, or drug side effects.

Discussion: The subjects in this clinical trial did not show significant decline over a 12-month period. This suggests that frontal lobe decline is not prominent among patients who meet inclusion criteria for a clinical trial. The mean cognitive score in the trial was higher than in a clinical cohort suggesting that patients enrolling in trials are less likely to be impaired than clinical patients. These results also differ from previous longitudinal research in a clinical cohort which revealed longitudinal cognitive decline (2).

Conclusion: Clinical trials may enroll patients with different cognitive profiles than those seen in routine clinic. This may relate to research patients needing to be motivated, having limited duration of disease due to inclusion criteria, or having higher FVC's which limits those enrolled with significant bulbar involvement. While the methodology does not allow us to generalize our conclusions, the lack of progression in this cohort also points towards a relative paucity of cognitive impairment in this group. The results add to increasing evidence that a screening tool is a practical way to monitor cognition and behavior longitudinally in large cohorts.

References:

• Woolley SC, York MK, Moore DH, et al. ALS 2010; in press.
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C51 COMPUTERIZED FRONTAL ASSESSMENT OF AMYOTROPHIC LATERAL SCLEROSIS

POLETTI B, LAFRONZA A, SOLCA F, TICOZZI N, MORELLI C, MESSINA S, SILANI V

Department of Neurology and Laboratory of Neuroscience, ‘Dino Ferrari’ Center, University of Milan, IRCCS Istituto Auxologico Italiano, Milan, Italy

E-mail address for correspondence: [email protected]

Keywords: frontal functions, computerized tools, neuropsychological assessment

Background: The study of cognition and behaviour as a feature of Amyotrophic Lateral Sclerosis (ALS) is an evolving field still lacking a full consensus on terminology, diagnostic criteria, and clinical significance of any detected abnormalities. Marked discrepancies remain regarding the incidence of abnormalities and characteristics of the impairments that define ALS, frequently depending on the kind of cognitive tool used. Frontal alterations in ALS have been variously described and assessed in literature. Moreover, different degrees of frontal involvement in ALS starting from minimal frontal changes to overt Fronto-Temporal Dementia (FTD) have been documented, thus revealing the importance of detecting the whole spectrum of frontal involvement characterizing motor neurone disease's cognitive pattern. The terms ALSci (ALS with cognitive impairment), ALSbi (ALS with behavioural impairment), and ALS-FTD are developing concepts that aim to capture the key differences between the various clinical phenotypes. Computerized neuropsychological tools seem to be ideal in exploring and characterizing frontal cognitive functions.

Objectives: The purpose of this study was to analyze frontal cognitive functioning of ALS patients with frontal cognitive computerized measures.

Methods: Fifteen patients fullfilling El Escorial Criteria for ALS and fifteen controls underwent an extensive neuropsychological and psychodiagnostic assessment. Patients received the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRSr). Attentional skills and frontal functioning have been investigated with a computerized neuropsychological battery named TEA (Test of Everyday Attention). This battery, focused on attention and frontal functions, assess the ability of patients to selectively attend, sustain their attention, divide their attention between two tasks, switch attention from one task to another, and withhold (inhibit) verbal or motor responses. 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 differences in cognitive performances between patients and controls, with higher difficulties in the more complex attentional tasks for the former and better scores for the latter. Significant differences of data emerge on different frontal measures, while a general cognitive slowness characterized patient's performances. Patients displayed lower performances on frontal task of cognitive functioning. Subjects differed significantly for the presence of anxiety symptoms. Neuropsychological and psychological data were correlated with functional and respiratory parameters.

Discussion and conclusions: Computerized neuropsychological assessment seems an ideal tool in detecting small ‘frontal’ cognitive changes frequently observed in the cognitive frontal spectrum of ALS. Implications for clinical purposes will be discussed.

SESSION 8C CLINICAL TRIALS

C52 ESSENTIALS OF PHASE II CLINICAL TRIAL DESIGN

RAVINA B

Clinical Trials Coordination Center, University of Rochester, Rochester, NY, United States

E-mail address for correspondence: [email protected]

Keywords: phase II trials, trial design

Phase II clinical trials encompass a broad range of goals. These goals may include: assessment of safety, tolerability, logistics and feasibility; characterization pharmacokinetics and pharmacodynamics in the target population; refinement of the target population; evidence of mechanism of action (‘target engagement’); prediction of efficacious doses, exposures, and responses; preliminary evidence of clinical efficacy or futility; characterization of selected biopharmaceutical and drug interaction and drug metabolism issues, as appropriate. Because of the broad range of goals, there is no single clinical trial design that is suitable for phase II. The design must be tailored to fit the goals of the study and the particular questions about the intervention. More than one study will generally be required to answer the key questions and lay the groundwork for phase III, comparative efficacy trials.

Phase II trials in neurodegenerative disorders pose unique challenges. These include problems with measuring target engagement and the pharmacodynamic activity of the intervention, and the relatively slow clinical evolution. These limitations may make phase II trials in neurodegenerative disorders longer and larger than desired or possible, leading to problems in the design and conduct of phase III trials. This is highlighted by the large number of negative phase III clinical trials and underscores the need for phase II clinical trials that are better able to predict subsequent success or failure in phase III.

This presentation will highlight critical questions in phase II using examples from other neurodegenerative disorders such as Parkinson's disease and Friedreich Ataxia: measurement of pharmacodynamic activity using biomarkers and strategies for dose selection. Additionally we will consider the predictive value of phase II designs. Certain phase II designs may be thought of as diagnostic tests that are meant to predict positive or negative results in phase III trials. In this framework, one should consider how the prior probability of success and the study design parameters, such as power and type I error rate, may affect the positive and negative predictive value of the phase II trial.

C53 THE ‘SMALL’ CLINICAL TRIAL: CHALLENGES IN DESIGN AND INTERPRETATION OF RESULTS

MCDERMOTT MP

University of Rochester, Rochester, NY, United States

E-mail address for correspondence: [email protected]

Keywords: phase II trial, sample size, research question

Clinical trials in rare diseases such as ALS pose many challenges given the limited available resources and numbers of willing trial participants. In this setting, feasibility constraints can lead to compromises in important principles of sound trial design. These principles include careful formulation of the research question, tailoring of the design to best answer the research question, and use of appropriate and efficient statistical methods for data analysis. Important design features include selection of an appropriate target population, choice of outcome measures, recruitment of an adequate number of trial participants, and use of methods to reduce or avoid bias (eg, randomization, blinding, and facilitating participant retention). The conduct of trials that are inadequately designed to address the research questions posed slows progress toward finding safe and effective treatments for ALS; this can be particularly problematic in middle development (Phase II). The lack of clear reporting and proper interpretation of trial results, especially for “small” trials with their inherent limitations, can likewise impede research progress. Properly designed Phase II trials and correct interpretation of their results can help avoid the inappropriate discarding of treatments from future consideration or inappropriate shift from equipoise in the research community in favor of a treatment.

This presentation will consider ways to address common difficulties encountered in the design and interpretation of clinical trials involving relatively small numbers of participants. These include precise formulation of a focused research question; carefully distinguishing between a ‘negative’ and an ‘inconclusive’ result concerning the potential efficacy (or activity) of a treatment; determining whether a seemingly ‘positive’ result concerning a treatment effect is to be believed; recognizing potential bias associated with the use of nonrandomized controls; and considering the impact of eligibility criteria on the generalizability of results. Examples from ALS and other disease areas will be used to illustrate the importance of formulating the right questions and addressing these questions in a rigorous manner to enhance decision-making in middle development.

C54 COMPARING ENDPOINTS: SLOPE OF ALSFRS-R HAS GREATER POWER THAN TIME TO LOSE SIX POINTS

MOORE DH, KATZ JS, MILLER RG, THE WALS STUDY GROUP

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

E-mail address for correspondence: [email protected]

Keywords: ALSFRS-R, linear mixed effects model, primary endpoint

Background: A recent randomized trial of lithium used time to an event, in this case losing ≥6 points in ALSFRS-R score or death, as the primary endpoint. A log-rank test was used to judge whether lithium treatment was efficacious or futile.

Objectives: To compare the power of the time to an event endpoint with that based on estimating rate of decline (slope) of ALSFRS-R over time.

Methods: We used data from 246 untreated patients in a recent controlled trial with monthly ALSFRS-R to generate simulated data to compare the two endpoints. Simulated data was based on linear fits to observed data from real patients. Simulated trials assigned patients to two groups: treated and placebo. For the treated, the slope of ALSFRS-R decline over time was reduced; for the placebo it was unaltered. The simulations recorded the numbers of patients with drops ≥6 points, or death, with slopes estimated in a linear mixed effects (lme) model fitted to the simulated data. For each simulation we recorded the P-value from the log-rank test with the P-value for testing a change in slope due to the treatment in the lme model.

Results: The results of the simulation clearly show that ability to detect a treatment effect is higher for the change in slope than for the time to event endpoint. For example, with 50 patients randomly assigned to each group (treatment and placebo) with up to 12 months followup, power was 0.67 for the slope change vs. 0.31 for the time to 6 point drop design when the simulated change in slope was a 40% reduction. When trial follow-up is shortened to 6 months, power was 0.85 for slope change vs. 0.15 for time to event.

Discussion: The time to an event endpoint is appealing because of its simplicity and lack of reliance on a specific model for rate of decline. However, power for time to event designs depend on the number of events so that patients that do not have drops ≥6 points contribute little to power. A slope analysis requires that rate of decline be reasonably linear but power increases with numbers enrolled. It appears that the advantage of estimating slopes over counting events is considerable when ALSFRS-R is the tool used to measure decline over time in ALS patients.

Conclusions: In ALS studies an endpoint based on slope comparisons is more powerful than one based on time to lose ≥6 points.

C55 ANALYSIS OF FUNCTIONAL SCORES DATA IN A STUDY OF DEXPRAMIPEXOLE FOR AMYOTROPHIC LATERAL SCLEROSIS IN THE CONTEXT OF NON-TRIVIAL DISCONTINUATIONS AND DEATHS

INGERSOLL E¹, ARCHIBALD D¹, SCHOENFELD D², MOORE DH³, GRIBKOFF V¹, MATHER J¹, CUDKOWICZ M⁴, SHEFNER J⁵, MILLER RG³, BOZIK M¹

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

E-mail address for correspondence: [email protected]

Keywords: dexpramipexole, clinical trial, ALSFRS-R

Background: Diseases of progressive deterioration like ALS present unusual challenges in analyzing clinical outcomes in interventional trials. The typical method, modeling slopes of functional change using a linear mixed effects model (LMEM), is conceptually valid, but fails to distinguish deaths from other discontinuations and does not reflect death as a worse outcome than survival with any degree of functional decline. Furthermore the LMEM assumes linearity of decline over time and that discontinuations and deaths are missing at random. An intent-to-treat analysis of mortality requires post-study assessments of vital status for subjects who discontinue.

Objective: To evaluate the clinical effects of dexpramipexole ([6R]-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine; KNS-760704) in a recent ALS study (KNS-760704- CL201; ‘CL201’) (1) in the context of imbalances in the rates of discontinuations and death between treatment groups by using multiple analytical methods.

Methods: In Part 2 of CL201, subjects were randomized (double-blind) to receive dexpramipexole 50 mg (n=48) or 300 mg (n=44) for 24 weeks. Clinical status was, in part, measured by ALSFRS-R and survival. Data were analyzed using 1) LMEM for slopes, 2) LMEM with imputation of zero scores for the first post-death visit of subjects who died (‘zero imputation’), 3) Kaplan-Meier life table estimates, and 4) analysis of subject rankings (2) on mortality and change from baseline in ALSFRS-R.

Results: Twenty-one subjects discontinued (14 in the 50 mg group and 7 in the 300 mg group), including 12 deaths (9 in 50 mg and 3 in 300 mg, P = 0.071). Three of the 12 deaths were in subjects who had previously discontinued for other reasons (2 in 50 mg and 1 in 300 mg). The unadjusted mean slope in Part 2 of CL201 for subjects who died was steeper than for survivors (-1.80 vs. 1.08, respectively). The same pattern was observed for all discontinuers vs. completers. The LMEM analysis showed a 20% difference in slopes favoring 300 mg (P = 0.178), which increased to 43% with zero imputation (P = 0.018). The joint rank test of mortality and ALSFRS R also favored the 300 mg group (P = 0.046).

Discussion and conclusions: In ALS trials, discontinuations and deaths complicate the interpretation of observed cases. Slope estimates per se do not reflect the seriousness of death. Zero imputation is useful only to illustrate effects of bias as a sensitivity analysis. A true intent-to-treat analysis of mortality requires post-study follow-up for all subjects who discontinue. The joint-rank test is based on an intuitively reasonable ranking of outcomes with appropriate weighting of death outcomes, which involves minimal statistical assumptions. This study demonstrates that the joint-rank test in ALS is a novel, powerful method that bears further investigation as a primary analysis method in future trials.

References:

• Bozik, et al. ALS 2009;10(S1):28.
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SESSION 9A AXONAL FUNCTION AND PATHOLOGY

C56 AXON DEGENERATION MECHANISMS: NEW INSIGHTS FOR SELECTIVE VULNERABILITY OF MOTOR UNITS IN AGEING AND DISEASE

COLEMAN MP, MILDE S, CONFORTI L, GILLEY J

The Babraham Institute, Cambridge, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Wallerian degeneration, dying back, axonal transport

Early loss of axons and synapses and axonal transport impairment are common to many neurodegenerative diseases including motor neuron disease but the molecular events that may link them are poorly understood. Axonal transport defects or axonal spheroids occur in amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Parkinsonism, multiple sclerosis, traumatic brain injury and glaucoma. Axonopathy in hereditary spastic paraplegia, peripheral neuropathy and motor neuron disease sometimes reflects loss or damage to the transport machinery itself and the substantial decline in transport during normal ageing could contribute to age-related ‘dying back’ disorders.

Intriguingly, a general disruption of anterograde delivery can lead to a specific axon degeneration pathway. Axon transection abolishes transport from the soma but addition of a single protein is sufficient to delay the ensuing Wallerian degeneration by tenfold. This ‘slow Wallerian degeneration’ protein (WldS) is an aberrant protein that arose in a healthy mutant mouse and combines sequences from nicotinamide mononucleotide adenylyltransferase Nmnat1 and ubiquitin ligase Ube4b. Mislocalisation or overexpression of Nmnat1 or Nmnat3 have similar protective effects.

In contrast, depletion of an endogenous Nmnat, the labile isoform Nmnat2, causes Wallerian-like degeneration of uninjured neurites in primary culture. WldS prevents this degeneration, probably by compensating for loss of Nmnat2 in an enzyme-dependent manner. However, the Nmnat function that promotes axon survival may not be NAD+ synthesis. Nmnat2 is abundant in the Golgi apparatus but live imaging shows it is also rapidly and bidirectionally transported along neurites. Our data suggest it is targeted to vesicular structures by palmitoylation, where it frequently comigrates with other Golgi-derived proteins but not with mitochondria.

In healthy axons, rapid and constant delivery of this essential protein seems to balance its degradation by the ubiquitin proteasome system, maintaining its level above the threshold needed for axon survival. When axonal transport or neuronal metabolism fails, we propose that failure to replace natural turnover of Nmnat2 limits axon survival. Deficiencies in other axonal cargoes take far longer to limit survival when loss of Nmnat2 is, indicating an important therapeutic window for some disorders if this pathway were appropriately targeted. It is essential now to extend these data in vivo and to understand the upstream and downstream events.

C57 CELLULAR TRANSPORT DYSFUNCTION IN SOD1G93A TRANSGENIC MICE AND NSC34 CELLS

FARG M¹, TURNER B², WALKER A¹, HORNE M², ATKIN J^1,2

¹Department of Biochemistry, La Trobe University, Melbourne, Australia, ²Howard Florey Institute, University of Melbourne, Melbourne, Australia

E-mail address for correspondence: [email protected]

Keywords: ER-Golgi, dynein, ER stress

Background: Motor neurons are large cells with long axons that have high synthetic and energetic requirements, placing a heavy demand on cellular transport processes. The motor protein dynein/ dynactin is involved in both axonal transport and transport of proteins from the endoplasmic reticulum (ER) to Golgi apparatus. Impaired cellular and axonal transport and disruption to dynein and dynactin function are linked to motor neuron degeneration in ALS. Dynein also physically interacts with mutant but not wildtype SOD1 and it co-localises with mutant SOD1 inclusions. Furthermore, impairment of dynein-dynactin function results in Golgi fragmentation. which occurs in both sporadic and SOD1-mediated familial human ALS and in transgenic SOD1^G93A animals. Recently there has been a surge of publications describing the importance of ER stress in ALS. We described induction of the whole unfolded protein response in both transgenic SOD1^G93A animals and in sporadic ALS patients. ER stress is triggered either by the presence of misfolded proteins in the ER or by inhibition of general protein transport from the ER.

Objectives: We hypothesised that disruption in ER- Golgi trafficking triggers ER stress in ALS. Anterograde and retrograde transport proteins were examined in transgenic SOD1^G93A mice and in NSC34 cell lines transfected with mutant and wildtype SOD1.We examined dynein, Rab1 and the coat protein complex II (COPII), which transports proteins from the ER to the Golgi apparatus in coated vesicles.

Methods: NSC34 cells transfected with SOD1 construct were immunostained with dynein intermediate chain, COPII and GM130 antibodies and confocal imaging was performed. Lysates from either NSC-34 cells or transgenic mice SOD1^G93A were coimmunoprecepitated with COPII,dynein and Rab1, followed by Western blotting with SOD1.

Results and conclusions: A physical interaction between mutant (but not wildtype SOD1) with both COPII and the dynein intermediate chain was detected in transgenic SOD1^G93A mice.This was detected in pre-symptomatic animals as early as postnatal age 10 days, which is 20 days prior to the onset of ER stress. This interaction was confirmed in NSC34 cell lines expressing SOD1 mutants A4V, G85R and G37R. COPII also co-localises with mutant SOD1 inclusions in NSC-34 cells. We also showed that secretion of brain derived neurotrophic factor (BDNF) was reduced in NSC-34 cell lines expressing mutant SOD1, thus implying dysfunction of the ER-Golgi secretory pathway in ALS. Mutant SOD1 also bound aberrantly to other proteins involved in ER to Golgi trafficking including Rab1, which is involved in the docking of vesicles between the ER and Golgi apparatus. These data suggest that impairment in cellular trafficking between the ER-Golgi apparatus occurs as an early event in ALS. These events occur prior to ER stress, suggesting that cellular trafficking dysfunction triggers ER stress in ALS.

C58 IN VITRO MODEL SYSTEMS FOR NMJ FORMATION UTILIZING MOTONEURONS FROM ADULT RAT, MOUSE AND SOD1 TRANSGENIC MOUSE

DAS M^1,2, BHARGAVA N¹, STANCESCU M¹, RUMSEY J^1,3, HICKMAN J¹

¹University of Central Florida, Orlando, FL, United States, ²Indian Institute of Technology, Kanpur, India, ³Sanford-Burnham Medical Research Institute, Lake Nona, FL, United States

E-mail address for correspondence: [email protected]

Keywords: in vitro, adult motoneuron, NMJ

Background: In vitro model systems for research in ALS and MND suffer from the limitation that they are composed of embryonic cells for the most part and there are few examples of neuromuscular junction (NMJ) formation in these culture systems. Model systems composed of adult motoneurons from rat, mouse, and SOD1 mouse, that exhibit neuromuscular junctions on the myotubes derived from adult satellite cells, would be a great benefit to the field. These systems could form the basis for basic research model systems as well as for high-content screens for drug discovery.

Objectives: To engineer defined, serum-free culture systems utilizing tissue or motoneurons from adult rat, mouse and SOD1 transgenic mice and demonstrate NMJ formation on myotubes derived from satellite cells from adult muscle. To then use these in vitro systems to study NMJ formation between diseased and healthy tissue or in the presence of factors known to cause ALS or MND type deficits.

Methods: Cultures were evaluated utilizing morphological analysis, immunocytochemical evaluation for phenotype and electrophysiology to determine electrical properties of the regenerated motoneurons as well as myotube function. NMJ formation was established by immunocytochemical and videography. All cell types, as well as co-cultures, were done in a defined, serum-free culture system utilizing a non-biological, silane based culture surface.

Results: Regeneration and recovery of electrical properties was achieved for adult rat motoneurons (1), adult mouse (2) and SOD1 transgenic mice. Single temporal doses of the neurotransmitters serotonin, glutamate (N-acetyl-DL-glutamic acid) and acetylcholine-chloride lead to the full electrophysiological functional recovery of approximately 60% of the adult spinal cord neurons. NMJ formation has also been clearly demonstrated in this defined, serum-free, in vitro system. The clustering of AChRs using alpha-bungarotoxin and their co-localization with synaptophysin vesicles was analyzed immunocytochemically and the appearance of striations after this time indicated that the co-cultures contained about twice the number of myotubes showing striations. Cultures survived for over 2 months with viable cellular properties.

Discussion and conclusions: The ability to routinely culture long-term the adult motoneurons of rat, mouse and SOD1 transgenic mouse opens new avenues to investigate in vitro NMJ formation. The ability to now culture the neurons from SOD1 mice after they begin expressing behavioral deficits could provide a new experimental tool to investigate this disease. This improved system supports the goal of creating a physiologically relevant tissue engineered motoneuron plus skeletal muscle construct to develop functional bio-hybrid devices to analyze NMJ activity. Furthermore, we believe this serum-free culture system will be a powerful tool in developing advanced strategies for regenerative medicine in ALS and MND.

References:

• Das M, Patil S, Bhargava, et al. Biomaterials, 2007; 28:1918–25.
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C59 INCREASED RATE OF MATURATION OF ELECTRICAL PROPERTIES OF SOD1 MOTONEURONS

QUINLAN K, SCHUSTER J, FU R, SIDDIQUE T, HECKMAN CJ

Northwestern University, Feinberg School of Medicine, Chicago, IL, United States

E-mail address for correspondence: [email protected]

Keywords: SOD1 mouse, electrophysiology, postnatal development

Background: Spinal motoneurons are highly vulnerable to ALS, rapidly degenerating while other neurons remain healthy. Previous research using standard animal models, mutant SOD1 mice, has revealed many deficits in cellular properties at different time points in the disease, starting in the early postnatal stages. However which properties in the motoneurons are the first to become abnormal has not been systematically investigated.

Objectives: Therefore we undertook a more comprehensive approach comparing the development of multiple markers excitability during the first 12 postnatal days (P0-12) in SOD1^G93A mice. We tracked more than 25 intrinsic properties associated with excitability in SOD1^G93A motoneurons to compare their postnatal development to non-transgenic and SOD1^WT motoneurons.

Methods: Using whole cell patch clamp of over a hundred motoneurons in acute slices from the lumbar spinal cord of mice P0 to P12, we tracked the developmental changes relating to excitability.

Results: We show changes in several properties with both age and SOD1^G93A mutation, including increases in both Na⁺ and Ca²⁺ components of the persistent inward current, increased input conductance, decreased action potential duration including faster rates of rise and fall, and faster decay of the afterspike after hyperpolarization. Though there were a few exceptions (current at which firing onset and offset occur, I_ON and I_OFF, and hyperpolarization of resting membrane potential), overall the changes in SOD1^G93A motoneurons were consistent with those observed during postnatal development. To test this, changes due to mutation were normalized and plotted compared to the normalized changes due to maturation. Results were significant (P < 0.0001), indicating that SOD1^G93A motoneurons are undergoing the same changes observed during postnatal development, except the SOD1 motoneurons seem to be progressing more rapidly towards the mature state.

Discussion and Conclusions: Overall, the differences in the SOD1^G93A motoneurons closely fit a profile of an increased rate of postnatal maturation. The resulting early achievement of the adult state in these motoneurons may trigger an overall accelerated rate of aging in motoneurons and thus increase motoneuron vulnerability.

C60 ASSESSING MOTOR NEURON LOSS IN THE ALS RAT WITH ELECTRICAL IMPEDANCE MYOGRAPHY

WANG L, LI J, RUTKOVE S

Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: MUNE, impedance, rat

Objective: To evaluate the relationship between electrical impedance myography (EIM) and standard electrophysiological measures, including motor unit number estimation (MUNE), and survival in the SOD1 G93A amyotrophic lateral sclerosis (ALS) rat.

Background: Current electrophysiological methods for quantifying motor neuron loss in ALS in both pre-clinical and clinical studies are limited. The most commonly used method, MUNE, is relatively unreliable early in the disease course, is time-consuming to perform, and can only be applied to a limited set of muscles. Past and ongoing studies in human subjects suggest that EIM may be a powerful indicator of disease progression. In addition to providing a sensitive measure of motor neuron loss, EIM is much easier to perform, may provide reliable data early in the disease course, and is readily applied to most muscles. How well EIM correlates to MUNE, identifies disease onset, and predicts survival, however, remains unknown.

Methods: Sixteen male ALS rats underwent repeated MUNE, EIM, weight and behavioral assessments starting from approximately 85 days of age. MUNE was performed using an incremental technique stimulating the sciatic nerve and recording from the entire distal leg. EIM was performed over the gastrocnemius muscle. Several different EIM parameters were evaluated. Animals were sacrificed when they became unable to right themselves after 15 seconds after being laid supine.

Results: EIM parameters effectively tracked disease progression in the animals and mirrored the MUNE. EIM's 50 kHz phase correlated strongly with MUNE, with r = 0.88 (P < 0.001). EIM 50 kHz phase was also a sensitive indicator of disease onset, showing reductions by 98 days, whereas MUNE and weight assessments did not demonstrate alterations until greater than 110 days. Additionally, the rate of decline in EIM 50 kHz phase from the time of disease onset until 140 days of age correlated strongly with the time of death several weeks later (r = 0.75, P<0.01) whereas MUNE obtained over the same time period did not.

Discussion: These results showed that EIM data strongly correlate with MUNE, supporting its essential nature as an indicator of motor neuron loss. However, EIM parameters also identified disease onset and predicted time of death earlier and with more accuracy than MUNE, supporting its clinical relevancy.

Conclusions: Since EIM can be applied to virtually any muscle or muscle group, is painless, and is rapid to perform with only minimal training, it deserves to play an important role in assessing drug efficacy in future clinical therapeutic studies in ALS.

C61 EVIDENCE OF EARLY SOMATO-DENDRITIC ALTERATIONS IN LUMBAR MOTONEURONS OF SOD1 JUVENILE MICE

FILIPCHUK A^1,2, PAMBO-PAMBO A¹, LIABEUF S¹, BROCARD C¹, KULAGINA I², KOROGOD S², GUERITAUD JP¹, DURAND J¹

¹CNRS UMR6196 and Université de la Méditerranée, Laboratoire de Plasticité et Physiopathologie de la Motricité, Marseille, France, ²Dnipropetrovsk National University, Dnipropetrovsk, Ukraine

E-mail address for correspondence: [email protected]

Keywords: morphology, modeling, development

Background: Recently we showed that lumbar motoneurons from superoxide dismutase 1 (SOD1) mutant mice (Amyotrophic Lateral Sclerosis, (ALS) mouse model) are affected in their electrical properties and have over-branched dendrites at an early postnatal age (P8-P10) (1–3). In this work, we addressed the questions of whether the abnormal branching is already present at birth and whether it might be due to changes in the synaptic organization to motor nuclei.

Objectives: To understand how and why SOD1 neonate motoneurons grow abnormally.

Methods: We identified and recorded lumbar motoneurons in wild type (WT) and in SOD1 juvenile mice at postnatal days P3-P10 using intracellular recording and staining procedures as previously described (1). The labeled motoneurons were then reconstructed using neurolucida system and morphometric and topologic parameters were analyzed. We used computer models of 3D reconstructed WT and SOD1 motoneurons to assess the functional consequences of the morphological changes (4). We also used specific antibodies directed against synaptophysin and GFAP and immunohistochemistry to study presynaptic terminals and glial processes in motoneuronal pools from ventral spinal cords.

Results: These experiments revealed that WT and SOD1 motoneurons have a comparable number of dendritic branches at P3-P4 while certain morphological parameters are already different at that early age. The number of branches in WT motoneurons did not increase between P3 and P9 indicating that motoneuron grows by elongation instead of developing new branches at that period. On the contrary, SOD1 motoneurons over-branched precisely between P4 and P8. The mean number of branches and the mean total dendritic length are significantly higher in SOD1 motoneurons compared to those measured in non transgenic WT motoneurons at P8-P9. The abnormal branching occurs in both SOD1 G85R and G93A mutant mice. Immunofluorescent labeling of synaptic terminal reveals significantly lower values at P3 in SOD1 motoneuronal pools compared with age-matched WT pools. Conversely, glial staining was lower in the ventral and lateral part of the spinal cord from SOD1 mice at P8 compared with age-matched WT mice. Some glial tissue might be replaced by supernumerary dendrites.

Discussion and conclusions: Our results show that lumbar motoneurons of SOD1 mice have an abnormal branching with an onset between P4 and P8 and suggest that this abnormal branching might be due to lower number of presynaptic terminals reaching the motoneuronal pools. These morphological changes might be among the earliest compensatory mechanisms detected in SOD1 motoneurons.

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SESSION 9B INTERNATIONAL PERSPECTIVES ON CARE PRACTICE

C62 PALLIATIVE CARE – ACCESS TO SERVICES AND SUPPORT FOR PROVIDERS – A PROJECT WITH OUTCOMES

HARRIS R

MND Association of Victoria, Melbourne, Victoria, Australia

E-mail address for correspondence: [email protected]

Keywords: palliative care, access, key worker

Background: People with ALS/MND were underrepresented in palliative care services in Victoria. Palliative care providers were reluctant to accept referrals of people with ALS/MND. The role and responsibility of palliative care providers appeared confused, and people with ALS/MND were reluctant to access services that could improve their quality of life.

Objectives: To establish a framework that triggers referral to a palliative care services; develop and describe mechanisms that support communication; articulate the roles of the palliative care provider, and identify gaps in current palliative service provision for people with ALS/MND and make recommendations for resolving them.

Methods: Data collection was based on a literature review and discussion with people living with ALS/MND, past and present carers, palliative care service representatives, and key opinion leaders within the palliative care sector. Topic specific information from the literature review was combined with interview themes to construct a series of recommendations for the coordinated integration of palliative care into the overall management of people living with ALS/MND.

Results: The project found that there was not an existing framework that could be implemented to integrate palliative care into the overall care received. Palliative care workers did not feel confident in their knowledge of ALS/MND, and people with ALS/MND had a distorted understanding of palliative care and the services it could offer. Palliative care workers and clients with ALS/MND reported that rarity and rapid progression of the disease caused problems with coordinating care between numerous agencies. Inpatient palliative care reported higher resource inputs were needed. This was supported by the literature showing other patients receive less care when a client with ALS/MND was admitted because time and resources were redirected. The lack of appropriate respite accessible in a timely manner was highlighted by carers as a significant unmet need, causing considerable emotional, psychological and physical demands. The presence of after hours palliative care support while not widely used offered carers and clients peace of mind as the disease progressed.

Discussion and conclusions: The research highlighted the fear of palliative care staff in working with ALS/MND of which they had little knowledge or understanding. It further highlighted the misapprehension that once a person was admitted to palliative care that the palliative care agency had responsibility for all of the clients needs. The report recommended the development of a key worker model within palliative care services to promote early referral, support and deliver education and coordinate between service providers. A comprehensive education and support program was to be developed. Guidelines and a mechanism for supplementary funding to address identified high care and ongoing needs need to be developed. The respite needs and after hours palliative care support be the subject of policy considerations.

C63 WWW.ALSHOME.DE: WEB-BASED SELF-ASSESSMENT OF SYMPTOMS IN ALS

MAIER A, LINKE P, HOLM T, DULLINGER J, MÜNCH C, BORISOW N, BAHRKE R, JANZEN C, MEYER T

Charité, Department of Neurology, Berlin, Germany

E-mail address for correspondence: [email protected]

Keywords: web-based self-assessment, self-management, outpatient care

Background: Self-assessment of symptom progression and quality of life in chronic diseases is of increasing importance in clinical research and specialised outpatient healthcare. Against this background, we developed the Internet portal www.ALShome.de which provides online access to the ALS Functional Rating Scale, revised (ALSFRSr) and other established self-assessment questionnaires. In home care, an internet-based self-assessment is a possible perspective for the monitoring of ALS-associated symptoms.

Objective: To survey web-based self-assessment of ALS symptoms using the ALS Functional Rating Scale, revised (ALSFRSr) and other established self-assessment questionnaires.

Method: www.ALShome.de was created as a secure and closed Internet portal for patients. The application was developed in c-sharp (c#) with the persistent data storage being realized via an MS-SQL database throughout. Data are captured by generic questionnaires, are visualised on the website and administrated via a content management system. Patients are assigned a discretionary number of online visits. In a prospective, controlled and stratified study, patients conduct a self-assessment of ALS-associated symptoms. The study protocol designates 300 patients to visit the website weekly over a 52-week period.

Results: We have already included 62 patients (25 female, 37 male) in this study, 93.5 % (n = 58) of whom still perform weekly visits to the website filling in the ALSFRSr questionnaire nine weeks on. Reasons for discontinuation were the psychological strain perceived by patients when being confronted with the later stages of the disease (n = 2) and an excessive time burden caused by analogue dial-up Internet connections at a speed of less than 56 kbytes/s (n = 2).

Discussion: The web-based self-assessment of ALS symptoms in a home care environment complements the well-established application of the ALSFRSr in outpatient departments. The current study was able to demonstrate the medical, logistical and technical feasibility. The low drop out rate expresses the high acceptance of this online self-assessment tool among ALS patients. The study supports the hypothesis that innovative elements of self-management perspectively gain significant relevance in outpatient care.

Acknowledgements: The study is supported by the Air Berlin fund for ALS therapy research at the Charité.

C64 VOICE BANKING TO FACILITATE COMPLIANCE WITH SPEECH GENERATING AUGMENTATIVE COMMUNICATION

SINGLETON E^2,3, ROSENFELD J¹

¹UCSF-Fresno Neurology, Fresno, California, United States, ²Neuroscience Outpatient Rehabilitation Center, Fresno, California, United States, ³Central California Neuroscience Institute, Fresno, California, United States

E-mail address for correspondence: [email protected]

Keywords: voice-banking, natural voice, AAC-compliance

Background: Patients living with ALS/MND often experience a partial or total loss of speech and may require the use of a speech-generating device (SGD) to communicate. Despite the need and significance of augmemtative and alternative communication (AAC), compliance is variable and issues contributing to optimal compliance are not well understood. Selection of the designated voice used with a SGD may directly affect compliance.

Voice banking uses a patient's own pre-recorded, natural voice and may increase motivation and compliance for use of their SGD. Voice banking requires planning as recordings are made prior to loss of functional speech. Pre-recorded messages can then be programmed onto most available SGDs.

We will be presenting the organization and phases of our voice banking program and efforts to determine the role of voice banking in enhancing AAC compliance.

Objectives: 1) To introduce the voice banking program at the UCSF-Fresno, Central California Neuroscience Institute; 2) To determine the role of voice banking in enhancing compliance with the use of speech generating AAC devices.

Methods: Phase 1 of our program is focused on exploring issues that both enhance and impede compliance of patients who are currently using speech generating devices. Selective emphasis is placed on the role of the SGD voice. The patient's satisfaction with that voice and its impact on compliance are evaluated by written questionnaire administered to both the patient and the caregiver. Phase 2 involves assessment of voice banking on improving AAC compliance. Compliance and satisfaction are initially measured in patients receiving speech generating AAC devices utilizing commercially available voices. This population of patients will also have pre-recorded (banked) their natural voice prior to onset of their dysarthria. The banked voice will then be replaced in their SGD and the patient's compliance and satisfaction are reassessed serially.

Results: We will present the results of our phase 1 and early phase 2 data from this ongoing longitudinal project. Data highlighting issues impacting compliance in our population will be presented. Methodology and early results on the logistics of voice banking in phase 2 will also be presented.

Conclusion: Our research program in voice banking at UCSF-Fresno, Central California Neuroscience Institute is an ongoing, multi-phase program designed to determine the role of voice banking in enhancing compliance with AAC. This presentation is the first phase of this novel ongoing initiative. The results of this program will identify key areas impacting compliance and the logistics of implementing a voice banking program at other institutions.

C65 AMYOTROPHIC LATERAL SCLEROSIS PATIENTS’ SELF-REPORTED SATISFACTION WITH ASSISTIVE TECHNOLOGY

GRUIS K¹, WREN P², HUGGINS J¹

¹University of Michigan, Ann Arbor, MI, United States, ²Oakland University, Rochester, MI, United States

E-mail address for correspondence: [email protected]

Keywords: rehabilitation, survey, assistive technology

Background: Clinical management recommendations for amyotrophic lateral sclerosis (ALS) patients with physical impairments include medical provider assessment and prescription of assistive devices to improve patients’ function, maintain independence, and decrease fatigue. However, there is little information about ALS patient reported usefulness of, and satisfaction with commonly prescribed assistive devices.

Objective: We assessed ALS patients’ self-reported satisfaction with thirty-three assistive devices using a telephone survey.

Methods: Ninety-six ALS patients were identified from our multidisciplinary clinic, and 63 were available via phone. A structured, telephone survey of those patients available by phone was conducted to assess demographics, ALS functional rating scale-revised (ALS-FRS-R), and assistive technology. Assistive technology frequency of use, perceived usefulness, and satisfaction with 33 devices was performed. Frequency of use was recorded on a scale of 1 – 5 (1=never, 2=rarely, 3=sometimes, 4=often, and 5=always) and designated as ‘high use’ if scored as often or always by ≥ 20% respondents. Usefulness and satisfaction were recorded on a 1 to 10 scale where 1, 2=very low; 3, 4=low; 5, 6=medium; 7, 8=high; and 9, 10=very high. Frequencies and percentages or medians and interquartile ranges (IQR) were calculated.

Results: Survey respondents median age was 62 years (52, 72); 37 (59%) were male and 52 (83%) reported limb-onset symptoms. The median duration between the diagnosis and survey was 26 months (17, 50), and the median ALS-FRS-R score was 25 (18, 33). Sixteen (48%) of the devices were designated high use. Of these, slip-on shoes and all bathroom devices received a very high rating for both usefulness and satisfaction; ankle braces and transfer boards were rated very high for usefulness with high satisfaction; speaker phones and electronic seating controls were rated high for both usefulness and satisfaction. Although walkers, motorized wheelchairs, personal digital assistants, and laptop computers were also high use and were given a high usefulness rating, they only received a medium satisfaction rating. The button hook and dressing stick received low ratings, and the long-handled reaching tool received very low ratings for both usefulness and satisfaction, despite being designated high use. Electronic speaking devices were used by only eight (13%) of ALS respondents, while writing on paper was used by forty-three (68%). Interestingly, both of these communication devices received the same, medium, rating for both usefulness and satisfaction.

Conclusions: This cross-sectional telephone survey of a cohort of ALS patients found that bathroom adaptive devices were uniformly the most frequently used and highly rated assistive devices for both how well the devices worked and satisfaction. Additionally, of those assistive technologies used often or always by ≥ 20% of respondents, the ankle brace, transfer board, slip-on shoes, speaker phone, and electronic seating controls were ranked highly for both usefulness and satisfaction.

C66 “THE DOCTOR SAID MND WOULDN'T AFFECT MY SEXUALITY … BUT IT HAS”

TAYLOR B

Oxford Brookes University, Oxford, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: sexuality, sexual expression, relationships

Background: Sexuality is a holistic concept that is individual and integral to each person throughout his or her lifetime (1). It influences how we perceive ourselves and is expressed in a variety of ways, including the way we dress, through touch, roles and relationships (1,2). Apart from studies on sexual function (3), little is known about the impact of MND on sexuality.

Objectives: This qualitative study explores the lived experiences of patients and partners of patients living with MND in the UK.

Method: One to one interviews were conducted with 13 patients and 10 partners of patients at different stages along the disease trajectory. Each person was interviewed twice and couples were interviewed separately. It was not a requirement of the study that people were in a partnered relationship.

Results: MND does have an impact upon peoples’ sexuality. It causes changes in peoples’ physical appearance and affects their self-concept and self-esteem. Participants have described how MND affects their self-confidence, and impacts upon their intimate and sexual relationships in a variety of ways, and also raises concerns about fertility. Health care professionals rarely speak about these issues, leaving individuals to find their own ways of managing change.

Discussion: Sexuality is an important aspect of peoples’ lives that means different things to different people. When MND affects peoples’ sexuality, some people are able to adapt and change, whilst others experience significant loss.

Conclusion: There is more to sexuality than erectile function. Telling people that their sexuality will not be affected does not recognise the psychosocial effects of MND or the practical and emotional interplay that is involved in sexual relationships. Recommendations are made for how this could be managed in healthcare practice.

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C67 THE NATION'S FIRST ALS GREEN HOUSE^® NURSING HOME RESIDENCE: A TECHNOLOGICAL APPROACH TO CARE

BERMAN B, KRAKOW M, SALING S

Chelsea Jewish Foundation, Chelsea, MA, United States

E-mail address for correspondence: [email protected]

Keywords: technology, nursing care

Background: The Leonard Florence Center for Living (LFCL) in Chelsea Massachusetts opened the first-in-the-nation ALS Residence in February 2010. The ALS Residence is part of the LFCL Green House® nursing home, a model which deinstitutionalizes care while providing skilled nursing care in a home environment. In the ALS Residence, ten residents have their own private bedrooms and bathrooms, and share a common living, dining and kitchen area. Universal care workers and clinical staff provide care and services on an as needed basis, and there are no institutional trappings. This fully automated house uses state-of-the-art technology to allow wheelchair-dependent individuals with little or no mobility to live their lives fully – open and close shades and doors, control lighting and heat/air conditioning, control audio-visual components, and communicate with others, all possible with hand or eye movement technology. The ALS Residence has a full ventilator program, available when needed.

Objectives: 1) Develop an ALS Residence to provide care in a home environment. 2) Develop technology that will be integrated throughout the building, allowing residents to maintain and/or regain independence. 3) Provide care to ten residents at a time, refining nuances of care in the first ALS Residence. 4) Develop a model that can be replicated in other areas of the country/world.

Methods: A landscape architect who has ALS worked with architects, contractors and engineers on all design elements of the building to create the most technological advanced ALS Residence possible, with the goal of providing independence and mobility to those living with ALS. This same individual consulted with the clinical care staff on all aspects of providing clinical care. The ALS Association Massachusetts Chapter provided ongoing consultation and input into the project.

Results: The first residents moved into the ALS Residence in late February 2010. Qualitative information indicates that residents are seizing control of their lives. For the first time in years, residents are able to open doors for themselves, turn lights on and off, and control the environment in their bedrooms and homes. This mobility and independence is promoting interaction among those living in the ALS Residence as well as among the ALS patients and those within the LFCL. We have received inquiries from all over the country requesting information about and/or admission into the ALS Residence.

Discussion and conclusions: The ALS Residence provides residents with unprecedented mobility, independence and dignity, providing a technological cure for ALS while we wait for the medical cure. This model can be replicated in other areas of the country, providing similar independence to others. Consultation with local clinics serving ALS patients as well as with the local and national chapters of the ALS Association will continue as we plan for project replication.

SESSION 9C CLINICAL TRIALS

C68 MORE POWER TO YOU: IMPACT OF TRIAL DESIGN ON ADHERENCE AND OUTCOME

CUDKOWICZ M

Massachusetts General Hospital Harvard Medical School, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: trial conduct, compliance, retention

Study conduct can greatly impact interpretability of trial results and the efficiency of developing new treatments for ALS. Study conduct includes adherence to the treatment intervention (medications, exercise, etc), study visits and study procedures. Using data from published clinical trials and trials conducting by the Northeast ALS consortium, adherence and study retention in ALS clinical trials will be explored. The goal is to learn from prior studies and identify modifiable factors in how studies are designed and conducted that can expedite therapy development for ALS.

C69 MODIFIABLE BARRIERS TO ENROLMENT IN AMERICAN ALS RESEARCH STUDIES

BEDLACK R¹, WICKS P², HEYWOOD J², KASARSKIS E³

¹Duke University, Durham, NC, United States, ²Patients Like Me, Cambridge, MA, United States, ³University of Kentucky, Lexington, KY, United States

E-mail address for correspondence: [email protected]

Keywords: enrolment, recruitment, advocacy

Background: A surprisingly small percentage of patients with ALS (PALS) enrol in research studies. Analysis of previously published ALS trials identified no obvious ‘trial factors’ that influence enrolment. ‘Doctor factors’ and ‘patient factors’ may play more prominent roles, as seen in other fields. Indeed, a small survey of North American ALS Clinic directors identified several physician behaviors that might be modified to improve enrolment.

Objective: To identify specific patient factors responsible for low enrolment in research studies.

Methods: Two online patient surveys and a cost analysis of previously published ALS studies.

Results: Survey responders were predominantly educated white males. Most got information about research studies from the Muscular Dystrophy Association or the ALS Association websites and support groups. Half the responders did not recall being invited to participate in a study; of those who were asked, 75% agreed to participate. The top reason for participation was to help others. Among those who participated in a study, 70% said they were never asked to do so again. Responders were confused about multiple aspects of studies, including a need to donate blood or spinal fluid more than once, and the amount of out-of-pocket cost that would be incurred. The main reason for declining participation was concern about physical and financial burdens. Indeed, our cost analysis suggested that these can be substantial; between 1992-2006, PALS made a total of 1,093,655 study visits, contributed 6.6 million hours of effort to published studies and faced a gasoline cost alone of $5,401,317. Limitations of our work include the small number of responders and inability to validate their diagnoses.

Discussion and conclusions: The above research has identified potentially modifiable patient factors which may influence enrolment in ALS research studies. These include failure to know about open studies, confusion over study methodologies and goals, and concerns about physical and financial burdens. The ALS Research Group (ALSRG) has several projects underway to try and affect these, including development of educational slides for use at support group talks, an ALS Clinical Research Learning Institute to empower a small group of PALS and caregivers to be research educators and advocates, an online trial registry similar to the Army of Women being used to invite large numbers of patients and healthy controls into new breast cancer studies, a video to standardize study presentations to patients, ALSUntangled to investigate alternative and off-label options, expansion of ALS Clinics offering research into more rural areas, and development of home based outcome measures.

C70 RESULTS OF A CLINICAL TRIAL OF TALAMPANEL IN PATIENTS WITH ALS

SHEFNER J¹, MEININGER V², THE TALAMPANEL ALS STUDY GROUP

¹SUNY Upstate Medical University, Syracuse, NY, United States, ²Hôpital de la Salpétrière, Paris, France

E-mail address for correspondence: [email protected]

Keywords: clinical trial, excitotoxicity, Talampanel

Background: Many studies have implicated glutamate mediated excitotoxicity as an important mechanism of cell death in ALS, and have suggested this pathway as a potential target for altering disease course. Evidence suggests that riluzole works in part by inhibiting glutamate release. Talampanel, a selective ampa receptor antagonist, showed promise in a phase II trial of 60 patients with ALS.

Objectives: To determine whether Talampanel is efficacious and safe in patients with ALS.

Methods: 559 patients with ALS were randomized to receive either placebo, 75 mg, or 150 mg daily. The primary outcome measure was change from baseline of the ALSFRS-R. Sample size was selected to provide 80% power to detect 20% decrease in deterioration slope. Secondary and exploratory outcome measures included survival, change in rate of decline of strength measured by manual muscle testing, and change in rate of decline of vital capacity. Multiple safety measures were also obtained.

Results: There were no significant differences in patient characteristics at baseline among the three groups. Subjects entered the study at a mean age of 55.9 years; 65% of subjects were male. 16.1% of subjects had bulbar onset; mean time from first symptom to study entry was 18.7 months. 83% of subjects were on riluzole while entering the study. The change in ALSFRS-R after 12 months of treatment was −12.4 pts in the placebo group, −13.1 points in the 75 mg/d group, and −12.6 pts in the 150 mg/d group. These differences were not statistically significant. Similarly, there were no differences in survival, strength, or pulmonary function between groups. While patients receiving active treatment had more adverse events than those receiving placebo, dropout rate was similar across groups; 28.6% dropout in placebo, 31.2% in the 75 mg/d group, and 30.6% in the 150 mg/d group. Dizziness, somnolence, and ataxia were noted more frequently in the active treatment groups in a dose related manner.

Discussion and conclusions: Talampanel was not effective in altering disease progression in this trial. Given the neurological nature of the adverse events, as well as evidence from various animal models, it is apparent that the drug did in fact reach CNS targets. This study does not suggest that the glutamate pathway is a poor target for new therapies; however, pure ampa antagonists are unlikely to be of benefit.

C71 PHASE II STUDY OF PIOGLITAZONE IN ALS

LUDOLPH AC, KASSUBEK J, MEYER T, THE GERMAN ALS STUDY GROUP*

University of Ulm, Ulm, Germany

E-mail address for correspondence: [email protected]

Keywords: study biological markers

Background: Because of independent positive results in ALS preclinical models, pioglitazone is seen as a potential candidate for human ALS therapy. We performed a double-blind placebo-controlled prospective study of pioglitazone as add-on therapy to standard therapy with riluzole in patients with ALS.

Methods: A total of 219 ALS patients were enrolled across 15 ALS centres, according to the revised El Escorial criteria (possible, probable (clinical or lab-supported) or definite ALS). Participants received pioglitazone (45mg/day) or placebo for 18 months. In addition, to commonly used primary and secondary outcome measures (survival, ALS FRS/R, quality of life) we measured pioglitazone blood levels and the effects of pioglitazone on cytokines and parameters of lipid metabolism, including quantitative MRI measurements, in a subgroup of participants.

Results: Interim analysis by the Data Monitoring Committee (DMC) determined the death rate in the pioglitazone group 27/109 (24.8%) compared with 18/110 (16.4%) in the placebo group. At the interim analysis point, 46 patients had completed the 18 month treatment period. In light of the interim analysis, it was considered extremely unlikely that the study would demonstrate significant therapeutic benefit of pioglitazone after the 18 month treatment period in the remaining trial participants.

Discussion: The study revealed that the application of pioglitazone to ALS-patients is safe. However, the results of this phase II study strongly suggest that pioglitazone is ineffective in slowing down the course of clinical ALS. We are presently performing detailed analyses of the data, including biological markers.

*AC Ludolph, Ulm; J Kassubek, Ulm; T Meyer, Berlin; T Grehl, Bochum; M Heneka, Bonn; A Storch, Dresden; J Weishaupt, Göttingen; S Zierz, Halle/Saale; S Petri, R Dengler, Hannover; GD Borasio, München; P Young, Münster; N Weidner, U Bogdahn, Regensburg; M Wittstock, R Benecke, Rostock; B Schrank, Wiesbaden; C Wessig, KV Toyka, Würzburg; J Grosskreutz, Jena

SESSION 10A GLIAL CELL BIOLOGY

C72 MECHANISMS AND THERAPY IN ALS: GLIA AS DETERMINANTS OF DISEASE PROGRESSION

CLEVELAND D

Ludwig Institute for Cancer Research, La Jolla, CA, United States, University of California, San Diego, La Jolla, CA, United States

E-mail address for correspondence: [email protected]

Since its description by Charcot in 1869, the mechanism of selective death of motor neurons in Amyotrophic Lateral Sclerosis (ALS) has remained elusive. An inherited form caused by mutation in superoxide dismutase (SOD1) triggers disease through an acquired toxicity unrelated to dismutase activity. Using mice carrying a deletable mutant gene or viral encoded siRNA to diminish mutant expression within motor neurons, disease onset is slowed but progression is not. Microhemmorages stemming from damage to the vasculature occur presymptomatically, albeit use of selective gene excision demonstrates this does not arise from mutant SOD1 acting within the endothelial cells of the capillaries. Reducing mutant SOD1 synthesis in two glial cell types, astrocytes or microglia, has little effect on disease onset, but strikingly slows disease progression. Reducing mutant synthesis in Schwann cells implicates a cascade of oxidative damage from the underlying axon to the Schwann cell as a component driving disease progression. Thus, toxicity is non-cell autonomous, with mutant SOD1 acting within motor neurons driving disease onset, while damage within neighboring glial (astrocytes and microglia) accelerates disease progression.

These findings validate therapies to slow disease progression that target astrocytes or microglia, including stem cell replacement or gene silencing approaches. One cell replacement therapy now underway is through injection of stem cell derived astrocytic progenitors. With discovery that peripherally administered Adeno Associated virus (AAV9) effectively transduces astrocytes after crossing the blood brain barrier, a final therapeutic approach now under development is use of AAV9-encoded shRNA to diminish mutant synthesis SOD1 within astrocytes.

Another third for targeting glial-driven SOD1-mediated ALS stems from administration after disease onset of Activated Protein C (APC), a signaling protease with anticoagulant and cytoprotective activities and which in the United States is already an FDA approved drug. APC is shown to cross the blood-spinal cord barrier where it activates Protease Activated Receptors 1 and 3 (PAR1 and PAR3) on the cell surfaces of motor neurons and microglia, thereby triggering transcriptional repression of SOD1 by inhibiting the Sp1 transcription factor. Delayed disease progression after APC administration supports its use as a therapy in ALS.

A final therapeutic approach targeted to glial cells exploits continuous infusion to deliver antisense oligonucleotides (ASOs). Infusion of such ASOs is shown to produce widespread gene silencing throughout the nervous system. Such ASOs catalytically target the mRNAs to which they hybridize for destruction by an endogenous, nuclear enzyme RNase H. Infusion of such an ASO to SOD1 slows SOD1 mutant-mediated disease progression, doubling disease duration. A clinical trial has now been initiated to test such ASO infusion as a treatment for SOD1-mediated inherited ALS.

C73 GENE EXPRESSION PROFILING OF ASTROCYTES IN PRE-SYMPTOMATIC G93A SOD1 MICE REVEALS DYSFUNCTION IN THE ASTROCYTE-MOTOR NEURONE LACTATE SHUTTLE

FERRAIUOLO L, HIGGINBOTTOM A, HEATH P, GREENALD D, KIRBY J, SHAW P

University of Sheffield, Sheffield, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: microarray, motor neurons, astrocytes

Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive death of upper and lower motor neurons (MN). Experiments on mice expressing mutant forms of the superoxide dismutase 1 (SOD1) enzyme have indicated that the development of the pathology requires interaction between MN and their non-neuronal neighbour, mainly astrocytes (1-2). Astrocytes play an important role in ALS, but the way in which the expression of mutant SOD1 in vivo alters their properties and their precise role in the generation of MN injury remains to be clarified.

Objectives: 1) To examine expression profile changes occurring within astrocytes in the spinal cord of G93ASOD1 mice at the presymptomatic stage of disease (60 days); 2) To identify pathways involved in the development of the neurodegenerative process at 60 days; 3) To validate with functional assays the involvement in the neurodegenerative process of the pathways identified through microarray analysis.

Methods: 1500 astrocytes were isolated using laser capture microdissection (LCM) from lumbar spinal cord sections stained with the astrocyte specific marker aldehyde dehydrogenase L1l (AldhL1l) (3). RNA was extracted, amplified and labelled and 15 μg cRNA was applied to the Affy Mouse 430 2.0 GeneChip. Transcription profiles of astrocytes from 3 60 day G93ASOD1 mice and 3 non-transgenic littermates were compared using ArrayAssist System 4.0 (Iobion). A threshold of fold change ≥ 2 plus P value ≤ 0.05 was used to identify differentially expressed genes.

Results: Initial comparison of the expression profile of astrocytes from 60 day G93ASOD1 mice and non-transgenic littermates showed a significant change in expression of 1109 genes. 526 transcripts are downregulated, and 583 are upregulated. The genes have been categorised according to their molecular function, and include genes involved in the transcription process, apoptosis, inflammation and metabolism. G93ASOD1 astrocytes show an increase in transcripts encoding for Ngf and several chemokines. One of the main features is an overall downregulation in genes involved in the carbohydrate metabolism and in particular the monocarboxylate transporter 5 (Mct5), in contrast to upregulation in the main genes involved in fatty acid synthesis and activation. Several transcripts involved in the WNT signalling cascade are also differentially expressed. This pathway is known to stimulate cell proliferation and cytoskeletal changes.

Discussion: Previous data obtained from the gene expression profile of MN isolated from SOD1G93A mice suggest that dysregulation of carbohydrate metabolism is one of the main features of the early stages of disease. Ngf upregulation has already been reported as potentially toxic for mature MN (4). We aim to investigate further the reported dysregulation as potential mechanism leading to motoneuronal damage.

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C74 COMPREHENSIVE GENETIC ANALYSIS OF ADULT REACTIVE ASTROCYTES IN RODENT MODEL (SOD1 G93A) OF ALS REVEALS MASSIVE MOLECULAR REORGANIZATION IN VIVO

YANG Y¹, ROTHSTEIN J²

¹Department of Neuroscience, Tufts University School of Medicine, Boston, MA, United States, ²Johns Hopkins University School of Medicine, Baltimore, MD, United States

E-mail address for correspondence: [email protected]

Keywords: RNA transcriptiome, reactive astrocyte, ALDH1L1

Background: Involvement of non-cell autonomous mechanisms has been demonstrated in the pathogenesis of ALS. In particular, astrocytes have been shown to significantly affect the disease progression, possibly by promoting the excitotoxicity through reduced glutamate uptake, altered metabolic support of neurons, and by secreting toxic factors that induces motor neuron death. However, the in vivo molecular mechanisms of astroglial dysfunction remain largely elusive, as embryonic/cultured astrocytes poorly reflect in vivo biology and until recently no reliable, accurate method was available to study in vivo astroglia.

Objective: This study aims to investigate the molecular changes in adult astrocytes, in vivo, during disease progression for a better understanding of the mechanisms/pathways of reactive astrocytes that may affect the pathogenesis/progression of ALS in rodent models.

Methods: Whole genome RNA transcriptome analysis was performed to investigate the molecular changes of astrocytes in diseases. Fluorescent activated cell sorting (FACS) and translating ribosome affinity purification approach (TRAP) approaches were employed to isolate the mRNA from astrocytes. For FACS approach, astroglial reporter mice GFAP eGFP or BAC ALDH1L1 eGFP were employed that selectively label astrocytes in vivo. Spinal cord astrocytes were isolated by FACS approach from GFAP eGFP X SOD1 G93A or BAC ALDH1L1 eGFP X SOD1 G93A mice at different ages (pre-symptomatic, symptomatic, and end stage). Total RNA was further isolated from these astrocytes and hybridized with mouse exon array for RNA transcriptome analysis. For TRAP approach, ribosomes of spinal cord astrocytes will be selectively labeled by an eGFP tag which allows specific pull-down of not only ribosomes but also the translating mRNA that binds to the ribosomes in astrocytes of BAC ALDH1L1 TRAP and BAC GFAP TRAP mice. This provides an alternative approach that purifies translating mRNA from spinal cord astrocytes without FAC sorting. These mice will be crossed with SOD1 G93A mice. Ribosomes and translating mRNAs will be isolated from BAC ALDH1L1 TRAP X SOD1 G93A or BAC GFAP TRAP X SOD1 G93A mice at different age (pre-symptomatic, symptomatic, and end stage), and will also be subject to the RNA transcriptome analysis.

Results: Various astroglial reporter and TRAP mice were characterized. We found that the newly identified astroglial marker ALDH1L1 expression (mRNA, protein, and promoter activity) is up-regulated in reactive astrocytes in SOD1 G93A condition. Initial RNA transcriptome analysis from the end-stage spinal cord indicates that expression levels of oligodendrocyte markers, including MAG, MOG, MOBP, MBP, and PLP are strongly induced in cells sorted from GFAP eGFP X SOD1 G93A mice.

Discussion and conclusions: Our initial results from RNA transcriptome analysis unexpectedly suggest that reactive astrocytes may lose their maturation identity. RNA transcriptome from other stages are ongoing for the overall gene expression change in astrocytes in SOD1 G93A spinal cord.

C75 MONOCARBOXYLIC TRANSPORTER (MCT1) BAC REPORTER AND OVER EXPRESSER MICE AS TOOLS TO REVEAL NOVEL GLIAL PATHOGENESIS IN ALS

LEE Y, JIN L, ROTHSTEIN J

Johns Hopkins University, Baltimore, MD, United States

E-mail address for correspondence: [email protected]

Keywords: astroglia, oligodendroglia, metabolism

Background: Monocarboxylate transporter (MCT) is the SLC16A family of membrane proteins that transport monocarboxylates such as lactate, pyruvate, and ketone bodies. Molecular MCT subtypes have unique tissue and cellular distribution with different kinetic properties. MCT-1 expressed in astrocytes has lower affinity for lactate than MCT-2 expressed in neurons, supporting the lactate shuttle hypothesis, in which lactate released from astrocytes is taken up by neurons as an energy substrate. However, there is no clear evidence which cell type of the central nervous system (CNS) expresses each MCT transporter in vivo. CNS energy demand is higher than other organs. Therefore, alteration of energy metabolism might be detrimental in many neurological diseases including amyotrophic lateral sclerosis (ALS). In preliminary experiments, MCT-1 expression is markedly reduced in ALS patients, and end stage of superoxide dismutase 1 (SOD-1) and mutant TDP-43ALS mouse models.

Objectives: We aimed at generating MCT-1 bacterial artificial chromosome (BAC) transgenic mouse and MCT-1 overexpression mouse models to better understand its expression and functions during the pathogenesis of ALS by cross-breeding with SOD-1 mice.

Methods: Mouse BAC RP23-208N5, containing the entire MCT-1 gene plus 50 kb upstream of the first exon and 132.2 kb downstream of the last exon, was modified using a double homologous recombination with the SV-RecA shuttle vector to insert a reporter gene, tdTomato. MCT-1 cDNA was inserted into human glial fibrillary acidic protein promoter (GFAP) driven internal ribosomal entry site (IRES) and EGFP sequences for its expression in astrocytes. The final modified MCT-1 BAC and GFAP-MCT-1-IRES-EGFP constructs were injected into mouse pronuclei for generation of transgenic mice.

Results: Surprisingly, tdTomato expresses predominantly in oligodendrocytes and specific type of neurons in brain and exclusively in oligodendrocytes in spinal cord. In addition, it expresses in blood, muscle, retina, lung, heart, liver, pancreas, spleen, stomach, intestine, colon, and kidney. Overexpression of MCT-1 was shown along with EGFP expression in astrocytes in the CNS. Interestingly, the most strong expression line showed clearly obese phenotype as well as aggressive behavior. The other lines have same phenotype with variable level depending on MCT-1 expression.

Discussion and conclusions: Unexpected discovery of MCT-1 expression in oligodendrocytes introduces new metabolic coupling among astrocytes, oligodendrocytes, and neurons in the CNS. Although the rate of lactate utilization in cultured oligodendrocytes was much higher than astrocytes and neurons, lactate transport in oligodendrocytes has not been studied in vivo. Our finding in mice shows clearly the existence of MCT-1 in oligodendrocytes, suggesting the possible effects of oligodendrocytes on ALS pathogenesis by alteration of MCT-1 expression.

C76 FOCAL IN VIVO TRANSPLANTATION OF MUTANT SOD1 GRP-DERIVED ASTROCYTES INFLUENCES WILDTYPE MOTOR NEURON VULNERABILITY

PAPADEAS S, KRAIG S, O'BANION C, LEPORE A, MARAGAKIS N

Johns Hopkins University School of Medicine, Baltimore, MD, United States

E-mail address for correspondence: [email protected]

Keywords: glia, stem cells, cell autonomy

Background: Evidence from in vitro studies, transgenic mutant (mSOD1) rodents, chimeric mouse models, and conditional CRE-LOX systems have identified astrocytes as key drivers of motor neuron (MN) degeneration and disease progression in ALS and has underscored the importance of astrocyte-specific disease mechanisms in ALS. However, a major shortcoming using in vivo rodent models is that mSOD1 is expressed in all cell types and is anatomically ubiquitous. As a result, these animals display a rapidly progressive disease that requires extrapolation of relevant pathways on animal survival measured in days or weeks. Furthermore, the specific in vivo influences of mSOD1 astrocytes on wildtype motor neurons, astrocytes, and microglia have not been elucidated.

Objectives: By transplanting mSOD1 glial restricted precursors (GRPs) focally into defined wildtype rat spinal cord levels, we studied regional influences of mSOD1 astrocytes on other wildtype (WT) cell types (including WT motor neurons, astrocytes, and microglia) and the role of mSOD1 astrocytes in disease progression and anatomical spread.

Methods: GRPs overexpressing human mSOD1^G93A, wildtype (WT GRPs), human wildtype SOD1 (SOD^WT), dead cells, and media were transplanted in cervical spinal cord ventral gray matter in 90 day old WT rats and then followed in vivo for up to 3 months. The influence of mSOD1 on GRP survival, differentiation, anatomical localization, and migration were assessed.

Spatial interaction of transplanted mSOD1^G93A GRP-derived astrocytes with host astrocytes and motor neurons (MNs) were examined, as well as pathohistological outcomes of mSOD1^G93A GRP transplantation, including host astrocyte and MN survival, ubiquitin aggregation, host astrogliosis and microglial activation.

To assess behavioural influences of mSOD1^G93A GRP-derived astrocytes, forelimb and hindlimb strength was assessed as we have described previously. We examined electrophysiological outcome by measuring phrenic compound muscle action potentials (CMAPs) following phrenic nerve stimulation.

Results: Animals were analyzed at 1 and 3 months following transplantation. We determined that mSOD1^G93A does not influences astrocyte survival, migration, or proliferation but does influence the capacity for astrocyte differentiation. SOD1^G93A GRP-derived astrocytes, but not WT or SOD^WT GRP-derived astrocytes, induced ubiquitin inclusions and death in host WT motor neurons. This was accompanied by ventral horn microgliosis. Interestingly, transplantation of SOD1^G93A GRPs was sufficient to result in focal forelimb weakness and electrophysiological declines in phrenic nerve CMAPs which declined slowly over time and then become static.

Discussion and conclusions: These findings suggest that the presence of mutant SOD1 in astrocytes has influences on astrocyte differentiation and also induces wildtype motor neuron pathology, motor neuron cell death, and a decline in behavioral and electrophysiological functions over a period of months which subsequently becomes static. These pathological findings are accompanied by microgliosis. Taken together, these data provide in vivo evidence that mSOD1^G93A astrocytes alone are sufficient to induce motor neuron vulnerability and functional motor decline.

SESSION 10B HOLISTIC CARE

C77 PRACTICAL IMPLICATIONS OF GENETICS IN ALS FOR PATIENTS AND FAMILIES

SIDDIQUE N

Northwestern University, Feinberg School of Medicine, Chicago, IL, United States

E-mail address for correspondence: [email protected]

Keywords: genetic testing, genetic counselling, inherited ALS

About 10% of ALS is known to be inherited, while the remaining 90% occurs as a single case within a family (sporadic ALS). Causative genes, including SOD1, TDP-43, FUS-TLS and others, have been identified for roughly 30% of inherited cases. Several genes, including the PON cluster, have also been associated with sporadic ALS. This explosion of information has made it difficult for both health care providers and families to determine its relevance for a given family. This session will review very basic genetics, delineation of sporadic and familial disease, genes currently known to cause ALS, genes associated with ALS. There will be recommendations for circumstances in which to do genetic testing and detailed discussion of implications for genetic testing in both patients and family members.

C78 TELEMEDICINE TO FACILITATE ALS RESEARCH

GRONKA S¹, WALKER M¹, WUU J¹, STANISLAW C¹, FANOS J¹, ATASSI N², JAFFA M², ANDERSEN P³, CUDKOWICZ M², BENATAR M¹

¹Emory University, Atlanta, GA, United States, ²Massachusetts General Hospital, Boston, MA, United States, ³Umea University, Umea, Sweden

E-mail address for correspondence: [email protected]

Keywords: telemedicine, remote evaluations, familial ALS

Background: Telemedicine, derived from the Greek ‘tele’ (at a distance) and the Latin ‘medicine’ (healing), refers to the use of telecommunication and other methods to perform remote medical evaluations. Telemedicine has the potential to enhance clinical research in a rare disease such as ALS and an ultra-rare disease such as familial ALS. It may improve recruitment by reducing the burden of travel for study participants and expanding the geographic scope and population from which participants may be recruited. It may also reduce costs.

Methods: Genetic counseling data were derived from the Pre-familial ALS (Pre-fALS) study, which enrolls healthy individuals at genetic risk for fALS. Participants who elect to undergo genetic testing with disclosure of results were randomized to receive pre- and post-test counseling either via telephone (TL) or in-person (IP). Respiratory muscle function is evaluated and blood collected remotely in the Arimoclomol trial, which enrolls affected individuals with fALS. Respiratory muscle function is evaluated using both forced expiratory volume in 6 seconds (FEV6), which can easily be measured in the home, and slow vital capacity (SVC), which is measured in the clinic. Blood for safety laboratory evaluations was collected in homes of Arimoclomol patients and analyzed in a central laboratory with results communicated via web and fax to the study center.

Results: Of the first 30 Pre-fALS participants who have elected to learn their genetic results, 15 have been randomized to each counseling group. Five TL and 3 IP subjects were SOD1+; the others were SOD1-. Fourteen subjects (3 SOD1+ and 4 SOD1- from each counseling group) were randomly selected for semi-structured interviews to formally assess TL and IP counseling. Among these 14 subjects, only 3 (2 TL and 1 IP) expressed preference to have received the other counseling modality. Regardless of positive or negative results, participants reported that they had adapted well to receiving results. Sixteen Arimoclomol patients yielded FEV6 and SVC data from 27 visits. Reproducibility of SVC and FEV6 were comparable with intraclass correlation coefficients of 0.98-0.99. There was high correlation between SVC and FEV6 expressed in liters (r=0.97, P <0.001) and percent predicted (r = 0.92, P < 0.001). In addition, all 64 remote blood collections in the homes of the 16 patients occurred within the designated visit window with no failed/missed collections.

Discussion: Pre-symptomatic genetic counseling was safely and effectively performed via telephone. Respiratory muscle function was reliably measured and blood was collected for safety laboratory tests, all without the need for participants to leave home. The use of these remote evaluations as part of a telemedicine approach has greatly facilitated our ability to undertake an observational study in people at risk for fALS and a clinical trial in patients already affected by fALS.

C79 IDENTIFYING THE NEEDS OF THE ALS/FTD CAREGIVER: DEVELOPMENT, IMPLEMENTATION, AND EVALUATION OF A CLINICAL ASSESSMENT INSTRUMENT

BROTHERS A¹, WALSH S², LYTER J², HOUSEMAN G³, KLAPPER J³, SIMMONS Z¹

¹Penn State College of Medicine/Hershey Medical Center, Hershey, PA, United States, ²ALS Association, Greater Philadelphia Chapter, Hershey, PA, United States, ³ALS Association, Greater Philadelphia Chapter, Philadelphia, PA, United States

E-mail address for correspondence: [email protected]

Keywords: frontotemporal dementia, caregiver needs, caregiver assessment

Background: Despite estimates that cognitive-behavioral change occurs in up to half of people with ALS, there are currently no guidelines for the clinical management of this subset of patients. ALS with frontotemporal dementia (ALS/FTD) is accompanied by unique clinical concerns, and the services provided to caregivers will require individualization. In order for ALS treatment teams to tailor their interventions accordingly, it will be important to accurately assess caregivers’ needs.

Objectives: 1) Understand the needs of family caregivers of patients with ALS/FTD; 2) Develop a questionnaire to identify caregiver concerns related to cognitive-behavioral changes; 3) Evaluate the assessment instrument for ease of completion and perceived usefulness.

Methods: An evidence-based practice approach was used to develop an ALS/FTD caregiver assessment instrument using three types of input: 1) comprehensive literature review; 2) expert clinician opinion from members of a multidisciplinary ALS clinic team; 3) ALS/FTD caregiver opinion through structured telephone interviews with six family caregivers. A process evaluation took place to gather caregivers’ feedback regarding the assessment instrument. Open-ended responses from the caregiver questionnaire and the process evaluation were qualitatively analyzed using a thematic analysis approach. This study was approved by the Penn State Hershey Medical Center Institutional Review Board.

Results: The assessment instrument consisted of 12 questions regarding patient safety concerns, financial and legal matters, and caregiver emotional well-being. Eight caregivers of patients with ALS/FTD completed the assessment. There were 4 men and 4 women, all spouses. All reported patient safety concerns, most commonly ‘Ability to find way home if lost’ (6 caregivers); ‘Risk of falling’ (6); ‘Taking medications as prescribed’ (5); and ‘Ability to be left alone or unsupervised’ (5). Half of the caregivers reported concerns related to the patient's financial behaviors. Six reported that the patient had designated a healthcare power of attorney, and five noted that an advance directive had been completed. Half reported feeling at least moderately burdened. Caregivers reported no difficulty in completing the questionnaire; 100% indicated that they thought the care they received from the ALS team would improve as a result of completing the questionnaire. The most commonly repeated qualitative themes related to caring for someone with ALS/FTD included a) sleep difficulties (patient and caregiver); b) desire for candid information about prognosis; and c) the difficulty of managing the ALS along with FTD symptoms.

Discussion and conclusions: Despite a small sample size, preliminary analyses show the ALS/FTD caregiver assessment to be a useful clinical instrument. It will be revised according to caregiver feedback, and implemented in the ALS Clinic for continued data collection. The results of this study will be used to design a psycho-educational intervention session for caregivers, as well as to develop a repertoire of individualized interventions for caregivers of patients with ALS/FTD.

C80 IDENTIFYING NEEDS AND INTERVENTIONS TO SUPPORT CAREGIVERS OF PATIENTS WITH ALS

BREMER B¹, STEPHENS HE², WALSH S³, FELGOISE S⁴, SIMMONS Z²

¹Penn State Harrisburg, Harrisburg, Pennsylvania, United States, ²Penn State College of Medicine, Hershey, Pennsylvania, United States, ³Greater Philadelphia Chapter, ALS Association, Philadelphia, United States, ⁴Philadelphia College of Osteopathic Medicine, Philadelphia, United States

E-mail address for correspondence: [email protected]

Keywords: caregiving, assessment, interventions

Background: As ALS progresses, an increasing burden is placed on caregivers to assist in activities of daily living and use of medical equipment, and to provide emotional support. Much of this care and support is provided by family members who must concurrently face the emotional strain of the impending loss of a loved one.

Objectives: A team of health care professionals and academic researchers used the evidence based practice approach to develop a comprehensive list of caregiving tasks, supportive activities, factors affecting caregiver physical and psychological health, and caregiver confidence, rewards, and concerns. These were incorporated into a Caregiver Assessment Questionnaire with both closed and open ended items which was mailed to caregivers.

Results: 102 caregivers of patients seen at a multidisciplinary outpatient ALS clinic in a four month period completed the questionnaire (65% women; 91% Caucasian, 78% spouses). Mean (SD) age was 58.9 (12.7) years. A mean (SD) of 14.79 (9.69) hours caregiving daily was reported. 17 concerns about caregiving tasks (0 none, 5 high) were combined (Chronbach's alpha 0.94) to produce a mean (SD) task concern score of 1.92 (1.17), range 0.12 to 4.88. Concern ratings ranged from a high related to providing emotional/spiritual support (mean 2.40) and injury prevention (mean 2.35) to lows related to medications (mean 1.47) and feeding (mean 1.21). Fear about the future was a common theme identified from the qualitative data. The supportive activity most commonly cited by caregivers was socializing (78.8%), followed by journaling/reading (50%) and spiritual practices (48%). Least likely to be reported as helpful were ALS education (1.0%), counseling, and support groups (6.7% each). Activities frequently discontinued while caregiving included socializing and hobbies. Love and character strengths were frequently cited as important for caregiving. 13.5% were positive on a depression screen, and 36.6% on a stress screen.

Discussion: 1) Specific concerns of ALS caregivers can be identified, with injury prevention, providing emotional and spiritual support, and fear of the future being among the top ones; 2) Support strategies for ALS caregivers most commonly include socializing, journaling or reading, and spiritual practices; 3) ALS caregivers often abandon supportive tasks such as socializing and hobbies; 4) Stress and depression are not uncommon among ALS caregivers. The use of a caregiver survey is one intervention that can be used to assess caregiver needs and guide support.

C81 A RANDOMIZED CONTROLLED TRIAL OF PHYSICAL ACTIVITY IN INDIVIDUALS WITH ALS: PRELIMINARY EXPERIENCE AND SHORT-TERM FOLLOW-UP RESULTS OF TREATMENT WITH DIFFERENT EXERCISE PROTOCOLS

LUNETTA C¹, SANSONE V², MAESTRI E¹, BELLA P¹, BETTINELLI M¹, PANZERI M², CELLOTTO N¹, MEOLA G², CORBO M¹

¹NEuroMuscular Omnicentre (NEMO), Fondazione Serena Onlus, Niguarda Ca’ Granda Hospital, Milano, Italy, ²Department of Neurology, IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

E-mail address for correspondence: [email protected]

Keywords: physical activity, cycloergometer, ALSFRS-R

Background: Physical exercise exerts a wide range of benefits on an organism's overall health and well-being. Exercise contributes positively toward an individual's healthy weight, muscle strength, immune system, and cardiovascular health. In addition, exercise has also been shown to be neuroprotective in both the central and peripheral nervous systems. Whether physical activity promotes or prevents progression of motor neuron degeneration in ALS is still debated. Vigorous exercise exacerbates the disease progression in ALS animal models and patients whereas moderate exercise regimens improve ALS patients’ functional scoring and disease symptoms.

Objective: 1) To evaluate the effects of active exercise program associated with cycloergometer exercise compared to only active or passive exercise programs on disease progression; 2) To define the impact of the different exercise programs on the patient's quality of life.

Patients and methods: Patients were randomized to receive active exercise program associated with cycloergometer activity (group A) or only active (group B) or passive (group C) excercise programs. Moreover, all patients were trained to home-based passive exercise programs. At baseline and after 2, 4, and 6 months, patients were assessed by manual muscle strength testing (MMT), ALSFRS-R, Forced Vital Capacity percentage (FVC%), and McGill Quality of Life (MGQoL) questionnaire.

Results: Sixteen ALS patients were screened and randomly assigned to one of three (group A, n= 5; group B, n= 5; group C, n= 6). Two patients did not complete the trial (one of group B and C, respectively). At 4 months, the group A had significantly higher ALSFRS-R score (A 36.5±0.7; B 29±2.8; C 33.5±2.1) and lower MGQoL physical symptoms subscale score (A 11±4.6; B 22.7±6.4; C 21.5±9.2) compared to the other two groups. No adverse events related to the intervention occurred, MMT and FVC indicated no negative effects.

Discussion: The patients treated with an active exercise program associated with cycloergometer activity demonstrated less motor deterioration and improvement of physical symptoms, as showed by ALSFRS-R score and MGQoL respectively, compared to the other two groups. Compliance with home-based exercise programs is a common problem, especially in old and disabled people. Effects of exercise on most examined parameters are probably short-lived if the program is not continued.

Conclusions: Our study, although small and short, showed that the active exercise program associated with the use of cycloergometer had significantly better function, as measured by ALSFRS-R and quality of life score without adverse effects as compared with subjects receiving exclusively active or passive exercise programs.

SESSION 11A CELL BIOLOGY AND PATHOLOGY

C82 NOVEL RNA BINDING PROTEINS IN ALS

GITLER AD

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, United States

E-mail address for correspondence: [email protected]

Keywords: RNA binding proteins, TDP-43, Ataxin-2

Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The causes of ALS are poorly understood, although the protein TDP-43 has been suggested to play a critical role in disease pathogenesis. Here we show that Ataxin-2, a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2 (SCA2), is a potent modifier of TDP-43 toxicity in animal and cellular models. The proteins associate in a complex that depends on RNA. Ataxin-2 is abnormally localized in spinal cord neurons of ALS patients. Likewise, TDP-43 shows mislocalization in SCA2. To assess a role in ALS, we analyzed the Ataxin-2 gene (ATXN2) in 915 ALS patients. We found intermediate-length polyQ expansions (27-33 Qs) in ATXN2 significantly associated with ALS (4.7% of cases, P=3.6310²⁵). These data establish ATXN2 as a new and relatively common ALS disease susceptibility gene. Further, these findings indicate that the TDP-43/Ataxin-2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

C83 MECHANISMS OF ALS RESISTANCE IN MOTOR NEURONS OF OCULOMOTOR AND ONUF'S NUCLEI

KAPLAN A¹, TOWNE C², SPILLER K¹, KANNING K¹, AEBISCHER P², HENDERSON C¹

¹Columbia University, Center for Motor Neuron Biology and Disease, New York, United States, ²Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland

E-mail address for correspondence: [email protected]

Keywords: oculomotor, resistance, gene profiling

Background: ALS patients retain eye movement and voluntary control of continence until terminal stages of the disease. This reflects the pathological observation that motor neurons in the oculomotor, trochlear, and abducens nuclei, which innervate extraocular muscles (EOMs), and motor neurons of Onuf's nucleus, which innervate pelvic sphincters, are mostly spared. If even a fraction of this resistance could be conferred on vulnerable spinal motor neurons, the benefits for patients would be significant.

Objectives: We wish to determine the molecular basis of ALS resistance in oculomotor and Onuf's nuclei as a strategy for identifying novel therapeutic targets.

Methods: Microarray analysis was performed on motor neuron populations isolated using laser capture microdissection from wild type P7 mice. MMP-9 expression was validated by in situ hybridization and immunostaining. Effects of MMP-9 on neuronal survival were quantified using ES-cell derived motor neurons (ES-MNs).

Results: A similar pattern of disease resistance was found in SOD1G93A mice. By endstage, only 13% of endplates in the fast tibialis anterior muscle were innervated. In marked contrast, this figure was nearly 100% in EOMs, and 80% in sphincter muscles. To uncover intrinsic molecular properties common to resistant nuclei, gene expression profiles of motor neurons in the oculomotor nucleus and the L6 DL nucleus (the homologue of Onuf's nucleus) of wildtype P7 mice were compared to that of vulnerable L5 lumbar motor neurons. Counting only >10-fold differences, nearly 100 genes were differentially expressed in either oculomotor vs. L5, or DL vs. L5. Of these, 17 genes were co-regulated in both resistant populations. One such gene was MMP-9 (matrix metalloproteinase-9), which is strongly expressed by many spinal and cranial motor neurons throughout postnatal life but is absent from oculomotor and DL nuclei. Even within disease-susceptible pools, a strong correlation exists between the fraction of motor neurons that initially express MMP-9 and the fraction lost at endstage in SOD1-G93A mice. Indeed, the ∼50% of motor neurons in these pools that survive to endstage do not express MMP-9. In vitro, activated MMP-9 (but not other MMPs) triggered death of motor neurons. MMP-9 levels have been reported to be elevated in ALS patients. Our data raise the possibility that motor neuron-derived MMP-9 may contribute to degeneration of vulnerable motor neurons in ALS, and that its absence may be one cause of resistance in oculomotor and Onuf's nuclei.

Discussion and conclusions: A set of molecular differences common to disease-resistant motor neurons distinguishes them from the majority of vulnerable motor neurons. These may constitute potential therapeutic targets but their functional role in vivo remains to be determined. We are currently testing the effects of MMP-9 knockdown and pharmacological inhibition on disease progression in SOD1G93A mice.

C84 INTERCELLULAR MISFOLDING OF HUMAN WTSOD1 PROPAGATED IN VITRO

CASHMAN N, GRAD L, YANAI A

University of British Columbia, Vancouver BC, Canada

E-mail address for correspondence: [email protected]

Keywords: SOD1, protein misfolding, prion-like activity

Background: Motor neuron cell death in ALS progresses through the neuraxis in a spatiotemporal manner reminiscent of prion disease. We have previously shown that expression of G127X and G85R, misfolded human mutants of superoxide dismutase-1 (SOD1), can induce misfolding of natively-structured wild-type SOD1 in human mesenchymal and neural cell lines, as determined by molecular surface immunoreactivity with misfolding-specific monoclonal antibodies (mAbs) and acquisition of protease sensitivity (suggesting structural loosening). Mouse wtSOD1 did not support the conformational conversion reaction, consistent with a structure-sensitive ‘species barrier’.

Objectives: To determine if misfolded human wtSOD1 is capable of supporting the SOD1 conformational conversion activity we have previously observed with mutant SOD1.

Methods: We transgenically over-expressed human wtSOD1 in HEK cells and monitored: 1) immunoreactivity with SOD1 disease-specific epitope (DSE) mAbs 3H1 (recognizing the misfolded electrostatic loop) and 10C12 (recognizing the oxidized C-terminal dimer interface domain); 2) proteinase K (PK) sensitivity; 3) incorporation into non-native disulfide-linked oligomers and multimers; and 4) generation of reactive oxygen species (ROS), as determined by immunodetection of protein carbonylation. Furthermore, we ascertained whether misfolding of wtSOD1 could be transmitted between cells via supernatant transfer from transfected to naïve HEK cells.

Results: Transfection-driven overexpression of human wtSOD1 in HEK cells was accompanied by detectable SOD1 misfolding, as monitored by DSE immunoreactivity, marked enhancement of PK sensitivity, formation of non-native disulfide bonds and generation of ROS. Misfolding of human wtSOD1 was transmitted to untransfected HEK cells by incubation in conditioned media from wtSOD1 transfected HEK cells. No misfolded human SOD1 was detected in mouse N2a cells incubated in transfected HEK media, and knockdown of endogenous SOD1 expression in human HEK cells by siRNA abrogated the effect, consistent with a concentration- and structure-dependent SOD1 conformational conversion driven by misfolded human wtSOD1. Pre-incubation of wtSOD1-transfected conditioned media with poly-specific SOD1 antibodies or DSE mAbs also blocked the SOD1 conformational transducing activity.

Discussion: Our data are consistent with intercellular exportation, uptake and conformational conversion activity of misfolded wtSOD1, similar to that observed for the natural SOD1 mutants G127X and G85R.

Conclusion: Misfolded wtSOD1 participates in a template-directed misfolding cascade which can be transmitted between cells, providing a possible SOD1 misfolding-dependent molecular mechanism for propagation of sporadic ALS in the nervous system.

C85 ALS MUTATIONS IN FUS ARE CLUSTERED IN THE NUCLEAR LOCALIZATION SEQUENCE AND INDUCE STRESS GRANULES

GAL J, ZHANG J, KWINTER D, ZHAI J, JIA H, JIA J, ZHU H

University of Kentucky, Lexington, KY, United States

E-mail address for correspondence: [email protected]

Keywords: FUS/TLS, nuclear localization sequence (NLS), stress granules

Mutations in FUS have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. Little is known about the regulation of FUS subcellular localization or how the ALS mutations alter FUS function. Here we demonstrate that the C-terminus of FUS constitutes an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS point mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. PABP1 formed large cytoplasmic foci that co-localized with the mutant FUS inclusions. No such foci, which resemble stress granules, were observed in the presence of wild-type FUS. In addition, processing bodies, which are functionally related to stress granules, were adjacent to but not co-localized with the mutant FUS inclusions. Our results suggest that the ALS mutations in the C-terminal NLS of FUS can impair FUS nuclear localization and induce cytoplasmic mislocalization, inclusion formation, and potential perturbation of RNA metabolism.

C86 MUTANT FUS PROTEINS THAT CAUSE AMYOTROPHIC LATERAL SCLEROSIS INCORPORATE INTO STRESS GRANULES

BOSCO D¹, LEMAY N¹, KO HAE K¹, ZHOU H¹, BURKE C¹, KWIATKOWSKI T², SAPP P¹, MCKENNA-YASEK D¹, BROWN R¹, HAYWARD L¹

¹University of Massachusetts Medical School, Worcester, MA, United States, ²Massachusetts General Hospital, Boston, MA, United States

E-mail address for correspondence: [email protected]

Keywords: stress granules, FUS, zebrafish

Background: Dominant mutations in the nucleic acid binding protein FUS (fused in sarcoma), also known as TLS or heterogeneous nuclear ribonucleoprotein (hnRNP) P2, cause ∼5% of familial ALS cases. FUS is a ubiquitously expressed protein normally concentrated in cell nuclei that has putative functions in DNA repair, transcription, and pre-mRNA splicing. In addition, FUS accompanies mRNA transcripts to the cytoplasm as a component of RNP granules in neurons and may influence the transport or translational regulation of specific mRNAs in dendrites. Initial clues suggest that the mutant variants of FUS may be localized abnormally in the cytoplasm of motor neurons under pathological conditions, but the mechanism(s) by which expression of the mutants injures motor neurons is not known.

Objectives: More than 15 ALS missense mutants are clustered within a non-classical nuclear localization signal (NLS) region that spans residues Pro508-Tyr526 at the C-terminus, while a truncation mutant (R495X) that causes a more aggressive phenotype removes the final 32 residues of FUS, including the NLS. We therefore tested whether ALS-linked FUS variants i) mislocalize upon expression in cell cultures and zebrafish embryos and ii) exhibit a mutant-specific phenotype in response to cellular stresses.

Methods: We engineered stable HEK-293 cell lines that express doxycycline-inducible GFP-tagged FUS constructs and also expressed human FUS variants in zebrafish embryos. Constructs encoding wild type or mutant FUS were injected into the yolk sac at the 1-2 cell stage, and the protein was detected at 24-72 h post fertilization either by live embryo imaging or by immunostaining using an anti-GFP antibody.

Results: We observed a striking redistribution from the nucleus into punctate cytoplasmic granules in both HEK-293 cells and zebrafish spinal cord for the clinically severe mutant (R495X) and an experimental truncation mutant (G515X). Quantitative measurements of GFP fluorescence intensity in live cells showed that the cytoplasmic:nuclear expression ratio per unit volume was significantly increased by 2-fold for GFP-R521G, by 29-fold for GFP-R495X, and by 48-fold for GFP-G515X in comparison to that for GFP-WT FUS. Furthermore, in response to translational arrest induced either by oxidative stress or by heat shock, the FUS mutants, but not WT FUS, assembled within minutes into larger perinuclear stress granules in proportion to their extent of cytoplasmic accumulation. In the case of heat shock, the focal accumulation of mutant FUS was partially reversible upon return to physiological temperature.

Discussion and conclusions: We report a novel ALS truncation mutant (R495X) that causes a relatively severe ALS phenotype compared to FUS missense mutations. Our findings also demonstrate a potential link between FUS mutations and cellular pathways involved in stress responses that may be relevant to altered motor neuron homeostasis in ALS.

C87 THE H63D HFE GENE VARIANT REDUCES SURVIVAL AND DISEASE DURATION IN SODG93A MICE

NANDAR W, NEELY E, SIMMONS Z, CONNOR J

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

E-mail address for correspondence: [email protected]

Keywords: iron, H63D HFE, SODG93A/HFE+/H67D

Background: There is considerable interest in identifying a genetic basis for amyotrophic lateral sclerosis (ALS). The H63D HFE gene variant has been identified as present at higher frequency in ALS patients, and a meta-analysis indicates that its presence increases the risk of ALS 4-fold. At the cellular level, H63D mutations are associated with iron overload, increased oxidative stress, increased glutamate release, tau phosphorylation and ER stress, each of which is under investigation as contributing to ALS. We have proposed that H63D mutations establish a permissive milieu for ALS pathogenic factors.

Objectives: To determine whether the H63D gene variant is associated with ALS pathogenesis using the SOD1 mouse model of ALS.

Methods: We crossed the SOD^G93A mice with H67D mice (homologous to H63D mutation in humans) to generate a mouse line (SOD^G93A/HFE^+/H67D), which is heterozygous for the H67D gene variant and carries the SOD^G93A mutation. We determined the survival, disease duration, age at disease onset and body weight loss in SOD^G93A/HFE^+/H67D mice. Disease onset was determined by monitoring the motor performance on a rotarod. Death was defined as the inability of the animal to right itself within 30 s after being placed on its side. Disease duration was the mean time from age at onset to death.

Results: We found that the survival of female SOD^G93A/HFE^+/H67D mice (128.3±1.9 days) was significantly reduced when compared with female SOD^G93A mice (138.2±4.9 days). Female SOD^G93A/HFE^+/H67D mice (18.07±1.4 days) also exhibited significantly shorter disease duration than SOD^G93A mice (33±5.9 days). The survival and disease duration of SOD^G93A/HFE^+/H67D males (126.8±1.6; 22.82±3.2 days) was not different from male SOD^G93A mice (123.3±1.6; 24±6.5 days). Age at disease onset and body weight loss in both male and female SOD^G93A/HFE^+/H67D mice were not significantly different from SOD^G93Amice.

Discussion and conclusions: The H63D HFE gene variant reduces the survival and disease duration in a transgenic mouse model of ALS. The epidemiological data, cell model based data and now animal model data all support HFE genotype as a factor in ALS pathophysiology. The shorter survival and disease duration observed in female SOD^G93A/HFE^+/H67D mice indicates that the effect of the H63D HFE gene variant on ALS may be gender-dependent, analogous to gender-dependent differences in ALS incidence in humans in some age groups. We now have an animal model to evaluate the pathogenic mechanisms by which an HFE gene variant contributes to the course of ALS, and to test how intervention strategies may be impacted by HFE genotype. Because the H63D gene variant occurs in as many as 30% of ALS patients, the mouse model presented here has meaningful implications for human disease.

C88 p-TDP-43 INCREASE IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS IS ASSOCIATED WITH CELLULAR STRESS: ROLE OF MITOCHONDRIA

AYALA V¹, NAUDÍ A¹, GRANADO A¹, CARABALLO-MIRALLES V², LLADÓ J², CACABELOS D¹, BOADA J¹, PAMPLONA R¹, PORTERO-OTÍN M¹

¹Department of Experimental Medicine, University of Lleida-IRBLLEIDA, Lleida, Spain, ²Department of Biologia/IUNICS, University of Illes Balears, Palma de Mallorca, Spain

E-mail address for correspondence: [email protected]

Keywords: phosphorylated TDP-43, oxidative damage, excitotoxicity

Background: Excitotoxicity plays an important role among the multifactorial mechanisms that lead to the death of motor neurons in ALS. As described with superoxide dismutase-1 and other proteins (eg ubiquitin), TAR-DNA-binding protein-43 (TDP-43) is aggregated in the cytoplasm in both the sporadic and SOD-associated forms of ALS. This abnormal localization can be associated to a lack of function of this protein in the nucleus, but the mechanisms involved in its mislocalization are unknown.

Objectives: Previous results in our laboratory suggest that excitotoxicity leads to an increase protein oxidation and endoplasmic reticulum stress contributing to motoneuron death in sporadic ALS (sALS). Hereby, we have examined what is the status of TDP-43 in a model of excitotoxic motor neuron death, reproducing features of sALS, and what could be the contributing factors for this mislocalization.

Methods: Excitotoxicity was induced by exposure to treohydroxyaspartate (THA) in the postnatal rat lumbar spinal cord organotypic cultures (1). We also used Neuro2a and SHSY-5Y cell lines as neuronal models. We measured content and distribution of TDP-43, p-TDP-43 (pS403/404 and pS409/410), ubiquitin and SMI-32 proteins, as well as typical ER and oxidative stress markers by Western blot, gas chromatography mass spectrometry (GC/MS) and immunochemistry techniques, as well as high resolution respirometry for mitochondrial function assessment.

Results: We show that p-TDP-43 is increased in a chronic excitotoxicity model along with the presence of fragments (≈25 kDa) and other hyperphosphorylated species, which have been previously detected in human ALS. Confocal measurements demonstrate that nuclear TDP-43 immunoreactivity decrease in the positive SMI-32 motoneurons after THA treatment. We also observed increase in both ubiquitin and phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by confocal microscopy as well as profound changes in lipid composition and protein oxidative damage markers under excitotoxicity, associated to changes in oxygen consumption suggesting defects in mitochondrial respiratory complexes. We also demonstrate that ER and oxidative stress lead to increased cytosolic location of TDP-43 with implication of ERK1 and ERK2. Finally, we provide evidence that mitochondrial impairment induces TDP-43 phosphorylation and mislocalization.

Discussion and conclusions: We have previously shown that chronic excitototoxicity in organotypic spinal cord cultures, an established model of sALS, leads to ER stress, ubiquitin alterations, protein oxidative damage and changes in fatty acid profile (2). Here, we demonstrate that a mitochondrial impairment could contribute to both factors, finally leading to an increase in cytoplasmatic TDP-43 and aggregate formation associated to oxidative and ER stress in a model of excitotoxic motor neuron death.

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C89 REPETITIVE NERVE STIMULATION TRANSIENTLY OPENS THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE IN MOTOR NERVE TERMINALS OF SYMPTOMATIC MUTANT SOD1 MICE

NGUYEN K, BARRETT J, GARCÍA-CHACÓN L, DAVID G, BARRETT E

University of Miami Miller School of Medicine, Miami, FL, United States

E-mail address for correspondence: [email protected]

Keywords: motor nerve terminal, mitochondria, permeability transition pore

Background: Mitochondria in motor nerve terminals temporarily sequester large Ca²⁺ loads during repetitive stimulation. In motor terminals of wild-type mice this Ca²⁺ uptake produces a small (∼1-2 mV), transient depolarization of the mitochondrial membrane potential (Ψ_m). This Ψ_m depolarization increases ∼5-fold in presymptomatic SOD1-G93A and -G85R mice, models of familial ALS (1).

Objectives: Investigate mechanisms underlying mitochondrial dysfunction in motor neurons of older, symptomatic mutant SOD1 mice by measuring stimulation-induced changes in Ψ_m depolarization in motor nerve terminals.

Methods: Mitochondria in levator auris longus neuromuscular preparations from symptomatic SOD1-G93A and -G85R mice were loaded with a potentiometric fluorescent dye, rhodamine-123 (Rh123). Changes in Rh123 fluorescence, indicating changes in Ψ_m, were measured in motor terminals stimulated with 3 successive trains of action potentials (100 Hz, 5 s trains separated by 30 s intertrain interval, neuromuscular transmission blocked with d-tubocurarine).

Results: Stimulation-induced Ψ_m depolarizations attain much higher amplitudes in motor terminals of symptomatic mutant SOD1 mice compared to presymptomatic mice. These large Ψ_m depolarizations decay more slowly and increment with successive stimulus trains. Additional Ψ_m depolarizations occur that are not synchronized with stimulation. These large Ψ_m depolarizations are reduced a) by cyclosporin A (CsA, 1 μM), which inhibits opening of the mitochondrial permeability transition pore (mPTP), or b) by replacing bath Ca²⁺ with Sr²⁺, which enters motor terminals and mitochondria but does not support mPTP opening. In wild-type mice stimulation-induced Ψ_m depolarizations resembling those in symptomatic fALS mice could be elicited following prolonged exposure to diamide (200 µM), which produces an oxidative stress, but could not be elicited simply by increasing Ca²⁺ influx with 3,4-diaminopyridine (0.1 mM), which prolongs the action potential.

Discussion and conclusions: These results suggest that the increased Ψ_m depolarizations evoked by repetitive stimulation in motor terminals of symptomatic mutant SOD1 mice have at least 2 components: a component not affected by CsA (also present in presymptomatic mice), and a CsA-sensitive component. Thus as disease progresses from presymptomatic to symptomatic stages, the already reduced ability of mitochondria to maintain Ψ_m during stimulation-induced Ca²⁺ entry is further compromised by increased activation of the mPTP. These transient mPTP openings may arise when Ca²⁺ influx is combined with damage produced by oxidative stress. The large, cumulating Ψ_m depolarizations are likely to disrupt multiple aspects of mitochondrial function, including ATP production and sequestration of Ca²⁺ loads, and may thereby contribute to motor terminal degeneration in fALS mice. Our results suggest that agents that reduce oxidative stress and/or mPTP opening might help preserve motor terminals. Supported by ALS Assn, MDA and NIH/NINDS.

Reference:

• Nguyen K, García-Chacón L, Barrett J, et al. Proc Natl Acad Sci USA 2009;106:2007–111.
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SESSION 11B RESPIRATORY AND NUTRITIONAL MANAGEMENT

C90 PREDICTING ENERGY NEEDS IN ALS: RESULTS OF THE ALS NUTRITION/NIPPV STUDY

KASARSKIS E^1,2, MENDIONDO M¹, KRYSCIO R¹, TANDAN R³, POTTER C³, MATTHEWS D³, MITSUMOTO H⁴, SIMMONS Z⁵, BROMBERG M¹, WELLS S¹, HEALEY M¹, THE ALS NUTRITION/NIPPV STUDY GROUP¹

¹University of Kentucky, Lexington, KY, United States, ²VA Medical Center, Lexington, KY, United States, ³University of Vermont, Burlington,VT, United States, ⁴Columbia University, New York City, NY, United States, ⁵Pennsylvania State University, Hershey, PA, United States, ⁶University of Utah, Salt Lake City, UT, United States

E-mail address for correspondence: [email protected]

Keywords: energy expenditure, predictive equations, nutrition

Background: PEG placement in ALS is indicated when the metabolic (energy, calorie) needs of the body cannot be met via the oral route. The equations currently used in dietetic practice do not accurately predict the energy needs of an individual ALS patient at various stages of their illness. Hence published guidelines emphasize indirect indices of nutritional status such as weight loss, prolonged meal times, and dysphagia rather than the direct measurement of energy balance, ie, Energy Intake matching Total Daily Energy Expenditure (TDEE).

Objective: The goal of this study is to develop ALS-specific equations to predict the energy (calorie) requirements for ALS patients during disease progression.

Methods: TDEE was determined using the ‘gold standard’ Doubly Labeled Water method every 16 weeks over a 48 week period. We measured other factors that might influence TDEE including food intake, body composition (DXA, BIS), resting metabolic rate, physical activity, and ALS clinimetric scales. ALS subjects were entered into two groups (≥80% and 50-79% of predicted FVC) and were further stratified within each group according to their ambulatory status into subgroups based on walking ability using the ALSFRSr. Thus TDEE was determined as subjects progressed through varying combinations of ambulatory and respiratory impairment. Modeling TDEE was accomplished using linear mixed model procedures.

Results: TDEE and the other factors were available for 249 independent determinations in 80 subjects. The mean number of visits was 3.2±1.2 with at least two sequential measurements in 82.5%. Subjects had a mean age of 59.0±11.7 years, limb-onset presentation in 72.5% (n = 58), bulbar onset in 26.3% (n = 21), and generalized onset in 1.3% (n = 1). The mean sitting FVC in Group 1 was 89.2±7.2% of predicted and in Group 2, 63.9±9.5%. At baseline, the mean ALSFRSr score was 36.1±5.8 and the BMI was 27.1±5.7 kg/m² and 27.1±4.0 kg/m² in females and males, respectively. The mean change in the ALSFRSr was 21.09±0.07 points/month.

Discussion and conclusions: Our final equations will be presented. We developed a ‘research’ equation to predict TDEE with the most important factors being food intake, physical activity, and body composition. A second set of ‘practical’ equations were developed by modifying the Harris- Benedict, Mifflin-St. Joer, and other equations for ALS. The results of this study will allow us to provide equations to predict the energy requirements of ALS patients as their disease progresses. The study was funded by the National Institute of Neurological Disorders and Stroke (NINDS) grant 1 RO1 NS045087, the ALS Hope Foundation, and the Cynthia Shaw Crispen Endowment.

C91 NON-INVASIVE VENTILATION IN ALS: A 10-YEAR POPULATION-BASED STUDY IN ITALY

CHIÒ A¹, CALVO A¹, MOGLIA C¹, SELLITTI L¹, BALMA M¹, MAZZINI L², MORA G³

¹ALS Center, Department of Neuroscience, University of Torino, Torino, Italy, ²ALS Center, Department of Neurology, Universrity of Eastern Piedmont, Novara, Italy, ³ALS Center, Fondazione Salvatore Maugeri, IRCCS, Scientific Institute of Milano, Milano, Italy

E-mail address for correspondence: [email protected]

Keywords: non-invasive ventilation, population-based study, outcome

Background: Recent guidelines (1,2) state that non-invasive ventilation (NIV) is the treatment of choice for respiratory failure (Class I evidence) in ALS. However, no data are available about the use of NIV in ALS patients and its impact on survival in a population-based setting.

Aim: To evaluate clinical characteristics and outcome of ALS patients who underwent NIV, using data from a prospective epidemiological register.

Methods: The Piemonte and Valle d'Aosta Register for ALS (PARALS) is a prospective epidemiological register established in 1995 collecting all ALS incident cases in two Italian regions. Data on NIV are prospectively collected using a standard form.

Results: Among the 1260 patients incident in the 1995-2004 period (mean annual crude incidence rate, 2.90/100,000 population), 243 (19.3%) underwent NIV. The number of NIV significantly increased from 88 (14.2%) in the period 1995-1999, to 161 (25.1%) in the period 2000-2004 (P =0.0001). The probability to undergo NIV was significantly higher for patients followed by a tertiary ALS centre (197; 32.8%) than those attending general neurological departments (52; 7.9%) (P =0.0001). Patients with spinal onset, younger than 69 and male had a significantly higher probability to undergo NIV.

The overall median survival time after NIV was 270 days. It was longer for patients followed by ALS tertiary centres (305 days vs. 150 days; P=0.0001). No difference in survival was found between bulbar and spinal patients, whereas age strongly influenced NIV outcome (median survival time: ≤49 years, 380 days; 50-69 years, 245 days ≥70 years, 210 days; P =0.0002).

Conclusions: The use of NIV is rapidly increasing in this population-based series, but mostly among patients attending tertiary ALS centers. Median survival time in our population is similar to that reported in clinical series and in controlled trials and is mainly related to patients’ age. Similarly to a previous survey on tracheostomy in the same population (3), younger age and male gender are strong predictors of the attitude to undergo NIV.

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C92 RISKS OF RESPIRATORY FAILURE, EMERGENCY HOSPITALIZATIONS AND UNPLANNED OUTCOMES OF ALS/MND PATIENTS: A 20 YEAR STUDY

CAZZOLLI P¹, BROOKS B², LEWARSKI J³, MCKIM D⁴, CHATBURN R⁵

¹ALS Care Project, Canton, OH, United States, ²Carolinas Medical Center Department of Neurology, Charlotte, NC, United States, ³Invacare Corporation, Elyria, OH, United States, ⁴Ottawa Rehabilitation Centre, Ottawa, ON, Canada, ⁵Cleveland Clinic Foundation, Cleveland, OH, United States

E-mail address for correspondence: [email protected]

Keywords: respiratory failure, noninvasive ventilation, emergency hospitalizations

Background: ALS/MND results in respiratory failure, unless breathing support is used. Noninvasive positive pressure ventilation (NPPV) is the treatment of choice, but severe bulbar impairment limits its use. Inability to tolerate NPPV may occur, as oral secretions increase. ALS/MND patients may not have timely pulmonary monitoring when respiratory impairment advances. Signs when to initiate or use NPPV may be overlooked. Unexpected acute respiratory failure (ARF) may cause panic, resulting in emergency hospitalizations, early mortality or unintended tracheostomy positive pressure ventilation (TPPV).

Objectives: To identify the risks contributing to unexpected respiratory failure, emergency hospitalizations, failed use of NPPV and commencement of TPPV in patients with or without prior NPPV use.

Methods: In this observational study, 166 ALS/MND patients who had emergency ARF during use of mechanical ventilation (MV) were followed prospectively. An Oral Secretion Scale (OSS) was used to determine the level of oral secretions (Score 4 = normal; Score 3=minimal; Score 2 = moderate; Score 1 = substantial; Score 0 = profound) of the patients when they began NPPV, died or transitioned to TPPV.

Results: In 81/166 (49%) MV patients, NPPV was initiated. 14/81 (17%) attempted NPPV and died. 13/81 (16%) started TPPV, after NPPV failed. 17/27 failed NPPV initiation with OSS scores of 0-1. 16/81 (20%) began emergency NPPV following a ‘good’ pulmonary report. 54/81 (67%) continued NPPV use, survived 1 to 86.5 months. 21/54 (39%) began NPPV with emergency hospitalization. 12/21 (57%) were ambulatory. 14/21 (67%) had OSS scores of 4. At start of NPPV, 6/21 (29%) were nonresponsive, but NPPV reversed symptoms. 23 (43%) of 54 NPPV users had emergency hospitalizations prior to death. 6/23 (26%) patients, who had OSS score of 4, experienced unexpected ARF when off NPPV. 3/23 (13%) with OSS score of 4, who tolerated NPPV, had failed use of NPPV, when hospice gave morphine/oxygen. 14/23 (61%) with excessive oral secretions (OSS scores of 0 or 1) no longer tolerated NPPV. In contrast, 118 patients began emergency TPPV. 104 /118 (89%) did not plan TPPV ahead. 14/118 (12%) began planned TPPV during ARF. 50/118 (42%) were ambulatory. 34/118 (29%) had early respiratory onset. 10/118 (12%) had ARF post-PEG placement. 20/118 (6%) previously used NPPV. 10/20 (50%) of previous NPPV users had OSS scores 0-1 when NPPV became intolerable. 9/20 (45%) with OSS score of 4 had ARF when off NPPV.

Conclusions: OSS score of 1 signaled intolerance of NPPV and need for hospice or planned TPPV. Risks for ARF, failed use of NPPV, emergency hospitalizations, early mortality and unplanned tracheostomy: excessive oral secretions, intolerance and inadequate use of NPPV, unawareness of pending ARF, overexertion, walking, respiratory onset, lack of pulmonary monitoring, use of CPAP, post-PEG tube placement and use of morphine/oxygen in NPPV tolerant patients.

C93 SLEEP CHARACTERISTICS AND THEIR PREDICTIVE VALUE IN ALS

PATEL S¹, PANKRATZ N³, IBER C^1,2, TIRYAKI E^1,2

¹University of Minnesota, Minneapolis, MN, United States, ²Hennepin County Medical Center, Minneapolis, MN, United States, ³Indiana University, Indianapolis, IN, United States

E-mail address for correspondence: [email protected]

Keywords: sleep, polysomnography, NIV titration

Background: Changes of sleep study characteristics in ALS have been previously described in small cohorts. While use of noninvasive ventilation (NIV) has been shown to extend survival, patient tolerance of NIV may vary. Little is known about sleep study characteristics and their predictive value for survival and respiratory complications.

Objective: The goal of this retrospective study was to describe clinical and sleep study characteristics in ALS patients and examine their predictive value for patient tolerance of NIV, respiratory complications and survival.

Methods: Consecutive patients with probable or definite ALS from a single center with polysomnography (PSG) from 2006 to 2010 were included. Standard PSG data and clinical characteristics were collected at multiple time points, including time from symptom onset to first respiratory complication (medical attention for dyspnea, aspiration or pneumonia).

Results: Fifty-six patients (52% male, mean age at onset: 57.5±3.1 years (range 30-82), 27% bulbar onset, mean BMI: 24.3±5.7) were studied. Median FVC at time of PSG was 47% of predicted. Sleep study characteristics were as follows: median respiratory rate during sleep was high at 16.3 (normal ≤14), median respiratory disturbance index (RDI) was 10.4, median apnea-hypopnea index (AHI) was 2.3, median respiratory event-related arousal (RERA) index was 4.6, median sleep efficiency was 61.8%, REM sleep was 6.9% (normal is >15%), nocturnal hypoxia (SpO2<88% >5 min) was 19.6% and percent of sleep related hypoventilation was 30.4%. Rate of disease progression was typical (median loss of 0.91 total ALSFRS points per month, median disease duration 2.9 years). Respiratory complications occurred in 16 individuals (29%).

Increased respiratory events (RERA and RDI) were significantly associated with occurrence of respiratory complication (P < 0.05). Each point of increase of these indices was associated with a 7% increase in risk of having a respiratory complication. Increased RERA and RDI and presence of sleep-related hypoventilation trended toward lower survival 6-months after first respiratory complication. Treatment recommendation with supplemental O₂ based on PSG significantly reduced 6-month survival after first respiratory complication (P = 0.01).

Suboptimal treatment with NIV was associated with a trend toward lower 6-month survival after first respiratory complication (P = 0.07). Of those patients who did not tolerate NIV, 70% had upper extremity onset. These patients were four times more likely to not tolerate NIV compared to LE or bulbar onset patients (P = 0.07).

Discussion and conclusions: Altered sleep characteristics indicative of respiratory distress with nocturnal tachypnea and poor sleep efficiency are observed in this population. Increasing frequency of respiratory events during PSG is associated with increased risk of respiratory complications. Inability to optimally titrate NIV appears to be linked to poor clinical outcome. As patients with upper extremity weakness are less likely to tolerate NIV, development of strategies to improve NIV tolerance is important for clinical management.

C94 THE ROLE OF AIRWAY CLEARANCE VERSUS HYPOVENTILATION AND RESPIRATORY MUSCLE FATIGUE IN ALS

TIRYAKI E^1,2, PATEL S¹, PANKRATZ N³, IBER C^{1, 2}

¹University of Minnesota, Minneapolis, MN, United States, ²Hennepin County Medical Center, Minneapolis, MN, United States, ³Indiana University, Indianapolis, IN, United States

E-mail address for correspondence: [email protected]

Keywords: airway clearance, hypoventilation, polysomnography

Background: Respiratory muscle involvement is a major predictor of survival in amyotrophic lateral sclerosis (ALS). Polysomnography (PSG) can be used to determine presence of hypoventilation and respiratory muscle fatigue and aid in titration of non-invasive ventilation (NIV).

Objective: To determine if impaired airway clearance is a stronger predictor of survival than hypoventilation or respiratory muscle fatigue.

Methods: Consecutive patients with probable or definite ALS studied with PSG from 2006 to 2010 were identified by chart review. Alteration in diet (dysphagia 1-3 or NPO as determined by formal speech evaluation) and drop in bulbar ALSFRS-R subscores for speech, salivation and swallowing at the time of PSG were used as proxies for impairment of airway clearance. Data regarding hypoventilation and respiratory muscle fatigue were captured at PSG, including CO₂ retention during sleep (delta TCM>10), increased respiratory rate during sleep (>14/min), hypoxia (SpO₂ <88% >5 min) and recommendation to initiate treatment. ALSFRS subscores for dyspnea and orthopnea were also used to determine respiratory involvement. Primary outcomes were time from onset to death and time to first respiratory complication (medical attention for dyspnea, aspiration or pneumonia).

Results: Data from 56 patients were analyzed. Rate of disease progression was typical (median loss of 0.91 total ALSFRS points/month, median disease duration 2.9 years). Rate of decline of forced vital capacity (FVC) accounted for 20% (P =0.02) of the variance in survival. A bulbar ALSFRS subscore ≤ 6 (of 12) at time of PSG explained 10% of variation in disease duration while a respiratory subscore ≤ 4 (of 8) explained 0%. Modification of diet explained 10% of the variation in disease duration (P = 0.08). Each respiratory PSG parameter explained less than 4%.

Respiratory complications occurred in 16 individuals (29%) and median time to first respiratory complication was 3.2 years. The rate of decline of FVC accounted for 24% of the variation in time to first respiratory complication (P = 0.09). Modification in diet explained 19%, while respiratory subscores accounted for 3%. None of the examined respiratory PSG parameters, including the recommendation to treat with NIV, accounted for more than 6%.

Discussion and conclusions: While most tests failed to reach statistical significance due to the small sample size, our preliminary data suggest that the risk for respiratory complications of ALS and survival are more strongly predicted by impairment in airway clearance than by hypoventilation or respiratory muscle fatigue. While important for titration of NIV, PSG does not capture airway clearance and as such does not fully assess variables that affect disease progression. Additional focus on aggressive management of airway clearance may need to be considered for optimal clinical management of ALS. The large Northeast ALS Consortium database will be analyzed to further test our hypothesis; these results will be presented at the meeting.

C95 USING LUNG VOLUME RECRUITMENT THERAPY TO IMPROVE SWALLOWING AND AIRWAY PROTECTION FOR INDIVIDUALS WITH ALS

CLEARY S¹, MISIASZEK J¹, WHEELER S², KALRA S¹, JOHNSTON W¹

¹University of Alberta, Edmonton, Canada, ²Misericordia Community Hospital, Edmonton, Canada

E-mail address for correspondence: [email protected]

Keywords: rehabilitation, respiration, swallowing

Background: Lung volume recruitment (LVR) is a manual breath stacking technique used to help patients with ALS cough with sufficient force to clear pulmonary secretions. LVR has been associated with positive treatment outcomes; however much of the evidence for its use is anecdotal and many questions remain about the nature of the treatment effect.

Objectives: The purpose of this study was to answer the following research questions: 1) What is the immediate intensity and duration of the LVR treatment effect on standard tests of pulmonary function? 2) Does LVR improve volitional airway clearance behaviours (ie, hawking, throat clearing, unassisted coughing and forced expiration)? 3) What is the effect of LVR on a specific compensatory swallowing technique (the supraglottic swallow maneuver)? 4) What are participants’ perspectives on the effects of LVR on their respiration and secretion management?

Methods: Participants were 29 individuals with ALS and were currently doing LVR. The mean forced vital capacity of the group was 58% of predicted norms and the median ALSFRS-R score was 28/48. The study involved a within-subjects, repeated measures cross-over research design. Measurements were collected on each participant under two conditions: LVR treatment and no treatment. The order of these conditions was counterbalanced to control for order effects. Assessments of pulmonary function, coughing, airway clearance and swallowing were conducted at three time points: baseline, immediately after the treatment, and 30 minutes after the treatment was provided.

Results: The results of this study were as follows: 1) LVR had a significant positive effect on forced vital capacity immediately post-treatment but did not have a facilitative effect on sniff nasal pressure at any time point; 2) LVR had an significant positive effect on peak cough flow (PCF) during unassisted coughing, hawking, throat clearing, and forced expiration immediately after treatment and 30 minutes post treatment; 3) LVR had a significant positive effect on PCF measured during the supraglottic swallow maneuver, immediately after and 30 minutes post-treatment; 4) The majority of participants expressed satisfaction with LVR, indicating that they tolerated the treatment well, and that they perceived positive effects on cough strength and the ability to clear the airway.

Discussion: Findings have important clinical implications for the symptom management of individuals with ALS. The current rationale for doing LVR is to clear secretions, but if scheduled prior to meals this treatment may provide enhanced airway protection and clearance over the length of a typical meal. In addition, the results help inform theoretical models of mechanisms of the LVR treatment effect. LVR is hypothesized to increase lung volumes and improve coughing effectiveness by recruiting collapsed lung segments, improving compliance and range of motion of the lung and chest wall and eliminating flow limitations within the airway.

C96 FINAL ANALYSIS OF PILOT TRIAL OF DIAPHRAGM PACING (DP) IN ALS/MND WITH LONG TERM FOLLOW-UP: NO SAFETY CONCERNS AND DP POSITIVELY AFFECTS DIAPHRAGM RESPIRATION

ONDERS R, KATIRJI B, SCHILZ R, ELMO M, KAPLAN C

University Hospitals Case Medical Center, Cleveland, Ohio, United States

E-mail address for correspondence: [email protected]

Keywords: respiratory failure, diaphragm pacing, diaphragm function

Background: Respiratory insufficiency is the major cause of mortality in patients with ALS. Ventilators, although life-saving, are only used by a very small percentage of patients. Alternate therapy to prevent or manage respiratory muscle decline in ALS is needed. The motor point diaphragm pacing (DP) pilot trial formed the basis for the multi-center pivotal trial. This reports the final outcome for the initial patients implanted.

Objective: Analyze safety, utility and long term use of the DP system from the initial prospective pilot FDA single site study.

Methods: Patients underwent outpatient laparoscopic diaphragm motor point mapping with electrode implantations. Stimulus/output characteristics of each electrode were determined and diaphragm conditioning initiated. Patients conditioned their diaphragms with 5 daily stimulation sessions of 30 minutes each but were allowed to increase usage. Each patient had three extensive lead-in assessments that were continued post implantation of the DPS system and included pulmonary function tests, fluoroscopic evaluation of diaphragm movement, ultrasound analysis of diaphragm thickness, and quality of life tests.

Results: From 3/05 to 3/07, 16 patients were implanted with no peri-operative or unanticipated device related adverse events. Seven had feeding tubes placements with or after DP. Average age was 50 (range 32-70) with 13 males. Patients were a median 35 months from symptoms at enrolment with a mean ALSFRr of 27 and FVC of 60% predicted at surgery. No patients stopped DP use because of pain, discomfort or failure of implanted electrodes. Four patients are still surviving with 3 on tracheostomy mechanical ventilation. There have been a total of 352 implant-months of follow-up with an average of 22 months per patient post implant. One patient was excluded due to an abdominal co-morbidity of colon cancer diagnosed post implant. Median tracheostomy-free survival was 18.6 months from DP implant, 43.4 months from diagnosis and 56 months from initial onset. In all patients, fluoroscopically observed diaphragm excursion was greater with diaphragm stimulation than under maximal voluntary effort. DP significantly increased muscle thickness when assessed with ultrasound (P-value 0.02). Paired FVC rate of decline (treatment – lead in) improved with DP 1.47±2.18% per month (P=0.03). NIV was never used by 7 of the 16 patients and 50% of patients used DP to overcome central sleep apnea. The cause of death or tracheostomy mechanical ventilation include respiratory failure(5 or 33% of all events), traumatic fall (1) aspiration (3), peri-operatively from a cervical spinal fixation (1), urosepsis (1), colon cancer (1) and three patients had to turn off DP to allow death during final hospice care.

Conclusion: Long term analysis of the DP system in ALS showed no safety issues. The pilot data suggests that DP can positively influence diaphragm physiology, respiratory function and survival in ALS/MND.

SESSION 12 JOINT CLOSING SESSION

C97 UNDERSTANDING THE SYNDROMIC NATURE OF MOTOR NEURON DISEASE

LEIGH PN

King's College London, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: phenotypes, biology, ‘omics’

Clinical practice requires a conceptual paradigm that is pragmatic above all else. The diagnosis must be correct (most of the time), and the advice given on prognosis and treatment must (more or less) be accurate. The clinicopathological revolution of Cruveilher, Charcot and their contemporaries and followers established the conceptual paradigm under which we, in the era of ‘Omics’, still labour.

The ‘Charcot worldview’ is certainly serviceable in the clinic. However, just as classical notions of the constellations and galaxies tell us little about astrophysics, so the identification of phenotype with theory may be more fanciful than helpful. How do we escape from this intellectual corral? First, we might try to agree on what phenotypes are important to study, although this is tautological! By defining the phenotypes, we perpetuate their (apparent) importance. Nonetheless, we can certainly improve our understanding of the (imperfect) descriptive tools in use (eg, the labels ‘typical’, bulbar onset’, flail arm, flail leg, PMA, and so on).

However, I am not convinced that this 19^th century solution is useful any longer. The labels we use are most likely misleading surrogates for the complex genetic and epigenetic factors that contribute to the development and manifestation of ALS. The key step therefore is to use basic indicators of biology (for example, age of onset, race, gender, genetic risk) and rate of spread (‘disease intensity’) and survival, to understand groupings that have undeniable biological significance. This is where unbiased approaches to analysis of phenotype are essential. For example, if we use a traditional phenotypic classification to study prognosis (ie, biology), we find interesting differences between ‘typical ALS’ and the flail arm and flail leg syndromes in terms of prognosis and sex distribution. However, taking an ‘unbiased’ approach – latent cluster analysis, provides a very different set of groups, with two factors accounting for most of the differences between groups.

An ALS study population can be divided up in all kinds of ways, depending on the prejudices of the investigators. To move beyond our prejudices (if such is possible) we may find that subdividing our population samples simply on the a few robust biological factors (eg, age of onset; groupings based on latent cluster analyses or similar approaches; short or long survival) is the most informative approach. It remains to be seen how biomarkers of disease fit into this paradigm, but traditional clinical phenotyping – bedside, pathology, has now outlived its usefulness for ALS research. A new consensus is needed.