P66 GUAMANIAN NEURODEGENERATIVE DISEASE: ULTRASTRUCTURAL STUDIES OF SKIN
NOMURA M, ISHIKAWA H, YASUI K, WATANABE T, MIKAMI H, YAMANO T, SUZUKI M, ONO S
Teikyo University Chiba Medical Center, Ichihara/Chiba, Japan
E-mail address for correspondence: [email protected]
Keywords: ultrastructural studies, skin, parkinsonism-dementia complex (PDC)
Background: Amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) have been highly prevalent in the Chamorro population of Guam. The cause of these disorders is unknown, although environmental neurotoxins with a long latency are thought to be possible causative factors. A remarkable neuropathological hallmark of PDC and Guamanian ALS is the constant and widespread distribution of neurofibrillary tangles (NFTs), suggesting these disorders have a common etiology or pathogenetic process. Numerous patients have been described with clinical features of both disorders. Studies of PDC patients have reported a high incidence of upper and lower motor neuron involvements. These findings support the view that Guamanian ALS and PDC constitute a single disease entity with a spectrum of clinical expressions. Several ALS studies of skin in patients with sporadic ALS have demonstrated morphological and biochemical abnormalities. However, there has been no study of skin in Guamanian ALS or PDC.
Objectives: We performed an ultrastructural study on skin biopsies in patients with Guamanian neurodegenerative diseases such as PDC and ALS.
Methods: Our subjects were 11 Guamanian patients with neurodegenerative disease, 11 clinically uninvolved Chamorro control subjects, 10 Japanese patients with sporadic ALS and 11 Japanese control patients with other neurologic or muscular diseases. Punch biopsy specimens of skin overlying the left biceps were taken. Electron micrographs were taken at 0.5 to 1.0 mm below the epidermal-dermal junction.
Results: For convenience, we have dealt with the Guamanian ALS and PDC cases as a single disorder. The diameter of collagen fibrils in sporadic ALS was significantly smaller (P<0.001) than in the Guamanian patients and controls and the Japanese controls. The diameter of collagen fibrils in Guamanian patients including one ALS patient was slightly smaller than that of Guamanian and Japanese controls, but this was not statistically significant (P=0.60). There was a significant negative correlation between the diameter of collagen fibrils and duration of illness in patients with sporadic ALS (r= −0.81, P<0.01), but there was no such correlation in Guamanian patients. In sporadic ALS patients, some areas showed that collagen bundles were widely separated by a large amount of amorphous material. The longer the duration of sporadic ALS, the more marked these findings. In the other three patient groups, all collagen fibrils were densely packed and parallel in collagen bundles without any increase in amorphous material.
Discussion and conclusions: It is suggested that Guama-nian ALS and PDC differ from sporadic ALS. Our limited biopsy data could provide key elements to our understanding of the pathogenetic mechanisms of Guamanian ALS, PDC and of the difference between sporadic ALS, Guamanian ALS and PDC. Therefore, the skin studies reinforce the view of a different disease mechanism in Guamanian ALS and PDC as compared to sporadic ALS.
P67 RAB5 AND ITS ACTIVATOR ALS2 REGULATE AUTOPHAGOSOME MATURATION AND AUTOPHAGOLYSOSOME-MEDIATED PROTEIN DEGRADATION
OTOMO A, KUNITA R, IKEDA J-E, HADANO S
Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan
E-mail address for correspondence: [email protected]
Keywords: ALS2, Rab5, autophagosome
Background: Rab5 is a small GTPase which acts as a key regulator of several different modes of endocytosis and endosome trafficking. ALS2, the causative gene product for juvenile recessive motor neuron diseases, regulates macropinocytosis following endosome fusion. Recently, we reported that loss of ALS2 in SOD1H46R mice resulted in an earlier death and accelerated accumulation of abnormal autophagosomes in the spinal axons, indicating that malfunction in autophagolysosome-mediated protein degradation underlies ALS2-linked neurodegeneration. Since ALS2 functions as an activator for Rab5, ALS2-mediated Rab5 activation might be crucial for autophagosome maturation and autophagolysosome-mediated bulk protein degradation. However, little is known about the mechanism of autophagosome maturation, despite the protein machinery involving in the synthesis of autophagosomes being well described. Furthermore, it remains elusive as to whether Rab5 regulates autophagosome maturation.
Objectives: To understand the molecular pathogenesis associated with ALS2-deficiency, we investigated the molecular linkages of Rab5 and/or its activators to autophago-some maturation and autophagolysosome-mediated protein degradation.
Methods: We performed the siRNA-mediated gene silencing in HeLa and NSC-34 cells. Rab5A, Rab5B, Rab5C, ALS2, or RIN1 were individually, or simultaneously knocked down by the transfection with respective siRNAs. Knock-down efficiency of the target genes was evaluated by either real-time PCR or Western blot analysis. Expression levels of autophagosome markers such as LC-3 and p62/SQSTM1 were examined by Western blot analysis. Subcellular localization of LC3-positive autophagosomes was analyzed by immunocytochemistry.
Results: Western blot analysis demonstrated that siRNA-mediated simultaneous depletion of Rab5A, Rab5B and Rab5C (Rab5s), but not a single Rab5 protein, resulted in a clear elevation of lipidated form of LC-3 (LC3-II) and p62/SQSTM1 in HeLa cells. Further, depletion of Rab5s changed the localization of LC-3-positive autophagosomes from cell periphery to juxta-nuclear region, and caused the abnormal accumulation of autophagosomes. As expected, ALS2 depletion also led to the accumulation of LC-3-positive autophagosomes in the juxta-nuclear region. By contrast, transfection of control siRNA or siRNA for RIN1, another activator for Rab5, did not affect the subcellular localization and expression levels of LC3-II and p62/ SQSTM1 in HeLa cells.
Discussion and conclusions: Our results indicate that Rab5s appear to function in autophagosome maturation and degradation, besides in the early endocytic pathway. Thus, attenuation of the Rab5 activity may impinge on autophagolysosome-mediated bulk degradation of cytoplasmic proteins and organelles. Interestingly, depletion of ALS2 but not RIN1 results in the autophagosome accumulation, suggesting that the Rab5 activation on a certain subtype of endosomes and/or autophagosomes by its regulator specifically functioning in autophagic pathway, such as ALS2, is required for the proper maturation and degradation of autophagosomes in cells. Further studies will provide insights not only into the mechanism of autophagosome maturation but also into the pathogenesis underlying ALS2-linked motor neuron diseases.
P68 DEVELOPMENT OF A NATIONAL ALS IPS CELL BANK FOR MOTOR NEURONS AND ASTROGLIA
ROTHSTEIN J1, MARAGAKIS N1, HENDERSON C3, EGGAN K2, MANIATIS T2
1Johns Hopkins University, Baltimore, MD, United States, 2Harvard University, Boston, MA, United States, 3 Columbia Unviersity, New York, NY, United States
E-mail address for correspondence: [email protected]
Keywords: iPS, astroglia, motor neurons
Background: To gain an understanding of the mechanisms underlying ALS requires access to relevant cell classes on the same genetic background. Living human motor neurons and astroglia have not been available for study. Mouse models although useful, employ highly over expressed human mutations in a non-human genetic background. iPS cells derived from human patients provide a potential but still largely unexplored approach to these shortcomings. As a first step toward the generalization of their use in the field, we are generating and validating new tools for the study of familial ALS. This will provide a strong basis for mechanistic and screening studies and for future extension of the approach to iPS cells from sporadic ALS patients.
Objective: We are generating 25 unique iPS lines from fALS skin biopsies, including various known ALS mutations: SOD1, TDP-43, FUS. Cell lines will be differentiated into motor neurons and astroglia, with a uniform and constant validation protocol. Finally all validated lines will undergo extensive genetic analyses including RNA seq. Final lines will be available to academic and commercial entities for pathogenesis research and drug screening.
Methods: A co-ordinated team approach is used to collect fALS skin biopsies from several academic sites. The fibroblasts generated from the biopsies are sent to iPierian for uniform generation of iPS cells with pMXS-VSVg retroviruses expressing KLF4, SOX2, OCT4 and cMYC. All final lines are chara-terized initially for pluripotency such as expression of 20+ pluripotency genes (NANOG, SOX2, OCT4, etc), ability to form all three germ layers, and normal karyotyping. Final lines are submitted to the astrocyte core lab – for generation of astroglia (eg expression of mature astroglial markers, functional glutamate transport, invivo astroglial differentiation), and to the motor neuron labs – for generation and characterization of motor neuron phenotype. Finally each validated line undergoes extensive RNA profiling employing Solexa deep sequencing (RNA-Seq).
Discussion and conclusion: This national ALS iPS consortium is generating a comprehensive collection of human iPS derived motor neuron neurons and astroglia – all efficiently created under identical production methods. The ability to actually have human cell lines – representing the natural disease in the most relevant cell types – motor neurons and astrocytes – will provide unprecedented tools to 1) study cell-cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models.
P69 MULTIPROTEIN BIOMARKERS OF AMYOTROPHIC LATERAL SCLEROSIS IN PERIPHERAL BLOOD MONONUCLEAR CELLS
BONETTO V1,2, NARDO G1,2, POZZI S1,2, PIGNATARO M1,2, LAURANZANO E1,2, SPANO G1,2, MONTEFORTE M2, TORRI V2, LUNETTA C3, CORBO M3, MORA G4, BENDOTTI C2
1Dulbecco Telethon Institute, Milano, Italy, 2Mario Negri Institute for Pharmacological Research, Milano, Italy, 3NEuroMuscular Omnicentre (NEMO), Niguarda Ca' Granda Hospital, Milano, Italy, 4IRCCS Fondazione Salvatore Maugeri, Milano, Italy
E-mail address for correspondence: [email protected]
Keywords: protein biomarkers, diagnosis/prognosis, PBMC
Background: For amyotrophic lateral sclerosis (ALS) there is still no diagnostic/prognostic test. The diagnosis is mostly based on clinical assessment with a history of progression of symptoms and is thus made with a delay of about a year from symptom onset, quite likely beyond the therapeutic window of a disease-modifying drug. Moreover, the clinical course varies widely. No ALS biomarkers are currently in clinical use but they would be valuable to support early diagnosis, monitor disease progression, and assess the efficacy of any new treatment.
Objectives : To identify a panel of reliable protein biomarkers of ALS in easily accessible peripheral blood mononuclear cells (PBMC) that exhibit traits of the disease in patients and animal models (1).
Methods: We used a proteome-based approach to examine PBMC of sporadic ALS (sALS) patients, non-ALS neurological patients and healthy individuals. In the analysis sALS patients were divided into two groups based on the levels of disease severity: low, with a functional rating scale score >24 and, high, with a score ≤24. The initial screening was done with pooled samples, then a panel of selected proteins was validated by dot blot on single samples of sALS patients (n = 60) and healthy (n=30) and non-ALS neurological (n = 23) controls, and on samples of PBMC and spinal cord of the G93A SOD1 transgenic rat model of ALS at different stages of the disease.
Results: We identified multiprotein biomarkers that are closely associated with ALS and can distinguish, with high discriminatory power, between two levels of disease severity (90%), ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%). Some of them showed similar changes in PBMC of the rat model already at a pre-symptomatic stage of the disease and had similar behavior in the spinal cord.
Discussion and conclusions : Our multiprotein biomarkers are easily measurable in large-scale immunoassays and thus suitable to develop a helpful test in clinical practice. Some of the proteins identified in PBMC of sALS patients were detected also in the rat model of ALS and were previously found as hallmarks of disease in the central nervous system of ALS patients. This overlap endorses the use of PBMC for in vitro studies and reinforces the theory that ALS is a multi-cellular/multi-systemic disease. Finally, this is the first study that provides a highly feasible strategy to identify and validate multiprotein translational biomarkers in PBMC, potentially applicable to several neurological diseases, both for diagnosis and treatment.
Reference:
- Nardo G, et al. Antioxid Redox Signal 2009;11:1559–67.
P70 SOD1 mRNA EXPRESSION INCREASE AS A BIOMARKER IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
GAGLIARDI S1, COVA E1, DAVIN A1, GUARESCHI S1, LAFORENZA U4, CASHMAN JR3, CERONI M2,1, CEREDA C1
1Laboratory of Experimental Neurobiology, IRCCS, Neurological Institute “C. Mondino”, Pavia, Italy, 2Division of General Neurology IRCCS, Neurological Institute “C. Mondino”, Pavia, Italy, 3Human BioMolecular Research Institute, San Diego, CA, United States, 4Department of Physiology, Section of Human Physiology, University of Pavia, Pavia, Italy
E-mail address for correspondence: [email protected]
Keywords: SOD1, mRNA
Background: Amyotrophic lateral sclerosis (ALS) is diagnosed on the basis of progressive symptoms in both the upper and lower motor neurons. The mutated Cu,Zn-superoxide dismutase gene (SOD1) is recognized as a pathological cause of 20% of the familial form of ALS. A mutation in the SOD1 gene can be considered as a genetic marker of ALS; no other specific biomarkers, as protein or gene expression, are available for ALS. Our previous results showed that lymphocytes from sporadic ALS patients possessed decreased SOD1 protein expression (1).
Objectives: mRNA SOD1 expression was examined in lymphocytes from SALS patients and controls to identify a possible SALS biomarker and to investigate whether protein expression decreasing was a consequence of decreased gene transcription (1). mRNA expression was evaluated in nervous tissues, in fibroblasts and in lymphocytes from SALS patients and in lymphocytes from Alzheimer's disease (AD) patients.
Methods: We examined SOD1 gene expression by Taq-Man Real-Time qPCR in lymphocytes, brain subregions affected (cervical spinal cord, brain stem) and unaffected (cerebellum and non-motor cerebral cortex) by the disease and fibroblast cell lines from SALS patients and controls. Also lymphocytes from AD patients were analysed. SOD1 protein levels in lymphocytes, spinal cord and brain stem tissues from SALS patients and controls were measured by Western blot. Spinal cord specimens from SALS patients and controls were used for immunohistochemical analysis to evaluate SOD1 protein expression.
Results: We described a high level of SOD1 transcript in spinal cord, brain stem and lymphocytes of SALS patients (P<0.001) with respect to controls and AD patients. No differences in SOD1 mRNA levels were detected in unaffected ALS tissues. No correlation was found with the site of disease onset, disease duration or disease severity. Protein expression studies showed a similar or lower amount of SOD1 in affected brain areas and lymphocytes, respectively.
Conclusions: The observation of elevated mRNA SOD1 expression in specific nervous tissues typically affected by the disease (brain stem and spinal cord) shows that SOD1 up-regulation is a pathological phenomenon and that lymphocytes have parallel motor neuron behaviour. The finding of normal levels of SOD1 mRNA in lymphocytes of AD patients indicates the specificity. A possible explanation for the discrepancy between mRNA and protein levels could be that SOD1 would change its structure and the misfolded protein could precipitate in the insoluble fraction as proteinaceous aggregates during protein extraction process (2). This hypothesis is in agreement with histochemical analysis showing higher SOD1 protein expression in SALS brain affected by disease. These findings provide new insight and understanding of the pathologic causes of SALS and allow a possible explanation for the molecular involvement of wild-type SOD1.
References:
- Cova, et al. Neurosci. Lett. 2006.
- Gruzman, et al. Proc. Natl. Acad. Sci. U. S. A. 2007.
P71 ALTERATION IN AMINO ACIDS IN MOTOR NEURONS OF THE SPINAL CORD IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
ISHIKAWA H, NOMURA M, YASUI K, MIKAMI H, WATANABE T, YAMANO T, SUZUKI M, ONO S
Teikyo University Chiba Medical Center, Ichihara/Chiba, Japan
E-mail address for correspondence: [email protected]
Keywords: amino acids, spinal cord
Background: Sporadic amyotrophic lateral sclerosis (SALS) is characterized pathologically by loss of motor neurons from the anterior horn of the spinal cord and a variable degree of corticospinal tract degeneration. However, the gracile and cuneate tracts of the posterior funiculus are well preserved in the spinal cord. Measurement of amino acids in precisely dissected regions of autopsied human spinal cord can provide useful information as to the biochemical basis of neurological disorders. Surprisingly little attention has been paid to possible abnormalities of amino acids in the spinal cords of SALS patients. At present, little is known concerning the changes in amino acid composition in different regions of the spinal cord in SALS.
Objectives: We describe here the results of quantitative amino acid analyses in precisely dissected regions of autopsied spinal cord from patients with SALS and compare the results with those in control subjects.
Methods: Our subjects were seven patients with SALS, seven patients with other neurologic diseases (control group A), and seven patients without neurologic diseases (control group B). At autopsy, blocks of cervical enlargement of the spinal cord were obtained from all the SALS patients and control groups A and B, and were immediately stored at −80°C. Continuous transverse sections of all blocks in each patient were separated by razor edge into the posterior funiculus, the lateral corticospinal tract, and the anterior horn. An aliquot of the hydrolysates was analyzed for its amino acid composition on a Varian 5500 liquid chromatograph configured as an amino acid analyzer.
Results: The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract (P<0.001, P<0.02, P<0.02, and P<0.02) and the anterior horn (P<0.01, P<0.02, P<0.01, and P<0.02) in SALS patients than in control groups A and B. The contents of the acidic amino acids glutamate and aspartate were significantly decreased in the lateral corticospinal tract (P<0.02 and P<0.05) and the anterior horn (P<0.02 and P<0.05) of SALS patients as compared with those of control groups A and B.
Discussion and conclusions: Our study indicates abnormalities of collagen-associated amino acids and excitatory amino acids in the spinal cord in SALS, and this alteration exhibits a marked regional specificity, underlying the involvement of motor neurons of the spinal cord in this disorder. So far there have been no reports indicating changes in these amino acids in precisely defined areas of the spinal cord in SALS. Whether the decreased level of these amino acids is merely a secondary phenomenon, or has some bearing on the pathogenesis of SALS, remains a subject for further study.
P72 SURVIVAL MOTOR NEURON PROTEIN DEFICITS OCCUR IN ALS
TURNER B1, TALBOT K2, ANSORGE O2, ATKIN J1, HORNE M1
1Florey Neuroscience Institutes, Melbourne, VIC, Australia, 2University of Oxford, Oxford, United Kingdom
E-mail address for correspondence: [email protected]
Keywords: spinal muscular atrophy, survival motor neuron, sporadic ALS
Background: ALS and SMA selectively target anterior horn cells of the spinal cord, suggesting common factors that confer neuronal vulnerability in these disorders. While the genetics of ALS is complex, SMA results from reduced dosage of survival motor neuron (SMN) protein encoded by SMN genes. Genetic association studies also provide compelling evidence that abnormal SMN genotypes producing low levels of SMN protein significantly increase risk or severity of sporadic ALS. We recently demonstrated that SMN protein was depleted early in the disease course of cellular and mouse models of familial ALS and genetic ablation of SMN worsens progression in mutant SOD1 mice (1). These studies collectively raise the important question of whether SMN protein deficiency occurs in sporadic ALS patients.
Objectives: To investigate the expression level and cellular localisation of SMN and its binding partners in spinal cords of sporadic ALS patients.
Methods: Post-mortem lumbar spinal cord tissues were obtained from familial ALS cases (n = 5), sporadic ALS patients (n = 15) and non-neurological disease controls (n = 5). Tissues were analysed by immunoblotting of protein extracts for SMN complex components and immunohistochemistry for SMN-positive nuclear gems in motor neurons.
Results: SMN and Gemin protein levels were severely depleted in spinal cord extracts of sporadic and familial ALS cases. SMN depletion correlated with disease progression, but not age of onset. SMN-immunoreactive gem counts were reduced accordingly in anterior horn cells of ALS patients.
Discussion and conclusions: These results establish that SMN protein loss occurs in motor neurons of sporadic ALS and correlates with clinical severity, consistent with genetic association data and observations from familial ALS models. Since SMN is functionally related to TDP-43 and FUS/TLS, these results emphasise the role of nuclear depletion of these RNA binding proteins as a potential common and upstream mechanism in ALS.
Reference:
- Turner B, Parkinson N, Davies K, et al. Neurobiol Dis 2009; 34:511–7.
P73 IMMUNOREACTIVITY OF SMAD UBIQUITINATION REGULATORY FACTOR-2 IN SPORADIC AND FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS AND ITS MOUSE MODEL
NAKAMURA M1, KANEKO S1, ITO H2, ASAYAMA S1, NISHII M1, FUJITA K1, KUSAKA H1
1Department of Neurology, Kansai Medical University, Osaka, Japan, 2Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan
E-mail address for correspondence: [email protected]
Keywords: Smad ubiquitination regulatory factor-2, phosphorylated Smad2/3, Tranforming growth factor-beta
Background: Smad ubiquitination regulatory factor-2 (Smurf2) is an E3 ligase belonging to the HECT-domain ubiquitin ligase family. Smurf2 interacts with Smad proteins and promotes their ubiquitin-dependent degradation, thereby controlling the cellular levels of these signaling mediators. Recently we reported that phosphorylated Smad2/3 (pSmad2/3), the central mediators of transforming growth factor (TGF)-beta signalling, abnormally accumulates in TAR DNA binding protein-43 (TDP-43) positive intracytoplasmic inclusions in sporadic amyotrophic lateral sclerosis (ALS) patients.
Objectives: As Smurf2 is an E3 ligase of phosphorylated Smad2, we aimed to determine the intracellular localization of Smurf2 in ALS.
Methods: Lumbar spinal cord sections from 8 sporadic ALS (SALS), 1 familial ALS (I113T:FALS), and 3 controls without neurological disorder were analyzed immunohistochemically. Lumbar spinal cord sections from G93A SOD1 transgenic (G93A Tg) female mice (mouse model of ALS) at 15-20 weeks of age and age-matched female wild-type litter-mates were also investigated immunohistochemically. The following primary antibodies were used: polyclonal antibodies against Smurf2 and pSmad2/3, and monoclonal antibodies (anti-phosphoserine 409/410) against phosphorylated TDP-43 (pTDP-43).
Results: Smurf2 immunoreactivity was hardly demonstrable in the cytoplasm of the control neurons. In all SALS patients, H&E staining revealed round hyaline inclusions (RHIs) and Bunina bodies. The RHIs and skein-like inclusions (SLIs) were immunopositive for Smurf2, but the Bunina bodies were devoid of labelling for it. A double immunofluorecence study for pTDP43 and Smurf2 revealed the co-localization of both within the RHIs and SLIs. In the FALS patient, neurofilamentous conglomerate inclusions (NFCIs) were present in the cytoplasm of the anterior horn cells, which were immunonegative for not only pSmad2/3 but also for Smurf2. In sections from control mice, Smurf2 immunoreactivity was observed in the cytoplasm of anterior horn cells. In the G93A Tg mice, the Lewy body-like hyaline inclusions (LBHIs) were present in these cells but not labelled with the Smurf2 antibody.
Conclusions: This is the first demonstration of the presence of Smurf2 immunoreactivity in the pSmad2/3- and pTDP-43-positive inclusions in ALS. Our present results imply that the pSmad2/3-Smurf2 complex may play an important role in the pathomechanism underlying ALS.
P74 HYPERACTIVITY OF THE MITOCHONDRIAL RESPIRATORY CHAIN IN FIBROBLASTS OF PATIENTS WITH ALS
CAMPOS Y1, MARTÍN R1, ESTEBAN J3, MORA J2, MENDAÑA L1, MERINO V1, FERNÁNDEZ E1, GARCÍA A2, ALVAREZ JL1
1Instituto de Salud Carlos III, Majadahonda/Madrid, Spain , 2Hospital Carlos III, Madrid, Spain, 3 Hospital 12 de Octubre, Madrid, Spain
E-mail address for correspondence: [email protected]
Keywords: mitochondrial respiratory chain, glucose, hyper-metabolism
Background: Amyotrophic lateral sclerosis (ALS) is a degenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and of lower motor neurons in the spinal cord and brainstem, culminating in respiratory insufficiency and death after 3–5 years. Despite extensive research, the cause of the disease is unknown in the majority of the cases, and the mechanisms of motor neuron injury are complex and are incompletely understood. A proportion of patients with ALS exhibit a generalized hypermetabolism, characterized in the transgenic mice with mutations in the superoxide dismutase gene 1 (mSOD1 mice) by an increased muscular expenditure of glucose. On the other hand, a significant percentage of patients show glucose intolerance and mSOD1 mice develop a low cerebral consumption of glucose prior to symptom and pathology onset.
Objectives: The aim of this work is to study mitochondrial function in fibroblasts of patients with ALS when they grow in low or high glucose medium, to determine whether it varies with the availability of the substrate.
Methods: Skin fibroblast cultures were established from age-matched controls and patients (n= 10 and n = 6, respectively) after their informed consent. Respiratory chain enzyme activities, mitochondrial membrane potential, cell viability and duplication time were determined in cells growing in a medium with low glucose (1 g/L) or high glucose concentration (4.5 g/L).
Results: No differences were observed in any studied parameter in fibroblasts from controls, when they grew in a medium with different glucose levels. We found that fibroblasts of patients growing in low glucose medium had a significant increase in the activity of mitochondrial respiratory chain complexes I and IV when compared with control cells. No differences were detected in patients’ cells cultivated in a rich glucose medium. However, the latter showed a significant drop in the membrane potential, cell viability and growth rate when compared to cells growing in low glucose concentration.
Conclusions: Fibroblasts from patients with ALS present an abnormal mitochondrial function when grown in different glucose concentrations. Considering that low glucose medium is the physiologic concentration in human blood (100 mg/100 mL) we hypothesise that fibroblasts of patients with ALS have an overactive mitochondrial respiratory chain in order to maintain membrane potential and the cell viability. This hyperactivity may be repressed by the presence of high glucose levels. Future studies will be necessary to explain this phenomenon.
P75 APOPTOSIS AND LOSS OF MITOCONDRIAL TRANSMEMBRANE POTENTIAL EVALUATED BEFORE AND AFTER INDUCTION OF OXIDATIVE STRESS IN PERIPHERAL BLOOD LYMPHOCYTES FROM SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
FROSALI S1, GAGLIARDI A1, LANDI C1, BONGIOANNI P2,3, ROSSI B2, BINI L1, DI MAIO G4, FEDERICO A4, DI STEFANO A1
1Department of Molecular Biology, University of Siena, Siena (SI), Italy, 2Azienda Ospedaliero-Universitaria Pisana, Pisa (PI), Italy, 3NeuroCare onlus, Pisa (PI), Italy, 4Department of Neurological, Neurosurgical and Behavioural Sciences, University of Siena, Siena (PI), Italy
E-mail address for correspondence: [email protected]
Keywords: mitochondrial transmembrane potential (MTP), apoptosis, lymphocytes
Background: Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease. Similar to other neurodegenerative disorders with incompletely defined etiology, such as Alzheimer's disease and Parkinson's disease, ALS appears to be a syndrome originating from diverse pathogenic processes. Only 10% of ALS cases seem to be associated with a familial course. Interestingly 20% of all ALS cases are caused by mutations of the copper-zinc superoxide dismutase (SOD1) gene. Multiple mechanisms have been implicated to explain motor neuron injury, including increased reactive oxygen species, altered mitochondrial function, glutamate excitotoxicity, altered calcium homeostasis, altered protein aggregation and proteosomal function, neuroinflammation and apoptosis. However, it is not clear which is the primary event or what the temporal relations are among these pathways. Recent evidence is building that actions on or originating in the mitochondria may be an important part of the disease. Mitochondrial dysfunction may cause motor neuron death by increasing generation of reactive oxygen species and by initiating the intrinsic apoptotic pathway.
Objectives: The present study was aimed to directly assess the spontaneous apoptosis and the susceptibility to undergo apoptosis of fresh human peripheral blood lymphocytes from 20 ALS patients compared to 20 healthy individuals. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent lymphocytes by interfering with cell redox status. The mitochondrial transmembrane potential (MTP) before and during dRib induced apoptosis was also investigated.
Methods: Isolated lymphocytes were cultured for 48 h and the time course (1 h, 24 h and 48 h) of both apoptosis and MTP modifications was monitored before and after dRib treatment by means of flow cytometry.
Results: Sporadic ALS lymphocytes showed increased levels of both spontaneous apoptosis and spontaneous MTP loss compared to normal lymphocytes. Interestingly, lymphocytes from patients with ALS showed a decreased susceptibility to apoptosis and a decreased tendency to lose MTP after dRib treatment. We observed a direct correlation between the severity of disease and minor propensity of lymphocytes to lose MTP during dRib -induced apoptosis in comparison to normal lymphocytes.
Discussion and conclusion: Our data reveal that lymphocytes from ALS exhibit: 1) increased level of spontaneous apoptosis and mitochondrial dysfunction, mirroring what has been found in motor neurons; 2) decreased susceptibility to oxidative stress induced by dRib that appears correlated to severity of disease. This could be likely due to a spontaneous adaptation of lymphocytes to oxidative stress, which has been already reported in ALS patients.
P76 THE BH3-ONLY PROTEIN BIM: POSSIBLE LINK BETWEEN ER STRESS AND APOPTOSIS IN CELLULAR MODEL OF ALS
SOO KY1,2, FARG M1, WALKER A1,3, HORNE M3,4, NAGLEY P2, ATKIN J1,3
1Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia, 2Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia, 3Brain Injury & Repair Group, Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia, 4Centre for Neurosciences University of Melbourne, Parkville, Victoria, Australia
E-mail address for correspondence: [email protected]
Keywords: SOD1, mitochondrial apoptosis, ER stress
Background: We previously showed that mitochondrial apoptosis was correlated specifically with cells bearing mutant Cu,Zn-superoxide dismutase 1 (SOD1) inclusions in ALS. However, dispersed SOD1 proteins, either wildtype (WT) or mutant, were found to partially protect cells against apoptosis, and this protection is upstream of mitochondria engagement in apoptosis. ER stress was also found to be linked to neurotoxicity associated with mutant SOD1 inclusions.
Objective: We demonstrate whether the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis in ALS.
Methods: Depletion of Bim by siRNA technology was used in Neuro2a cells, a mouse neuroblastoma cell line, expressing either WT or mutant SOD1 at 72 h post transfection. Immunostaining and confocal microscopy were performed to investigate both mitochondrial apoptotic and ER stress markers. Cells expressing WT SOD1 were treated with apoptotic inducers for a further 24 h and changes of nuclear morphology were determined. For kinetic studies, NSC-34 cells expressing WT or mutant SOD1 were examined from 10 to 24 h post transfection and before inclusion formation. Proapoptotic markers, such as translocation of CHOP to nucleus and recruitment of Bax to mitochondria, were investigated by immunostaining and confocal microscopy.
Results: In Neuro2a cells, Bim knockdown by siRNA significantly reduced nuclear apoptotic features in cells bearing mutant SOD1 inclusions. After Bim knockdown, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased in such inclusion-bearing cells. However, CHOP translocation to nucleus, a marker of ER stress, was not reduced by Bim knockdown. Significantly, the neuroprotection afforded by dispersed WT SOD1 was substantially enhanced by Bim-depletion, observed in Bim-depleted cells exposed to various apoptotic insults. In cells not subjected to Bim knockdown, kinetic studies indicated CHOP translocation to nucleus to occur prior to formation of mutant SOD1 inclusions. Interestingly, Bax recruitment to mitochondria (but not apoptotic nuclei) was also observed before formation of mutant SOD1 inclusions.
Discussion and conclusion: These data provide the evidence that Bim plays an important role in SOD1-linked ALS. Furthermore, the findings from this study also suggest that neurotoxicity in ALS is induced by a toxic structure derived from mutant SOD1 that activates stress responses in cells much earlier than the appearance of grossly aggregated SOD1 in inclusions.
P77 ELEVATION OF ANGIOPOIETIN RECEPTOR TIE-2 IN PLASMA FROM PATIENTS WITH SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
ZHANG R1, HONRADA R1, MADISON C2, CHAMPION S2, KATZ J2, FORSHEW D2, MILLER RG2, MCGRATH MS1
1University of California, San Francisco, San Francisco, CA, United States, 2California Pacific Medical Center, San Francisco, CA, United States
E-mail address for correspondence: [email protected]
Keywords: monocyte/macrophage activation, inflammation/activation markers, Tie-2
Background: Our previous studies demonstrated elevated blood levels of abnormally activated monocyte/macrophages in sALS. Elevated plasma Lipopolysaccharide (LPS) levels correlated with degree of monocyte/macrophage activation and levels of plasma monocyte/macrophage activation markers sCD14 and sCD163. These data confirmed the inflammatory nature of sALS. Recent studies have implicated angiopoietin receptor Tie-2 as well as vascular endothelial growth factor (VEGF) in the pathogenesis of inflammatory and autoimmune diseases. Although Tie-2, is mainly expressed by endothelium, it is also expressed in monocytes participating in the development of angiogenic and inflammatory disease processes. Angiogenic molecules known to control blood vessel growth and proliferation appear also to regulate neuronal process development. These data linking sALS as an inflammatory disease with inflammation associated markers of angiogenesis suggest that Tie-2 and its level may be involved in sALS pathogenesis.
Objectives: 1) To quantify levels of plasma Tie-2 in sALS patients as compared to control groups; 2) To determine if plasma levels of Tie-2 correlate with clinical stage of disease in sALS.
Methods: Tie-2 ELISA was performed to quantify plasma levels of Tie-2 in heparinized blood samples from 15 sALS, 10 Alzheimer's (AD), and 15 healthy controls (HC). Results from this immune study were evaluated in light of the severity of neurological impairment as determined by ALSFRS-R scores.
Results: Compared to HC (16.7±2.7 ng/ml), significantly higher levels of plasma Tie-2 were identified in sALS (20.4±5.2 ng/ml, P <0.05) and AD (22.2±6.0 ng/ml, P <0.05). Plasma Tie-2 levels between two disease groups, sALS and AD, were similar. Similar elevated patterns were observed in our previous studies on monocyte/macrophage activation markers sCD14 and sCD163 in sALS and AD patient specimens. Plasma Tie-2 and levels of monocyte/macrophage activation markers in sALS, AD and HC were significantly correlated (sCD14: r=0.5346, P =0.0004; sCD163: r=0.4544, P = 0.0079). There was a trend associating increased levels of plasma Tie-2 with ALS disease severity: patients with more marked impairment had the highest Tie-2 levels (ALSFRS-R score=25–36, n = 9, P < 0.05) compared with those with mild impairment (ALSFRS-R score=37–48, n=6); but no difference was found between HC and sALS patients with mild impairment.
Conclusions: This study, for the first time, reveals that plasma levels of Tie-2 are significantly elevated in patients with sALS, and plasma Tie-2 levels might be associated with ALS disease severity. Increased levels of Tie-2 were significantly correlated with degree of monocyte/macrophage activation markers. These findings not only demonstrate a role for angiopoietin/Tie system in ALS pathogenesis but also suggest that a complex array of inflammation/activation-related markers might be able to be used to monitor progressive sALS disease and potential response to therapeutic intervention in patients with sALS.
P78 MODIFICATIONS AND AGGREGATION OF TRANSTHYRETIN IN ALS
COLLINS M, KOLARCIK C, BOWSER R
University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
E-mail address for correspondence: [email protected]
Keywords: transthyretin, protein aggregation, cerebrospinal fluid
Background: Transthyretin is a homotetrameric protein found in the blood and cerebrospinal fluid (CSF) that regulates the transport of biological molecules, including thyroxine and vitamin A. Mutations in the TTR gene result in familial amyloidoses, whereby TTR protein is deposited extracellularly. This can induce peripheral neuropathy and autonomic dysfunction. These protein deposits contain TTR amyloid-beta structured fibrils. Post-translational modifications to TTR influence the protein's propensity to aggregate and have been observed in Alzheimer's disease. Our prior studies have identified a decrease in the native monomer form of transthyretin (TTR) in the cerebrospinal fluid (CSF) of ALS patients. In addition, we detected increased levels of specific post-translational modifications to TTR in the CSF of ALS patients. The current study was performed to further characterize TTR in the CSF and spinal cord tissue of ALS patients.
Objective: To characterize modifications and protein aggregation state of TTR in CSF and spinal cord tissue of ALS patients.
Methods: CSF was obtained from ALS and healthy control subjects. TTR was isolated from CSF by immunoprecipitation or column chromatography. For immunoprecipitation experiments, TTR was incubated with polyclonal TTR antibody (DAKO) and conjugated to paramagnetic protein A/G micro-beads which were then loaded into an elution column. The labeled immune complex was eluted with SDS sample buffer. For column chromatography experiments, CSF was first run through a multiple affinity removal column (Agilent) to remove abundant CSF proteins. The depleted fractions containing TTR were then run through a size exclusion column to separate TTR species of various molecular weights. Fractions from the size exclusion column were saved, desalted, and analyzed by Western blot. Fresh frozen and fixed spinal cord tissue was obtained at time of autopsy from 12 ALS and 5 non-neurologic disease control subjects. We measured levels of TTR in the CSF by ELISA and mass spectrometry. We also performed immunohistochemistry for TTR in post-mortem spinal cord tissue sections. Immunoblotting was performed on 10-12% BIS/TRIS gels. Proteins were transferred from the gels to polyvinylidene fluoride membranes. Membranes were blocked in 2% nonfat milk for 1 hour, incubated in primary antibody overnight (TTR polyclonal; DAKO), incubated in secondary antibody (donkey anti-rabbit), and visualized by ECL.
Results: We verified our prior results indicating decreased levels of native TTR monomer in the CSF of ALS patients, with concurrent increased levels of TTR containing specific post-translational modifications. We observed increased levels of high molecular weight TTR in the CSF of ALS patients.
Conclusions: Within the CSF, TTR post-translational modifications are altered during ALS. These findings suggest that TTR aggregation occurs during ALS. The presence of extracellular TTR aggregates in ALS would be a novel pathologic finding in ALS, suggesting novel pathogenic mechanisms of disease.
P79 EXTRACELLULAR MATRIX AND CELL ADHESION ALTERATIONS IN ALS
COLLINS M, BOWSER R
University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
E-mail address for correspondence: [email protected]
Keywords: extracellular matrix, mass spectrometry, neuropathology
Background: The extracellular matrix (ECM) forms a critical component of cellular communication, protection and adhesion with neurons and non-neuronal cells within the central nervous system. The ECM provides important structure to the synapse, provides guidance during neuronal migration and axonal outgrowth/regeneration, binds numerous growth factors and modulates their release and delivery to cell surface receptors. In addition, the ECM forms perineuronal nets that provide neuronal protection and regulate synaptic plasticity. Little work has been done to examine the ECM and perineuronal nets in the human spinal cord and how their disruption may affect neuronal survival. Therefore, we examined the ECM in the spinal cord of ALS and control subjects.
Objective: To characterize extracellular matrix proteins in the CSF and spinal cord tissue of ALS patients and control subjects.
Methods: CSF was obtained from 250 ALS, disease control and healthy control subjects. Liquid chromatography based mass spectrometry (LC-MS/MS) was performed to identify ECM proteins and extracellular matrix proteases in the CSF of ALS and control subjects. Fresh frozen and formalin fixed spinal cord tissues were obtained at the time of autopsy from 12 ALS and 5 non-neurologic disease control subjects. We measured levels of ECM proteins in frozen spinal cord tissue homogenates by immunoblot. Immunohistochemistry for ECM proteins was also performed in post-mortem spinal cord tissue sections.
Results: By LC-MS/MS, we identified altered levels of specific ECM proteins in the CSF of ALS patients, including tenascin-R, specific collagen isoforms, fibronectin, and matrix metalloproteinases (MMPs). We also observed altered distribution of ECM proteins in the spinal cord of ALS patients by immunohistochemistry. Perineuronal nets appeared disrupted around spinal cord motor neurons in ALS patients. We also detected altered subcellular distribution of cell adhesion proteins within motor neurons during ALS.
Conclusions: Our results indicate that the extracellular matrix within the central nervous system (CNS) is significantly altered during ALS. Disruption of the ECM may directly impact neuronal survival, synaptic integrity, neuronal plasticity and regeneration, and affect the vasculature within the CNS.
P80 THE EFFECT OF ALS IGG ON INTRACELLULAR CALCIUM IN CULTURED PYRAMIDAL NEURONS AND ASTROCYTES
MILOSEVIC M1, STEVIC Z2, ANDJUS P1
1Institute for Physiology and Biochemistry, School of Biology, 2Institute of Neurology, School of Medicine; University of Belgrade, Belgrade, Serbia
E-mail address for correspondence: [email protected]
Keywords: IgG, Ca2+ homeostasis, excitotoxicity
Background: It was suggested that IgG of ALS patients cause excitotoxicity by acting on voltage-gated Ca2+ channels. We have previously shown that ALS IgGs increase the frequency of spontaneous glutamatergic synaptic activity in rat hippocampal neurons in culture (1) and of glycine-mediated IPSCs in hypoglossal motor neurons in rat brain-stem slices (2). It was also shown that ALS IgGs can modify neuronal Ca-transients (3).
The aim of the study : to measure the direct effect of the IgG fraction from sera of sporadic ALS patients on intracellular calcium in hippocampal pyramidal neurons and cortical astrocytes in culture.
Patients and methods: The IgG fraction was isolated from 6 sporadic ALS patients and 5 controls (pathologic and normal). IgGs were purified by affinity chromatography (protein A-sepharose) as described in (1). ALS IgGs dialized towards the experimental medium were applied with a hand micropipette to the measuring chamber by adjusting the applied volume to reach 0.1 mg/ml of final concentration. Pharmacological agents were added to the chamber by perfusion. Hippocampal neurons and cortical astrocytes were dissociated from PND 2-5 Wistar rats and cultured on glass coverslips. Ca-imaging was performed with the fluorescent dyes Fluo 3 and 4 AM.
Results: Most of the ALS IgG samples regularly induced an acute Ca2+ response with a fast transient often followed by a slow and prolonged phase lasting several minutes. Depending on the IgG sample the second phase could be transient or result in a stable increase in intracellular Ca2+. In cases of recovery Ca2+-rise could be induced again by KCl. The response in cultured cortical astrocyte was more of the transient nature. The transients were shown to be dependent in neurons on the state of lysosomes (by their disruption with 0.2 mM Gly-Phe-beta-naphtylamide), and in neurons as well as astrocytes on Ca2+-stores (by the use of 1 μM thapsigargine), but also on Ca2+ influx (with Ca2+-free external solution).
Conclusion: Although qualitative, these observations confirm the general excitotoxic effect of ALS IgGs on Ca2+homeostasis in non-motor neurons as well as in non-neuronal cells. The efect is compelx involving intracellular Ca2+ stores as well as trans membrane Ca2+ influx.
References:
- Pagani MR, Reisin RC, Uchitel OD J. Neurosci. 2006;26:2661–72.
- Andjus PR, Stevic-Marinkovic Z, Cherubini E. J Physiol. 1997;504(Pt 1):103–12.
- Andjus, PR. Arch. Biol. Sci. 2007;59:251–544.
- Andjus PR, Khiroug L, Nistri A, et al. Neuroreport 1996; 7:1914–6.
P81 NFL MICRORNA EXPRESSION PROFILE IN SPORADIC ALS
STRONG M1,2, HE Z1, CAMPOUS D1
1Robarts Research Institute, 2Department of Clinical Neurological Sciences; The University of Western Ontario, London, Ontario, Canada
E-mail address for correspondence: [email protected]
Keywords: RNA, neurofilament
Background: A neuropathological hallmark of ALS is the formation of neurofilamentous aggregates due to alterations in the stoichiometry of the individual neurofilament (NF) subunit proteins that includes a selective suppression of the low molecular weight NF (NFL) steady state mRNA levels relative to those of the intermediate molecular weight (NFM) and high molecular weight (NFH) steady state mRNA levels. We have shown that degenerating ALS motor neurons (identified by increased TDP-43 expression) express elevated levels of P-bodies (RNA granules that target mRNA for degradation) and that these granules contain NFL mRNA. Recognizing that the trigger for P-body formation is the conversion from a stress granule (translationally silent RNA granules), we sought to determine whether the NFL miRNA expression profile differs between ALS and control spinal cord.
Objectives: To characterize the expression profile of miRNA that interact with NFL mRNA 3’ UTR and determine whether this profile differs between ALS and control.
Methods : We used two different prediction algorithms (Target Scan, MiRanda) to develop a panel of highly conserved miRNAs that recognize the human NFL mRNA 3’ UTR. This yielded 103 distinct miRNAs that would potentially interact with the NFL mRNA 3’ UTR. Of these, 19 were highly conserved. We extracted miRNA from ventral lumbar spinal cord lysates of control (3) or sALS (7) using the MirVana miRNA isolation kit and then examined miRNA copy number in the ventral lumbar spinal cord for each of the 103 predicted NFL mRNA interacting miRNAs. Copy levels were determined by quantitative real-time PCR (RT-qPCR; SYBR green), quantified using known standards, and expressed as high (107 – 108 copies), intermediate (104 – 106 copies), low copy number (101 – 103 copies) or absent. Differences between miRNA expression levels amongst cases were analyzed using Real-Time StatMiner version 3.0 (Integromics). Physical interaction between the miRNA of interest and NFL mRNA was confirmed by EMSA, and functional interaction using the luciferase assay.
Results : From among the 103 miRNAs predicted to interact with NFL mRNA, we observed only 4 differentially expressed between ALS and control. This included a reduction in copy number for miR 188–3p (P<0.01) and miR 92–2* (P < 0.05) and an increased copy number for miR23a* and miR23b* (P ≤ 0.01). Both EMSA and luciferase assays are in progress, although for the latter, 2 of the 4 miRNAs induce an enhanced rate of degradation of NFl mRNA when co-expressed with the luciferase construct in HEK 293T cells.
Conclusions: These observations suggest that the suppressed levels of NFL mRNA observed in ALS spinal motor neurons is related to an alteration in miRNA expression favoring an enhanced rate of mRNA degradation mediated by P-bodies. Research supported by the ALS Society of Canada Senior Scientist Bridge Grant (MJS).
P82 HETEROGENEITIES OF MUSCLE PATHOLOGY FROM ALS/MND PATIENTS
AL-SARRAJ S1, CLEVELAND M2, PRADAT P-F3, CORSE A4, ROTHSTEIN J4, LEIGH N1, BATES S2, WURTHNER J2, MEININGER V3
1King's College Hospital, London, United Kingdom, 2GSK Biopharm R&D, Stevenage, United Kingdom, 3Hôpital de la Salpêtrière, Paris, France, 4Johns Hopkins University, Baltimore, United States
E-mail address for correspondence: [email protected]
Keywords: mitochondrial abnormalities, myofibrillary architecture, muscle pathology
Background: Amyotrophic lateral sclerosis (ALS) is a primary degeneration of upper and lower motor neurones. However, it is now recognised that the degeneration is more widespread throughout the CNS with spectrum of changes involving, in addition, the brainstem and cerebral cortex. Muscle biopsies from patients with ALS are known to show neurogenic changes with de-enervation and re-enervation processes. The extent of pathology depends on the degree of loss of anterior horn cells. There is no recent major review of myopathology of ALS patients.
Objectives: To comprehensively examine muscle pathology from a cohort of clinically confirmed ALS patients and to investigate various markers on inflammation, complement activation and deposition of abnormal proteins and compared them with an age matched control.
Methods: Thirty one muscle biopsies from clinically confirmed ALS patients alongside 20 normal controls were stained for comprehensive protocol of histochemical stains in addition to HLA-ABC, utrophin, neo-myosin, C5b-9, p62, TDP-43 and desmin.
Results: The neurogenic changes of various degree and extent were confirmed in 29/31 cases with another case of possible neurogenic changes. In one case there were no features of neurogenic changes. 3/31 cases showed necrosis of muscle fibres with inflammatory infiltration and HLA-ABC over-expression. A further 2 cases showed over-expression of HLA-ABC without evidence of inflammatory cell infiltration. In 4/31 cases there was significant deposition of complement (C5b-9) in the small endomysial capillaries. Mitochondrial abnormalities were seen in 3/31 cases more than those expected in normal ageing. Many biopsies show severe disruption of myofibrillary architecture and 13/31 cases show cytoplasmic bodies; some of these were faintly and focally stained with p62 and desmin but none were immunoreactive to TDP-43.
Discussion and conclusions: Although neurogenic changes are confirmed in the majority of ALS patients, there are additional pathologies including possible low-grade inflammation and mitochondrial abnormalities which may give an alternative insight into the management of ALS.
P83 AUTOPHAGY IN MOTOR NEURONS OF THE SPINAL CORD IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
SASAKI S
Tokyo Women's Medical University, Tokyo, Japan
E-mail address for correspondence: [email protected]
Keywords: autophagy, autophagosome, autolysosome
Background: Autophagy is a cellular pathway in the bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. Autophagy is induced in response to cellular stress, and may either serve as a cell survival mechanism or play a role in cell death. Dysregulation of autophagy is considered to play a role in neurodegeneration. Of autophagy in amyotrophic lateral sclerosis (ALS), very little is known, and in particular, the precise fine structural evidence of autophagy and involvement of autophagy in the pathomechanism of neurodegeneration of motor neurons remain unknown.
Objectives: To examine electron-microscopically if autophagic processes are observed in the cytoplasm of motor neurons of the spinal cord in patients with sporadic ALS, and if autophagy is involved in the pathomechanism of the neurodegeneration of motor neurons in this disorder.
Methods: Autopsy cases were examined in which postmortem investigations had been performed within 6 h of death to avoid delayed postmortem artifacts to the utmost. Electron-microscopically, 16 patients with sporadic ALS (aged 49–83 years; mean age 68.7 ± 8.5 years) and 15 age-matched control patients (aged 44–80 years; mean age 62.7±11.2 years) were studied who died without having any known neurological disease. The lumbar spinal cord (L1–L5) was fixed in 2% glutaraldehyde with phosphate buffer (pH 7.40) at the autopsy. After conventional procedures, ultrathin sections, which were stained with uranyl acetate and lead citrate, were studied by electron microscope.
Results: In ALS, all patients showed autophagic processes in the cytoplasm of normal-appearing motor neurons and more frequently degenerated motor neurons with central chromatolytic change. In particular, the patients with a short clinical course and relatively well preserved anterior horn neurons showed autophagy more frequently. Autophagosomes surrounded by a unique double-membrane and autolysosomes isolated by a single membrane contained the sequestered cytoplasmic organelles such as mitochondria and ribosome-like structures. Autophagy was also found in frequent association with ALS-characteristic inclusions: Lewy body-like inclusions contained autophagosomes and autolysosomes inside and/or at the periphery; autophagosome or autolysosome engulfed tiny or relatively large skein-like inclusions; Bunina bodies contained autophagic structures. Moreover, other abnormal structures such as the honeycomb-like structure showed autophagy occasionally. In the controls, normal-looking motor neurons showed no autophagic processes in the cytoplasm.
Conclusions: These findings suggest that moderate up-regulation of autophagic activity at the early stage of motor neuron degeneration may play a protective role, whereas at the later stage, excessive autophagy beyond the ability of clearance of protein aggregates may contribute to neurodegeneration of motor neurons, eventually leading to autophagic cell death in sporadic ALS.
P84 INTRANUCLEAR AGGREGATES OF SUPEROXIDE DISMUTASE-1 IN GLIAL CELLS OF PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
FORSBERG K, ANDERSEN P, MARKLUND S, BRÄNNSTRÖM T
Umeå University, Umeå, Sweden
E-mail address for correspondence: [email protected]
Keywords: nucleus, glial cells, superoxide dismutase-1
Background: The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). There is accumulating evidence for involvement of non-neuronal cells in the pathogenesis of ALS. In murine SOD1 models, it has been shown that down-regulation of SOD1 expression in astrocytes and microglia slow progression after disease onset, whereas down-regulation in motoneurons delays onset of disease. The exact role of non-neuronal cells in the pathogenesis of ALS is unknown but all cells implicated in the transgenic models show signs of activation or alteration in the human disease.
Objective: Here we searched for signs of involvement of SOD1 in the pathogenesis of ALS with special focus on glial cells.
Methods: Spinal cord from nine ALS-patients carrying SOD1-mutations, from 51 patients with sporadic or familial ALS who lacked such mutations, and from 46 controls, 20 non-neurological diseases and 26 neurodegenerative diseases, were examined by immunohistochemistry. An in-house set of anti-peptide antibodies with very high specificity for misfolded SOD1 was used.
Results: Immunoreactivity for misfolded SOD1 was regularly detected in the nuclei of ventral horn astrocytes, oligodendrocytes, and microglia both in ALS patients carrying SOD1 mutations and in sporadic and familial patients lacking such mutations. Misfolded SOD1 were seen as numerous immunopositive intranuclear granular aggregates approximately measuring 0.5–2 μm. There was only negligible staining in control patients with neurodegenerative or non-neurological disease. Staining for misfolded SOD1 staining was also seen occasionally in nuclei of motoneurons of ALS patients.
Discussion and conclusion: The results suggest that mis-folded SOD1 present in glial and motoneuron nuclei may be involved in the pathogenesis of ALS.
P85 SOD1 PROTEIN LEVELS IN THE CSF OF ALS PATIENTS
WINER L1, CHUN S1, JAFFA M2, LACOMIS D3, BOWSER R3, CUDKOWICZ M2, MILLER T1
1Washington University, St. Louis, MO, United States, 2Massachusetts General Hospital, Boston, MA, United States, 3University of Pittsburgh, Pittsburg, PA, United States
E-mail address for correspondence: [email protected]
Keywords: CSF, SOD1, antisense oligonucleotides
Objective: To determine levels of SOD1 in the CSF of ALS patients versus controls.
Background: Mutations in SOD1 cause a proportion of familial ALS and there is increasing evidence that SOD1 is involved in sporadic ALS. Treatments are being developed to decrease levels of SOD1 as a treatment for familial ALS. Understanding whether SOD1 levels are different in ALS patients versus controls will provide an important background for understanding the effect of medications that decrease SOD1 and for understanding whether SOD1 may be part of the pathogenic process in ALS.
Design and methods: In CSF from 100 patients with sporadic ALS, 42 healthy controls, and 50 neurological disease controls, we measured SOD1 protein levels by ELISA assay and total protein by colorimetric assay. CSF samples were provided by the Northeastern ALS Consortium Sample Repository. Neurological disease controls included Alzheimer's (11), peripheral neuropathy (15), multiple sclerosis (19), and upper motor neuron only (5). In a separate group of 7 patients, CSF was collected every 6 months for approximately 2 years. SOD1 protein levels were measured.
Results: SOD1 protein levels were increased (182 +/− 78 ng/ ml) in sporadic ALS patients compared with healthy controls (135 +/− 54 ng/ml) or neurological disease controls (140 +/−31 ng/ml), (P<0.05). Total protein levels were increased in ALS patients (72+/−20 mg/dL vs healthy controls (58 +/− 15 mg/dL), P<0.05, but not significantly changed relative to neurological disease controls (68+/−22 mg/ml), (P>0.05). Repeat measurements of SOD1 in the same patient at different time points showed less than 20% variation in all patients.
Conclusions: SOD1 protein levels are elevated in CSF from sporadic ALS patients compared with healthy controls and neurological disease controls, suggesting that SOD1 may be part of the pathological process in sporadic ALS. Total protein is also increased in CSF from ALS patients compared with healthy controls. These results will be considered in terms of ALS disease characteristics. In repeat samples at different time points, SOD1 protein levels appear relatively stable. Supported by The Barnes-Jewish Hospital Foundation (BJHF) and the Washington University Institute of Clinical and Translational Sciences (ICTS).
P86 EVIDENCE OF NUCLEAR RELOCALIZATION OF WILD-TYPE SOD1 UNDER STRESSFUL CONDITIONS
GUARESCHI S1, MILANI P1, BIGI R1, ALVISI E2, BIANCHI M1, CERONI M1,2, CEREDA C1, COVA E1
1Laboratory of Experimental Neurobiology, 2Division of General Neurology; IRCCS, Neurological Institute “C. Mondino”, Pavia, Italy
E-mail address for correspondence: [email protected]
Keywords: SOD1, nucleus, oxidative stress
Background: It has been suggested that wild-type SOD1 (WTSOD1) may acquire, following oxidative damage, aberrant and/or toxic properties of mutant SOD1 and be implicated in a fraction of sporadic ALS cases (SALS), which represent 90% of ALS patients. We have previously demonstrated that SALS patients’ lymphocytes express decreased SOD1 protein albeit mRNA expression is significantly higher compared to control. These findings prompted us to investigate the discrepancy between the presence of low protein amount and high messenger expression by assaying the hypothesis of a different compartmentalization of SOD1 in patients.
Aim: In this work we assayed the presence of an altered distribution of WTSOD1 between nucleus and cytosol in SALS lymphocytes. Moreover SOD1 distribution was studied in a cell line under stressful conditions and compared with WTSOD1 in SALS lymphocytes.
Methods: Lymphocytes were isolated from SALS and control subjects by Ficoll gradient. SH-SY5Y (SH) cells were maintained in DMEM/F-12 medium and treated with 1 mM H2O2. Confocal analysis: cells were plated and fixed using 10% paraformaldehyde. Samples were incubated o/n with anti-SOD1 antibody and then with secondary antibody. Flow cytometry: increased free radicals in nucleus and cytoplasm and cell damage were evaluated by dihydro-rhodamine 123 and Apocons kit, respectively. Nuclear and cytosolic SOD1 protein expression was analyzed by Western blot (WB). Immunoprecipitation: after pre-clearing samples were incubated with an anti-SOD1 antibody. The antibody-antigen complexes were analyzed by WB.
Results: Flow cytometry experiments showed a significantly higher amount of free radicals (ROS) both in nucleus and cytosol of SALS lymphocytes compared to controls. A significantly larger fraction of SALS lymphocytes was apoptotic. Higher SOD1 protein expression was found in SALS nuclei than in control cells, as demonstrated by WB and confocal microscopy. Cytosolic SOD1 protein level was unchanged in SALS and controls. SH cells, upon treatment, showed an expression pattern similar to SALS. Immunoprecipitation experiments demonstrated that in SALS lymphocytes SOD1 is more oxidized than in control cells as detected in SH cells upon oxidative stress.
Discussion: In this work we proved an altered SOD1 intra-cellular distribution in SALS lymphocytes compared to control cells. The same pattern of expression was detected in a human neuroblastoma cell line after H2O2 treatment, suggesting that this redistribution could be a physiological response to increased oxidative stress. Higher apoptosis rate and ROS amount in nuclear and cytosolic compartments of patients sustain this hypothesis and indicate that WTSOD1 role in the nucleus need to be investigated. Although the observation of high level of oxidised SOD1 in SALS does not solve the question if it is the cause or the consequence of increased oxidative stress, it proves higher WTSOD1 oxidation in SALS patients.
P87 EVIDENCE OF ABNORMAL OXIDATION OF SOD1 PROTEIN IN LYMPHOBLASTS OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS PATIENTS AND IN AN ALS CELLULAR MODEL BY SELDI-TOF-MS TECHNOLOGY
GHIROLDI A1,2, COVA E1, BARBARINI N3, LISTA S1, GUARESCHI S1, CERONI M1,4, MAGNI P3, CEREDA C1
1Laboratory of Experimental Neurobiology, IRCCS, National Neurological Institute “C. Mondino”, Pavia, Italy, 2Department of Neurosciences, University of Pavia, Pavia, Italy, 3Department of Computer Science and Systems, University of Pavia, Pavia, Italy, 4Division of General Neurology IRCCS, Neurological Institute “C. Mondino”, Pavia, Italy
E-mail address for correspondence: [email protected]
Keywords: SELDI-TOF-MS, SOD1, oxidation
Background: Recent studies suggest that superoxide dismutase (SOD1) may represent a major target of oxidative damage in neurodegenerative diseases. It has also been demonstrated that WT-SOD1 may acquire toxic properties of SOD1 mutant forms through oxidative damage (1). These modifications could alter protein stability, enzyme activity and redox state (2). We used a proteomic approach to study SOD1 post-translational modifications (PTM): SELDI-TOF-MS is a proteomic technique that combines chromatography and mass spectrometry, it is used for biomarker discovery and to study PTMs (3).
In this study we highlighted the presence of increased SOD1 oxidation in sALS patients’ lymphoblasts with SELDI-TOF-MS approach and advanced bioinformatic analysis of peak decomposition.
Methods: Oxidative stress: To identify the SOD1 peak corresponding to oxidation, purified SOD1 from human erythrocyte (hSOD1) and SH-SY5Y (SH) cells were incubated for 1 hour with hydrogen peroxide 1 mM. SELDI-TOF-MS analysis: 5 micrograms of protein extracted with RIPA buffer from lymphoblasts of sALS patients, healthy controls and human neuroblastoma cell line SH were loaded onto IMAC30 array functionalized with Cu2+ and analyzed using SELDI-TOF-MS. hSOD1 was employed as standard to properly recognize SOD1 forms. A bioinformatic innovative procedure was applied to decompose the SOD1 broad low resolution peak, in order to determine overlapping isotopic distributions (peaks) and to identify SOD1 PTMs.
Results: Analysis of hSOD1 peaks showed the presence of seven different peaks representing seven protein PTMs. Analysis of oxidized hSOD1 presented the same pattern of decomposition, but showed an increase in the peak at 15.885 Da that differed 30 Da compared to the main SOD1 peak at 15.855 Da, indicating the SOD1 oxidized form. Analysis of SOD1 peaks in lymphoblasts of both patients and controls, showed a pattern of PTMs similar to hSOD1. However differences between patients and controls were observed. In patients, the oxidized SOD1 peak was increased, indicating more oxidized SOD1 in sALS lymphoblast. In untreated SH cells, decomposition of SOD1 peak was similar to healthy control lymphoblasts and hSOD1, whereas in cells undergoing oxidative stress, analysis of SOD1 peak showed an increase of SOD1 oxidized form as observed in sALS patients and hSOD1 treated with hydrogen peroxide.
Discussion: These data suggest that in sALS patients a large portion of SOD1 is oxidized demonstrating the hypothesis of increased oxidized WT-SOD1 in sALS cells. This alteration could confer to WT protein, toxic and pathogenic properties of ALS-linked mutant SOD1, as previously suggested (1). Data on treated SH-SY5Y confirm the validity of SOD1 oxidation peak. Moreover the innovative bioinformatic analysis of peak decomposition proved to be a useful tool in proteomic analysis with SELDI-TOF-MS.
References:
- Ezzi, et al. J Neurochem 2007.
- Harraz, et al. J Clin Invest 2008.
- Cuccurullo, et al. J Mass Spectrom 2007.
P88 MISFOLDED SOD1 IN CEREBROSPINAL FLUID FROM ALS PATIENTS
ZETTERSTRÖM P1, ANDERSEN PM2, BRÄNNSTRÖM T1, MARKLUND SL1
1Umeå Universtity, Medical Biosciences, Umeå, Sweden, 2Umeå Universtity, Pharmacology and Clinical Neuroscience, Umeå, Sweden
E-mail address for correspondence: [email protected]
Keywords: misfolded SOD1, cerebrospinal fluid, interstitial space
Background: It is still not known how mutant SOD1 is toxic to motor neurons or how the disease is spread throughout the motor system. The SOD1 species toxic to motor neurons remain elusive with the dominating hypothesis being that they are un/misfolded SOD1. Only a misfolded conformation could be common to all the over 150 different SOD1 mutations known today. Therefore, a misfolded SOD1 species could exert a mechanism common for all mutants. Recent data shows that SOD1 can be secreted extracellularly, the mechanism is still unknown but may involve chromogranins A and B binding misfolded SOD1 and transporting it out of the cell. Extracellular SOD1 is toxic to primary motor neuron cultures and can cause gliosis and other kinds of non-cell autonomous damage. Therapeutic strategies involving active and passive immunization against SOD1 in mice have also shown promising results arguing for an extracellular SOD1 factor. That SOD1 exists in the cerebrospinal fluid (CSF) is well known, but nothing is known about misfolded SOD1 in CSF.
Objectives: The objective of this study was to develop an ELISA method to be able to measure the levels of misfolded SOD1 in CSF samples from ALS patients (11 SOD1 mutation carriers, 9 non-SOD1 fALS, 31 sALS, total n=51) and neurological controls (n=52).
Methods: We have created a set of anti-peptide antibodies that are highly specific for misfolded SOD1. Three of these antibodies, towards amino acids 23–39, 57–72 and 111–127 in human SOD1 respectively, were used to set up three different ELISAs highly specific for misfolded SOD1 (misELISA). The secondary antibody used in the misELISAs has 8-fold larger reactivity towards misfolded SOD1 than native SOD1. We have also measured total amounts of SOD1 and total protein levels in the CSF samples.
Results: We have for the first time been able to demonstrate and quantify misfolded SOD1 in CSF samples. The concentrations are very low, only about 1/2,000 of the SOD1 molecules in CSF are misfolded. We found no significant difference in contents of misfolded SOD1 between controls and ALS cases with any of the three misELISAs. When corrected for amounts of total SOD1 there were still no differences, nor did we find any differences between sALS patients and fALS patients with and without SOD1 mutations.
Discussion: The low levels of misfolded SOD1 found in the CSF argue against a direct role of misfolded SOD1 in pathogenesis of ALS. The concentrations are a thousand times lower than the concentrations toxic to cultured primary motor neurons. Even low concentrations of extracellular misfolded SOD1 could trigger non-cell autonomous effects and/or function as a seed in a prion-like process leading to protein aggregation and thereby be part of spreading the disease in the motor areas.
P89 MISFOLDED SOD1 IS A CHARACTERISTIC OF BOTH FAMILIAL AND SPORADIC FORMS OF ALS
GRAD L1, MACKENZIE I2, O'NEILL M1, CASHMAN N1
1Brain Research Centre, Department of Medicine, 2Department of Pathology and Laboratory Medicine; University of British Columbia, Vancouver, BC, Canada
E-mail address for correspondence: [email protected]
Keywords: superoxide dismutase, protein misfolding, antibodies
Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive death of motor neurons. The sporadic form of the disease (SALS) makes up ∼90% of all cases of ALS, with the remainder being an inheritable, or familial, form (FALS). 10–20% of all FALS cases are due to mutations in superoxide dismutase 1 (SOD1), a soluble ubiquitously-expressed free-radical defense enzyme. Mutations in SOD1 promote misfolding and/or aberrant oxidation of the protein, leading to its subsequent aggregation and resulting in a cytotoxic gain-of-function. There is increasing evidence that oxidation/misfolding of wild-type SOD1 acquires many of the same cytotoxic properties as mutant SOD1, suggesting mutation in SOD1 is not essential for its participation in ALS pathogenesis.
Objective: To determine if misfolded SOD1 participates in a common pathological mechanism in both sporadic and familial forms of ALS.
Methods: To address the role of misfolded SOD1 in different forms of ALS, we utilized monoclonal antibodies that specifically recognize misfolded/oxidized forms of SOD1 to test for their presence in human spinal cord from SOD1-FALS and non-SOD1 SALS patients. These disease-specific epitope (DSE) antibodies were used in immunoprecipitation and immunohistochemistry of human ALS spinal cord tissue. We assessed SOD1 misfolding by protease sensitivity, as native SOD1 is highly resistant to digestion. We further analyzed FALS/SALS spinal cord tissue by non-reducing electrophoresis and immunoblotting to detect other biochemical similarities.
Results: Our results confirm the presence of misfolded/oxidized SOD1 in spinal cords from both FALS and SALS by both immunoprecipitation and immunohistochemistry, using our DSE antibodies. This misfolded SOD1 is highly protease-sensitive, suggesting a structural loosening of the enzyme from its normally protease-resistant conformation. We also detect a significant increase in the abundance of disulfide cross-linked SOD1-containng hetero-oligomers in both FALS and SALS spinal cords, further confirming the presence of misfolded/ oxidized SOD1. In addition, high molecular weight SOD1-containing species are associated with a nitrotyrosine signature, suggesting pathological SOD1 is in complex with a protein other than itself, and advanced glycation end-products, post-translational modifications associated with oxidative stress and progression of age-related neurodegenerative disease.
Discussion: The clinical presentation of ALS shows similarities between SOD1-related FALS and typical SALS suggesting a possible common mechanism for both. Moreover, there is increasing evidence in the literature implicating a role for SOD1 in non-SOD1 SALS; other studies have found common molecular signatures of SOD1 in cases of FALS and SALS. The evidence is consistent with the hypothesis that aberrant oxidation and protein misfolding of SOD1 is a primary molecular mechanism common to all forms of ALS.
Conclusions: We conclude that misfolded/oxidized SOD1 is a pathological characteristic in both SOD1-related FALS and non-SOD1 SALS and likely participates in a pathogenic mechanism common to both types of ALS.
P90 JUVENILE AMYOTROPHIC LATERAL SCLEROSIS WITH FUS/TLS PATHOLOGY AND MUTATION
HUANG E1, ZHANG J1, GESER F2, TROJANOWSKI J2, STROBER J1, DICKSON D3, BROWN R4, SHAPIRO B5, LOMEN-HOERTH C1
1University of California San Francisco, San Francisco, CA, United States, 2University of Pennsylvania, Philadelphia, PA, United States, 3Mayo Clinic, Jacksonville, FL, United States, 4University of Massachusetts, Worcester, MA, United States, 5Case Western Reserve University, Cleveland, OH, United States
E-mail address for correspondence: [email protected]
Keywords: FUS/TLS, mutation, pathology
Background: Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a well-recognized entity. However, the molecular underpinnings of this devastating disease are poorly understood.
Objectives: The goal of this study is to provide a comprehensive genetic and neuropathological characterizations in two young women with fatal rapidly progressive ALS with basophilic inclusions.
Methods: Immunohistochemistry, immunogold electron microscopy and analyses of the FUS/TLS (fused in sarcoma/translocated in liposarcoma) gene to identify potential mutations.
Results: A germline mutation (P525L) was detected in the FUS/TLS gene in one of the two patients. Postmortem examinations in both cases revealed severe loss of spinal motor neurons with remaining neurons showing basophilic inclusions that contain abnormal aggregates of FUS proteins and disorganized intracellular organelles, including mitochondria and endoplasmic reticulum. In both patients, the FUS-positive inclusions were also detected in neurons in layers IV-V of cerebral cortex and several brainstem nuclei. In contrast, spinal motor neurons in patients with late-onset sporadic ALS showed no evidence of abnormal accumulation of FUS protein.
Discussion: These results underscore the importance of FUS mutations and pathology in rapidly progressive juvenile ALS. Furthermore, our study represents the first detailed characterizations of neuropathological findings in rapidly progressive juvenile ALS patients with a mutation in the FUS/TLS gene.
Conclusions: Our results indicate that FUS/TLS mutation and FUS pathology are important part of the work-up for juvenile ALS with basophilic inclusions.
P91 ABNORMAL TDP-43 AND FUS PROTEINS IN MUSCLES OF SPORADIC IBM: SIMILARITIES IN A TARDBP-LINKED ALS PATIENT
MILLECAMPS S1, LAIN AH2, DUBOURG O3, SALACHAS F4, BRUNETEAU G4, LEGUERN E1, SEILHEAN D3, DUYCKAERTS C3, MALLET J1, MEININGER V4, PRADAT P-F4
1Research Center of the Brain and Spinal Cord Institute, Pitié-Salpé trière Hospital, Paris, France, 2Department of Neuropathology, Madrid, Spain, 3Department of Neuropathology, 4Department of Neurology, Pitié-Salpé trière Hospital, Paris, France
E-mail address for correspondence: [email protected]
Keywords: inclusion body myositis,TDP-43,FUS
Background: TAR DNA-binding protein (TDP-43) and FUS (Fused in Sarcoma) are two components of protein aggregates in amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). Both proteins are ubiquitously expressed and share striking functional and structural homology. TDP-43 aggregates have also been described in muscles of sporadic inclusion body myositis (s-IBM) patients, but whether this is associated with FUS abnormalities remains unknown.
Objective : We studied TDP-43 and FUS expression in patients with s-IBM and ALS.
Results: Protein levels of TDP-43 and FUS were evaluated in muscle biopsies of five patients with s-IBM, five patients with sporadic ALS (SALS) (including one patient with TARDBP gene mutation) and five controls with myalgia. All s-IBM patients showed abnormalities on immuno-blots for TDP-43, in particular accumulation of c-terminal truncated TDP-43 fragments. In these patients, there was also a consistent decrease in FUS protein levels with a concomitant increase of a high molecular weight FUS immunoreactive protein. Interestingly, the TARDBP-linked ALS patient showed very similar abnormalities in contrast to the 4 SALS cases which were indistinguishable from controls. Sarcoplasmic TDP-43 inclusions, and loss of nuclear TDP-43 protein were observed only in muscles of s-IBM patients but neither in SALS nor in TARDBP-linked ALS patients.
Discussion and conclusion : s-IBM is associated with abnormalities in TDP43 immunoreactivity and loss of normal FUS in muscle. Similar mechanisms might be at work in TARDBP-linked ALS.
P92 CLOSE ASSOCIATION OF TDP-43 PATHOLOGY WITH LOSS OF RNA EDITING ENZYME ADAR2 IN MOTOR NEURONS IN SPORADIC ALS
AIZAWA H2, SAWADA J2, HIDEYAMA T1, YAMASHITA T1, KWAK S1
1Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 2Asahikawa Medical College, Asahikawa, Japan
E-mail address for correspondence: [email protected]
Keywords: TDP-43, ADAR2, AMPA receptor
Background: Motor neurons of normal subjects express GluR2, a subunit of the AMPA receptor, with arginine (R) at the Q/R site, whereas considerable proportion of those of sporadic ALS patients express GluR2 with glutamine (Q) at the Q/R site. Conversion of Q (CAG) to R (CGG) at the Q/R site of GluR2 pre-mRNA is specifically catalyzed by adenosine deaminase acting on RNA 2 (ADAR2). Therefore, it is likely that ADAR2 activity is not sufficient to edit this site of all the GluR2 mRNAs in motor neurons of sporadic ALS. Because failure to edit the Q/R site of all the GluR2 is a direct cause of death of motor neurons, we have proposed that GluR2 Q/R site-underediting due to ADAR2 underactivity is a neuronal death-inducing cause in sporadic ALS. Formation of TDP-43-positive inclusion bodies with loss of TDP-43 in the nucleus is another disease-specific molecular abnormality in sporadic ALS motor neurons.
Objectives: To investigate whether there is a molecular link between reduced ADAR2 activity and TDP-43 pathology, we immunohistochemically investigated localization of ADAR2 and phosphorylated TDP-43 in the motor neurons of sporadic ALS.
Methods: This study was conducted using lumbar spinal cords from seven cases of sporadic ALS and six disease-free control cases. Western blot analysis was performed on nuclear and cytoplasmic fractions of human brains. Formalin-fixed paraffin-embedded sections were double-immunostained with anti-ADAR2, anti-TDP-43 and anti-phosphorylated TDP-43 antibodies. In addition, effects of paraffin-embedding and postmortem delay on the immunoreactivity was tested.
Results: ADAR2a and ADAR2b were localized predominantly in the nucleus in Western blot analysis and immunohistochemical examination of frozen spinal cords. The number of anterior horn motor neurons (AHCs) was reduced to 40% of that in control cases (P < 0.0001). We found that all AHCs examined (n = 380) were ADAR2-positive in the control cases, whereas more than half of them were ADAR2-negative in each of the ALS cases examined. All ADAR2-negative neurons (n = 98 out of 170 AHCs in ALS cases) had cytoplasmic inclusions that were immunoreactive to phosphorylated TDP-43 but lacked non-phosphorylated TDP-43 in the nucleus.
Discussion and conclusions: Our results indicate a molecular link between reduced ADAR2 activity and TDP-43 pathology in motor neurons of sporadic ALS. Because TDP-43 knockout mice are embryonic lethal, and because motor neurons undergo slow death in conditional ADAR2 knockout mice, it is likely that either one of the two ALS-associated abnormalities induces neuronal death via causing the other, rather than both of them occur simultaneously as a result of other upstream abnormalities.
P93 REGULATION OF TDP-43 BY NR2A-CONTAINING NMDA RECEPTOR/PTEN SIGNALING: ITS ROLE IN GLUTAMATE TOXICITY-INDUCED NEURONAL INJURY
ZHENG M1,2, WAN Q2, FAN D1
1Peking University Third Hospital, Beijing, China, 2University of Nevada School of Medicine, Reno, United States
E-mail address for correspondence: [email protected]
Keywords: TDP-43, PTEN, glutamate injury
Background and objectives: Two DNA/RNA binding proteins TDP-43 (TAR DNA binding protein) and FUS/ TLS (fused in sarcoma/translocation in liposarcoma) were recently found to be hallmarks of amyotrophic lateral sclerosis (ALS). Abnormal TDP-43 and FUS/TLS comprised pathological inclusions which extensively distributed in sporadic ALS (SALS) and non-SOD1 mutation familial ALS (FALS). Both of them played an important role in RNA processing, which implied the high metabolic demand of motor neurons, placed great stress on transcription leading to preferential vulnerability of this cell type. Enhanced transcription activity might be a pro-survival response of motor neurons to environmental injury. Here we demonstrated that glutamate neurotoxicity increased TDP-43 expression, and this enhanced expression was negatively regulated by PTEN.
Methods : Downregulation of PTEN expression induced by glutamate injury could be subdued by inhibition of NMDAR activity, which implicated NMDAR activity involved in regulation of PTEN expression.
Results : Application of different NMDAR subunits antagonist showed that inhibition of NR2AR activity could decrease PTEN downregulation and TDP-43 upregulation induced by glutamate injury. Inhibition of NR2BR, however, had little influence on PTEN and TDP-43 expression. Comparing with enhanced expression of TDP-43 by inhibition of PTEN alone, inhibition of NR2AR and PTEN together did not prominently decrease this upregulation of TDP-43 expression, which indicated that NR2AR and PTEN were in the same intercellular pathway, and NR2AR located at the upstream of PTEN to regulate TDP-43 expression. We also tested the effect of TDP-43 on neuron injury induced by glutamate neurotoxicity. Result showed that knockdown of TDP-43 expression promoted glutamate induced neuronal death and TDP-43 overexpression provided a protective effect to damaged neurons.
Conclusions : Consistent with neurotoxicity early in the disease process, glutamate injury upregulates TDP-43 expression through NR2A-containing NMDA receptor/PTEN signaling, and increased TDP-43 protects neurons from glutamate toxicity-induced injury. In early stage of ALS, activation of the NR2AR/PTEN/TDP-43 pro-survival pathway in glutamate induced neuron injury might be a prospective therapeutic target.
P94 HFE H63D CONTRIBUTES TO INCREASED TDP-43 LEVEL AND CYTOSOLIC LOCALIZATION
LIU Y1, SIMMONS Z2, CONNOR J1
1Department of Neurosurgery, 2Department of Neurology; Penn State University, M.S. Hershey Medical Center, Hershey, PA, United States
E-mail address for correspondence: [email protected]
Keywords: HFE, TDP-43, iron
Background: Genetic studies on different populations have reported an increased frequency of a specific form of HFE polymorphism, H63D, in individuals with amyotrophic lateral sclerosis (ALS). Mutation of the HFE gene is best known as being associated with cellular iron overload, but the mechanism by which H63D HFE might increase the risk of motor neuron degeneration is unknown. The TAR DNA binding protein 43 (TDP-43) is present in neuronal inclusions in ALS, as well as in frontotemporal dementia. In ALS, the expression of TDP-43 is up-regulated and in contrast to its normal nuclear localization, becomes cytosolic. Several TDP-43 mutations have been discovered that may increase TDP-43 aggregation and promote neuronal toxicity. However, in most ALS cases without TDP-43 mutations, the factors that transform the wild type (WT) endogenous TDP-43 into its pathological form remain elusive. In this study, we sought to examine the link between HFE H63D and TDP-43 aggregation.
Objective: To determine whether and how the presence of the HFE H63D mutation, a putative risk factor for ALS, contributes to the up-regulation and cytoplasmic aggregation of TDP-43.
Methods: We assessed the expression and localization of TDP-43 in two models: a cell culture model expressing inducible HFE wild type (WT) or H63D protein and a knock-in mouse model, carrying the H67D variant of HFE which is the mouse analogue of the H63D mutation.
Results: Expression of WT HFE protein decreased TDP-43 expression. In contrast, the presence of the iron overload mutant HFE H63D was associated with an up-regulation of TDP-43 expression. Moreover, cytoplasmic localization of TDP-43 was detected in HFE H63D expressing cells. Iron challenge in both HFE WT and H63D expressing cells resulted in an increase of TDP-43. Treatment with desferroxamine mesylate (DFO), an iron chelator, did not completely reverse the increased TDP-43 associated with HFE H63D, or change the TDP-43 expression in the WT HFE cells. The relationship between HFE H63D and TDP-43 in the cell culture model was also found in an in vivo model. In the lumbar spinal cord homogenates from HFE H67D mice at age 6-months, there were elevated levels of TDP-43 expression compared to the WT littermates. At 12-months of age, the difference in TDP-43 expression between the WT and H67D mice was increased.
Discussion and conclusions: These data strongly support our hypothesis that HFE H63D contributes to the abnormal expression and localization of TDP-43. Increased intracellular iron might be the mechanism that underlies this association. HFE H63D and an associated increase in iron may transform the WT endogenous TDP-43 into pathological aggregates in ALS patients without TDP-43 mutations.