THEME 8 HUMAN CELL BIOLOGY AND PATHOLOGY

P181 SERUM SIGNATURES OF MICRORNAS IN GENETIC ALS PATIENTS AND PRE-MANIFEST MUTATION CARRIERS

Freischmidt A¹

Müller K¹

Zondler L¹

Weydt P¹

Volk AE¹

Lošdorfer Božic A²

Walter M³

Bonin M³

Mayer B¹

Otto M¹

Dieterich C²

Holzmann K¹

Andersen P^1,4

Ludolph AC¹

Danzer KM¹

Weishaupt JH¹

^aUlm University, Ulm, Germany

^bMax Planck Institute for Biology of Ageing, Cologne, Germany

^cUniversity of Tübingen, Tübingen, Germany

^dUmeå University, Umeå, Sweden

Email address for correspondence: [email protected]

Keywords: microRNA, mutation carrier, serum

The nature and timepoint of patho-molecular alterations preceding onset of symptoms in amyotrophic lateral sclerosis (ALS) is unknown, but might govern future concepts of pre-manifest disease modifying treatments. MicroRNAs (miRNAs) are central regulators of transcriptome plasticity and participate in pathogenic cascades and/or mirror cellular adaptation to insults. We obtained comprehensive expression profiles of miRNAs in the serum of familial ALS patients (fALS), asymptomatic mutation carriers and healthy controls. We observed a strikingly homogenous miRNA profile in fALS patients that was largely independent from the underlying disease gene. Moreover, we identified 24 significantly downregulated miRNAs in pre-manifest ALS mutation carriers up to 20 years before the estimated time window of disease onset. 91.7% of miRNA alterations in mutation carriers overlapped with the fALS patients. Our data thus suggest specific common denominators regarding molecular pathogenesis of different ALS genes. We describe the earliest pathomolecular alterations in ALS mutation carriers known to date, which provide a basis for the discovery of novel therapeutic targets and strongly argue for studies evaluating pre-symptomatic disease-modifying treatment in ALS.

P182 THE INFLUENCE OF NEUROFILAMENT PROTEINS ON TRAFFICKING OF RNA BINDING PROTEINS LINKED TO ALS

Durham H

Tibshirani M

Gentil B

McGill University, Montreal, QC, Canada

Email address for correspondence: [email protected]

Keywords: RNA binding proteins, neurofilaments, preferential vulnerability

Background: The high content of neurofilaments (NFs) in motor neurons, including NF bundles in dendrites, is a factor in their preferential vulnerability to disease. The relative expression of NF proteins (NFL, NFM, NFH, peripherin) is altered in sporadic ALS and is influenced by mutant proteins associated with familial disease, including SOD1 and RNA-binding proteins such as TDP-43, which decrease stability of NEFL mRNA (1). Trafficking of RNA binding proteins can be altered in both sporadic and familial forms of ALS, leading to their accumulation in cytoplasmic inclusions in affected neurons. This finding is reproduced in cultured motor neurons ectopically expressing these proteins (2, 3).

Objectives: In this study, we tested the hypothesis that NFs promote cytoplasmic accumulation and aggregation of ALS-associated mutant RNA binding proteins, TDP-43 and FUS, thereby contributing to preferential vulnerability of motor neurons to dysfunction.

Methods: Spinal cord-DRG cultures were prepared from E13 embryos of Nefl^−/− mice or wild type Nefl^+/+ mice. NFL is a core protein required for NF assembly, thus Nefl^−/− mice lack NFs. Plasmids encoding flag-tagged human FUS^WT, FUS^R521H, TDP-43^WT, or TDP-43^A315T were expressed in motor neurons of mature cultures by intranuclear microinjection. Distribution of endogenous or ectopically expressed protein was assessed by immunocytochemistry after three days and the distribution was quantified as nuclear, cytoplasmic or both. NF formation was induced in Nefl^+/+ neurons by expressing NFL.

Results: Distribution of mutant FUS and TDP-43 was much more nuclear in Nefl^−/− compared to Nefl^+/+ motor neurons, the percentage of neurons with cytoplasmic protein and inclusions being reduced by about 50% in Nefl^−/− neurons (p < 0.05). This was not due to variability among cultures as cytoplasmic protein and inclusions were increased in Nefl^−/− neurons by ectopically expressing NFL. Interestingly, expression of NFL^Q333P, a mutant causing Charcot-Marie-Tooth disease 2E, promoted redistribution of endogenous FUS and TDP-43 from the nucleus to the cytoplasm, affirming the influence of NF proteins on distribution of RNA binding proteins and their involvement in pathogenesis of multiple disorders affecting motor neurons.

Discussion and conclusions: These data reinforce NFs as a factor in preferential vulnerability of motor neurons to disease, at the mRNA, protein, and structural level. NF proteins affect a wide variety of cellular processes through structural and signalling mechanisms, including common pathways affected in multiple forms of ALS (inclusion formation, mitochondrial dynamics, calcium handling, RNA metabolism, dendritic structure/function, and response to stress).

Acknowledgements: Supported by MDA, ALSA and an E-RARE-2 grant funded by CIHR, FRQS and ALS Canada

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P183 OPTINEURIN AND MYOSIN VI-RELATED TRAFFICKING DEFECTS IN SPORADIC ALS

Sundaramoorthy V

Walker AK

Farg M²

Soo KY²

Atkin J^1,2

^eAustralian School of Advanced Medicine, Macquarie University, NSW, Australia

^fDepartment of Biochemistry, La Trobe University, Victoria, Australia

Email address for correspondence: [email protected]

Keywords: myosin VI, autophagy, optineurin

Background: Defects in vesicular trafficking are increasingly implicated in ALS. Mutations in optineurin cause a proportion of familial ALS cases, and wild type optineurin is misfolded and forms inclusions in sporadic ALS motor neurons. However it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes.

Objectives: To (i) determine how optineurin mutation leads to ALS and (ii) determine whether the same defects are present in sporadic ALS.

Methods: Autophagy was examined using immunoblotting and immunocytochemistry. Sporadic ALS patient tissues were examined by immunohistochemistry and immunoblotting.

Results: Here we demonstrate that ALS-linked mutations Q398X and E478G cause optineurin to dissociate from myosin VI, leading to an abnormal diffuse cytoplasmic distribution rather than the normal vesicular appearance. Furthermore, in control human patient tissues optineurin displayed its normal vesicular localization but in sporadic ALS patient tissues, vesicles were present in a significantly reduced proportion of motor neurons. In addition, optineurin binding to myosin VI was decreased in tissue lysates from sporadic ALS spinal cords, implying that disruption of optineurin-myosin VI function is present in sporadic as well as familial disease.

Expression of Q398X and E478G mutant optineurin in motor neuron-like NSC-34 cells also inhibited secretory protein trafficking, induced endoplasmic reticulum (ER) stress and caused Golgi fragmentation. These defects were not present in control NSC-34 cells expressing another optineurin mutation, E50K, which causes primary open angle glaucoma. We also provide further insight into the role of optineurin as an autophagy receptor. Wildtype optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin in these cells, or siRNA-mediated knockdown of endogenous optineurin, blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation.

Discussion and conclusion: Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI mediated intracellular trafficking processes. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi, and induction of ER stress, to disruption of optineurin function. It also implies that optineurin-myosin VI dysfunction is a common and novel pathogenic mechanism of both sporadic and familial ALS.

P184 EXTRACELLULAR VESICLES ARE IMPLICATED IN THE TRANSMISSION OF PROPAGATED SOD1 MISFOLDING IN ALS TISSUE

Fernando S

Silverman J

Grad L

Cashman N

University of British Columbia, Vancouver, BC, Canada

Email address for correspondence: [email protected]

Keywords: protein misfolding, extracellular vesicles, SOD1

Background: A prion-like propagated protein misfolding mechanism has been observed for SOD1, which may help to explain the clinical observation that ALS disease pathology spreads spatiotemporally through the neuroaxis from one contiguous area to the next (1). We have previously shown that natively folded wild-type human SOD1 (WTSOD1) in cultured cells can be induced to misfold by physical contact with mutant misfolded SOD1 (misSOD1) (2), and that cell-to-cell transmission can occur between cells via the uptake of exosomes (3). However, the mechanism by which misSOD1 is propagated in vivo remains ambiguous.

Objective: To determine if extracellular vesicles (EVs), including exosomes, microvesicles, and other small secreted vesicle populations, are a mode of transportation for cell- to-cell propagation of misSOD1 in ALS mouse models and patient tissue, and to characterize the biology of EV populations from these tissues.

Methods: EVs were isolated from whole neuronal tissues harvested from transgenic mouse models of ALS or post-mortem tissue from ALS patients by dissociation with papain and serial centrifugation of resultant supernatant. Microvesicles and exosomes were isolated at separate centrifugal speeds, and exosomes were further purified in a sucrose density gradient. Both EV populations were examined via direct immunoblot to determine cargo and composition, and used in immunoprecipitation experiments to detect misSOD1.

Results: Isolated exosomes and microvesicles were positive for EV-specific markers such as prion protein, flotillin-1, and native WTSOD1. Immunoprecipitation using misSOD1- specific antibodies showed that misSOD1 is detected in EV fractions and is preferentially localized to the vesicle surface. Application of the secreted EV-containing fraction from cells onto wild-type cells in culture induced misfolding of SOD1 in those cells, a phenomenon which was abolished by heat-denaturing the EV-containing fraction prior to treatment of cells.

Conclusion: Pathogenic misSOD1 is present on EVs isolated from ALS patient and murine neuronal tissues and is abnormally localized to the surface of vesicles, whereas WTSOD1 is normally found in the lumen of EVs (4), suggesting that misSOD1 may be sorted into EVs in a manner different than WTSOD1. Our results also indicate that the secreted EV-containing fraction is competent to induce misfolding of WTSOD1 in cells, implicating the EV transport system in the propagation of SOD1 misfolding seen in disease.

Acknowledgements: Funding provided by ALS Society of Canada and University of British Columbia Four-Year Doctoral Fellowship. Misfolded SOD1-specific antibodies provided by Amorfix Life Sciences.

References:

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P185 RNA METABOLISM AND MITOCHONDRIAL DYSFUNCTION IN FIBROBLASTS FROM FAMILIAL ALS PATIENTS

Colombrita C^1,2

Onesto E²

Gumina V²

Dusi S³

Borghi O²

Tiranti V³

Silani V^1,2

Ratti A^1,2

^gDepartment of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy

^hIRCCS Istituto Auxologico Italiano, Milan, Italy

ⁱFondazione IRCCS Istituto Neurologico “Carlo Besta”, Milan, Italy

Email address for correspondence: [email protected]

Keywords: RNA metabolism, mitochondria, fibroblasts

Background: Defects in RNA metabolism represent an important and still not well-defined pathogenic mechanism in ALS and frontotemporal dementia (FTD) in association to TDP-43 and FUS RNA-binding proteins, as well as to the recent discovered C9ORF72 gene. Both loss- and gain-of function hypotheses have been suggested to explain the pathogenic role of these three genes in ALS and FTD. In particular, the loss of TDP-43 and FUS RNA-binding activities is supposed to greatly impair post-transcriptional regulatory mechanisms associated to pre-mRNA splicing, mRNA transport, stability and translation of target transcripts.

However, some recent literature data indicate that TDP-43 may also localize at mitochondria and that mitochondria functionality is altered in disease cellular models obtained by over-expressing both wild-type and mutant TDP-43. Mitochondrial dysmorphology and dysfunction have indeed been largely described in muscle and brain tissues from ALS patients and also in experimental cell and animal models expressing mutant SOD1 gene. To date the potential link between RNA metabolism defects and mitochondrial activity has not been explored.

Objectives: To investigate whether mitochondrial function is altered in physiological disease cell models such as human fibroblasts derived from familial ALS patients carrying mutations in genes involved in RNA metabolism.

Methods: Primary fibroblasts were obtained from ALS patients carrying mutations in TARDBP and C9ORF72 genes and from age- and sex-matched healthy controls. Mitochondrial morphology was analyzed by measuring specific parameters upon DsRedMito transfection. Several parameters referring to mitochondrial functionality and dynamics were analyzed and compared to control fibroblasts.

Results: Our results indicate that mitochondrial morphology and activity are altered both in TARDBP- and C9ORF72-mutated fibroblasts although in a different and gene-specific manner. Mitochondrial network appeared highly fragmented in mutant TDP-43 fibroblasts, as also sustained by changes in fission/fusion protein levels and in mitochondrial mass. Interestingly, over-expression of wild-type TDP-43 in mutant fibroblasts was able to rescue the observed mitochondrial morphology alterations. Mutant TARDBP and C9ORF72 fibroblasts also exhibited different and specific alterations in mitochondrial activity by measuring mitochondrial membrane potential, respiration rate and ATP synthesis. Moreover, our immunofluorescence and sub-cellular fractionation data do not suggest that TDP-43 is localized at mitochondria and that it directly induces mitochondrial dysfunction in mutant TARDBP-expressing cells.

Discussion and conclusion: Mutant TDP-43 and C9ORF72 may affect not only RNA metabolism, but also mitochondrial function in peripheral disease cell models obtained from familial ALS patients. Mutant fibroblasts may recapitulate the mitochondrial dysfunction occurring in affected neurons and therefore may represent a suitable disease cell models to study pathogenic mechanisms and for biomarker discovery and drug screening.

Acknowledgements: The Italian Ministry of Health (grant RF-2009-1473856).

P186 ALTERATIONS IN SARCO/ENDOPLASMIC RETICULUM PROTEINS IN HUMAN SKELETAL MUSCLE IN ALS AND OTHER NEUROPATHIC CONDITIONS

Derusso A¹

Biliciler S³

Kwan J²

Chin E¹

^jUniversity of Maryland, College Park, MD, USA

^kUniversity of Maryland, Baltimore, MD, USA

^lUniversity of Texas Health Sciences Center, Houston, TX, USA

Email address for correspondence: [email protected]

Keywords: skeletal muscle biomarkers, ER stress, Ca²⁺ regulation

Background: Recent studies in the SOD1^G93A mouse model identified alterations in skeletal muscle intracellular Ca²⁺ regulation due to decreased expression of sarcoplasmic reticulum (SR) proteins (SERCA1 and SERCA2) (1). This was associated with increased expression of endoplasmic reticulum (ER) stress proteins (2) and proteins involved in regulating protein synthesis. These protein changes tracked with disease progression. Alterations in SR/ER function, possibly due to protein mis-folding, and the downstream changes in SR/ER protein expression may provide useful biomarkers of cellular pathology and disease progression in denervated muscle.

Objectives: To assess the changes in SR/ER proteins and markers of protein synthesis in skeletal muscle from ALS and other neuromuscular diseases.

Methods: Muscle biopsy samples were obtained from a tissue bank at the University of Texas Health Sciences Center in Houston. These samples were from normal (no evidence of neuromuscular disease, NORM; n = 3), disease control (myopathies, MYOP; n = 3) and ALS (ALS; n = 3) as determined by clinical evaluation and histopathology. Samples were homogenized and then analyzed for target protein expression by western blot. Images were quantified by densitometry using Image J.

Results: The SR Ca²⁺ pump protein SERCA1 was not different across groups and SERCA2 was increased in ALS, 1.7-fold vs. NORM and 2.3-fold vs, MYOP. ER stress markers were also increased: GRP78/BiP by 6.8 -fold in ALS vs. NORM (p < 0.05) and CHOP 2.2 -fold in ALS vs. NORM and 2.1 -fold vs. MYOP (p < 0.05). GRP78/BiP was also elevated in MYOP vs. NORM (6.4 -fold; p = 0.068). Both total and phosphorylated p70S6K, markers of muscle hypertrophy and protein synthesis, were elevated in ALS (2.1 and 1.9 -fold, respectively, in ALS vs. NORM; p < 0.05) but unchanged in MYOP. The ratio of phosphorylated/total p70S6K, however, was decreased in ALS (to 90% vs. NORM; p < 0.05) and in MYOP (to 86% vs. NORM). Total Akt was also elevated in ALS (3-fold vs. NORM).

Discussion and conclusion: These preliminary data indicate that ER stress is activated in skeletal muscle under denervation conditions, with increased expression of chaperone protein GRP78/BiP and ER stress apoptotic marker CHOP. Markers of protein synthesis are also elevated suggesting possible futile cycling of protein synthesis and breakdown. SR Ca²⁺ handling proteins were not reduced, however, in contrast to the SOD1^G93A model.

Conclusions: Quantitative analysis of muscle protein expression may be useful for discriminating between ALS and other myopathies.

References:

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P187 UPREGULATION OF GLUTAMMINASE IN MICROGLIA OF SPINAL CORDS FROM SPORADIC ALS PATIENTS

Inose Y¹

Shibata N¹

Niida-Kawaguchi M¹

Kakita A²

^mDepartment of Pathology, Tokyo Women's Medical University, Tokyo, Japan

ⁿDepartment of Pathology, Brain Research Institute, Niigata University, Niigata, Jordan

Email address for correspondence: [email protected]

Keywords: microglia; oxidative stress; tumor necrosis factor-α (TNF-α)

Background: Previous studies have suggested implications for inflammation (1) and excitotoxicity (2) in amyotrophic lateral sclerosis (ALS). Glutaminase (GLS) is an intracellular enzyme that catalyzes the deamination of glutamine to glutamate, and it has been shown to be upregulated in the presence of tumor necrosis factor-α (TNFα) (3).

Objectives: To determine the involvement of GLS in ALS.

Methods: Cervical segments of spinal cords from 10 sporadic ALS patients and 10 age-matched control subjects were examined. Formalin-fixed, paraffin-embedded or frozen sections of each case were used for immunohistochemistry, using specific antibodies to GLS (Abnova), TNFα (CST), ionized calcium-binding adaptor molecule 1 (Iba1) (Wako), glial fibrillary acidic protein (Dako), and N-methyl-D-aspartate receptor (NMDAR) (CST) . Antibody binding was visualized by the immunoperoxidase and immunofluorescence methods. Fresh materials of each case were used for immunoblotting, using antibodies to GLS and β-actin. Immunoreaction was detected by the chemiluminescence method, and optical densities of GLS-immunoreactive signals were statistically compared between the ALS and control groups by unpaired Student’s t-test.

Results: Immunohistochemical analysis revealed that immunoreactivities for GLS and TNFα were mainly localized in Iba1-identified microglia, and staining was more intense in the ALS group. NMDAR immunoreactivity was localized in lower motor neurons. Immunoblot analysis disclosed a significant increase in β-actin-normalized GLS signals in the ALS group as compared to the control group (p < 0.05).

Discussion and conclusion: The present results suggest the involvement of TNFα-induced GLS upregulation in microglia and NMDAR-mediated glutamate neurotoxicity in ALS in an autocrine or paracrine manner.

Acknowledgements: The authors wish to thank M Karita, H. Takeiru, N. Sakayori, F. Muramatsu and S. Iwasaki for technical assistance.

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• Rizzo F. et al. Cell Mol Life Sci. 2014; 71:999–1015.
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P188 IDENTIFICATION OF TRANSTHYRETIN AGGREGATES IN THE CSF OF ALS PATIENTS

Bowser R¹

Collins M²

An J¹

Peller D^1,3

Ladha S¹

^oBarrow Neurological Institute, Phoenix, AZ, USA

^pUniversity of Pittsburgh, Pittsburgh, PA, USA

^qArizona State University, Tempe, AZ, USA

Email address for correspondence: [email protected]

Keywords: cerebrospinal fluid, protein aggregation, biomarkers

Background: Many proteins have been identified within intracellular inclusions in motor neurons and glia of ALS patients. These include the RNA binding proteins TDP-43 and FUS, as well as SOD1. Misfolded SOD1 has also been proposed to be neurotoxic by cell-to-cell transfer via synaptic connections and exosome transport between cells. It may also be toxic via activation of neighboring microglia. We have a long standing interest investigating the CSF proteome in ALS patients, and have explored the possibility that misfolded or aggregated proteins may also exist in the CSF of ALS patients and contribute to the pathobiology of the disease.

Objectives: To identify aggregated proteins in the CSF of ALS patients and characterize potential neurotoxicity of these protein aggregates.

Methods: CSF was collected from ALS, healthy control and subjects with disease mimics, using our standard operating procedures for collection, processing and storage. The six most abundant proteins were removed from the CSF by column chromatography using HPLC (Agilent Hu-6HC column). The CSF samples were then fractionated by size exclusion chromatography on an Acquity UPLC (BEH200 SEC), and each fraction was analyzed by non-denaturing gel electrophoresis. Western blot analysis was performed on the resulting blots, probing the blots with antibodies to proteins of interest. To examine potential neurotoxic nature of the protein aggregates, we cultured primary motor neurons generated from rat embryonic spinal cord. Protein aggregates were generated in vitro and incubated with cultured cells to examine effects on cell viability and cell morphology.

Results: We determined that transthyretin (TTR) exists in a high molecular weight aggregate in the CSF of approximately 75% of ALS patients. TTR aggregates were present in a very small proportion of healthy or disease control subjects. These results have been confirmed by filter binding assays.

Discussion and conclusions: We identified a novel extracellular protein aggregate in the CSF of ALS patients. Aggregation of TTR may disrupt its normal function and aggregated TTR has neurotoxic potential to motor neurons. TTR aggregates may also activate glia via cell surface receptors. Patients containing TTR aggregates did not have mutations in the TTR gene known to cause amyloidosis. It is possible that extracellular aggregates of TTR and other proteins may help facilitate spread of ALS to distal regions of the CNS. Our results highlight a novel route of disease pathogenesis/spread and a new therapeutic target.

Acknowledgments: This work was supported by NIH grant NS061867 and a Pfizer research grant to RB.

P189 ALS/FTLD WITH SQSTM1 MUTATIONS; LESSONS FROM PAGET’S DISEASE OF BONE

Rea S¹

Goode A^2,3

Majcher V²

Searle M³

Layfield R²

^rCentre for Medical Research, University of Western Australia, and Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia

^sSchool of Life Sciences

^tCentre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, UK

Email address for correspondence: [email protected]

Keywords: SQSTM1, p62, autophagy

Background: Recently mutations affecting the SQSTM1 gene, which encodes the p62/SQSTM1 protein, have been genetically associated with a small number of cases of ALS and FTLD. SQSTM1 mutations have previously been identified as a common cause of the skeletal disorder Paget’s disease of bone (PDB) and we have extensively studied them in this bone context. Significant coexistence of ALS/FTLD with PDB has not been recognized to date. However, although many mutations are ALS/FTLD-specific, several SQSTM1 mutations are in fact common to both ALS/FTLD and PDB.

Objectives: To reassess how previous knowledge of the impact on p62/SQSTM1 function, of specific PDB-associated SQSTM1 mutations which are now known to be relevant in ALS/FTLD, may provide new insights into ALS/FTLD disease mechanisms.

Methods: A literature analysis was performed to identify p62/SQSTM1-dependent pathways which SQSTM1 mutations common to both PDB and ALS/FTLD impact upon, and also the functional significance of different domains of the p62/SQSTM1 protein which ALS/FTLD-specific mutations map to. Published data was reinterpreted in the context of previously proposed pathophysiological mechanisms in ALS/FTLD.

Results: SQSTM1 mutations common to both PDB and ALS/FTLD have been reported to affect p62/SQSTM1- dependent NF-kappaB signalling, Keap1/Nrf2 signalling and autophagy pathways in various cell-based and animal model systems. Further, ALS/FTLD-specific mutations directly map to regions within the p62/SQSTM1 primary sequence shown to be essential for all three of these pathways. Changes associated with the three pathways are all consistent with proposed pathophysiological mechanisms in ALS/FTLD.

Discussion and conclusion: Investigation into the effects of disease-associated SQSTM1 mutations on p62/SQSTM1-dependent pathways in model systems directly relevant to ALS/FTLD is now merited.

Acknowledgements: RL, MSS and AG are supported by the UK MND Association (Ref:6095).

Reference:

• Rea SL. et al. Exp Cell Res. (2014) 325, 27–37.
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P190 PROTEIN DISULPHIDE ISOMERASE IS PROTECTIVE AGAINST THE PATHOLOGIES INDUCED BY MUTANT SOD1, TDP-43 AND FUS BOTH IN VITRO AND IN VIVO IN AMYOTROPHIC LATERAL SCLEROSIS

Parakh S^1,2

Spencer DM²

Halloran M^1,2

Arachchillage N²

Soo KY²

Farg M²

Walker AK²

Turner BJ³

Cole NJ¹

Atkin J^1,2

^uMacquarie University, Sydney, NSW, Australia

^vLa Trobe University, Melbourne, Vic, Australia

^wThe Florey Institute, Melbourne, Vic, Australia

Email address for correspondence: [email protected]

Keywords: ER stress, protein disulphide isomerase, SOD1

Background: Superoxide dismutase (SOD1), Fused in Sarcoma (FUS) and Tar-DNA binding protein-43 (TDP-43) are key proteins linked to amyotrophic lateral sclerosis (ALS) pathology. They share similar pathological hallmarks such as protein aggregation, inclusion formation, Endoplasmic Reticulum (ER) stress and ER-Golgi trafficking defects. Whilst neurodegenerative mechanisms are not fully defined in ALS, dysfunction to the ER is increasingly implicated in pathology (1). Protein disulphide isomerase (PDI) a key protein induced during ER stress, catalyses the formation of protein disulphide bonds and it also possesses general chaperone activity. It is primarily located in the ER but it is also found in other cellular locations. Our laboratory previously showed that over-expression of PDI is protective against mutant SOD1 pathology and a molecular mimic of PDI - BMC reduces SOD1 inclusions in neuronal cells(2). PDI also co-localises with FUS and TDP-43 positive inclusions in ALS patients. However, the mechanism of the protective action of PDI remains unclear.

Objectives: To investigate: (i) whether over-expression of PDI is protective against mutant FUS and TDP-43 cellular pathologies; (ii) how an ER chaperone can be protective against mutant ALS proteins which are not generally present within the ER; (iii) the mechanisms underlying the protective activity of PDI; (iv) effect of PDI on transgenic FUS^R521H fish and effect of BMC on SOD1^G93A mice.

Methods: Mutations in the active site of PDI were designed and expressed in motor neuronal cell lines with ALS-linked mutant proteins. Cellular pathologies were examined using immunofluorescence and immunocytochemistry. Transgenic zebra fish expressing FUS^R521H embryos were microinjected with constructs containing PDI-mkate and observed for FUS aggregation 24 h post fertilization. BMC was administered to SOD1^G93A mice by ICV using mini osmotic pumps and counted at P100 (40 days infusion). Motor neurons in every third section were counted from a total of 30 ventral horns per mouse.

Results: PDI was protective against mutant FUS and TDP-43 induced pathology - ER stress, ER-Golgi trafficking defects and mis-localisation into cytoplasm. The disulphide activity, in contrast to its chaperone activity, was found to be the key factor necessary for its protective activity. However, we also found that the protective activity is enhanced when PDI is localised in the cytoplasm rather than the ER. Interestingly, PDI significantly decreased aggregation in FUS^R521H transgenic zebra fish. Furthermore, BMC was found to rescue motor neuron loss in SOD1^G93A mice.

Conclusion: We demonstrate that PDI is protective against the major misfolded proteins linked to ALS. Hence small molecule mimics of PDI activity could be effective as a novel and broadly acting therapeutic agent in multiple forms of ALS.

References:

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P191 THE IMMUNOMODULATORY EFFECTS OF HUMAN MESENCHYMAL STEM CELLS ON PERIPHERAL BLOOD MONONUCLEAR CELLS IN ALS PATIENTS

Kwon M²

Noh M-Y¹

Oh K-W¹

Cho K-A¹

Kang B-Y¹

Kim K-S³

Kim S-H¹

^xHanyang University, Seoul, Republic of Korea

^yCHA University, Gyeonggi-do, Republic of Korea

^zCoreStem Inc., Seoul, Republic of Korea

Email address for correspondence: [email protected]

Keywords: mesenchymal stem cell, regulatory T lymphocyte, cytokines

Background: In a previous study, we reported that central injection of MSCs slowed disease progression in SOD1^G93A mice. In the present study, we found that central MSC administration vastly increased the infiltration of peripheral immune cells into the spinal cord of SOD1^G93A mice. Thus, we investigated the immunomodulatory effect of MSCs on peripheral blood mononuclear cells (PBMCs) in ALS patients, focusing on regulatory T lymphocytes (T_reg; CD4⁺/CD25^high/FoxP3⁺) and the mRNA expression of several cytokines (IFN-γ, TNF-α, IL-17, IL-4, IL-10, IL-13 and TGF-β).

Methods: Peripheral blood samples were obtained from nine healthy controls (HC) and sixteen patients who were diagnosed with definite or probable ALS. Isolated PBMCs from the blood samples of all subjects were co-cultured with MSCs for 24h or 72h.

Results: Based on a FACS analysis, we found that co-culture with MSCs increased the T_reg/total T lymphocyte ratio in the PBMCs from both groups according to the co-culture duration. Co-culture of PBMCs with MSCs for 24h led to elevated mRNA levels of IFN-γ and IL-10 in the PBMCs from both groups. However, after co-culturing for 72h, although the IFN-γ mRNA level had returned to the basal level in co-cultured HC PBMCs, the IFN-γ mRNA level in co-cultured ALS PBMCs remained elevated. Additionally, the levels of IL-4 and TGF-β were markedly elevated, along with GATA mRNA, in the both HC and ALS PBMCs co-cultured for 72h. The elevated expression of these cytokines in the co-culture supernatant was confirmed via ELISA. Furthermore, we found that the increased mRNA level of indoleamine 2,3-dioxygenase (IDO) in the co-cultured MSCs was significantly correlated to the increase in Treg induction.

Conclusion: These findings of Treg induction and increased anti-inflammatory cytokine expression in co-cultured ALS PBMCs provide indirect evidence that MSCs may play a role in the immunomodulation of inflammatory responses when MSC therapy is targeted to ALS patients.

Acknowledgements: This study was supported by grants from the Korea Healthcare Technology R&D Project of the Ministry for Health & Welfare Affairs of the Republic of Korea (A101712 and A120182).

P192 CALCIUM CHELATING ANTICOAGULANTS SIGNIFICANTLY AFFECT MONOCYTE/MACROPHAGE ACTIVATION ANTIGEN EXPRESSION IN ALS PATIENT BLOOD SPECIMENS

Zhang R¹

Miller RG²

Katz J²

Forshew DA²

McGrath MS¹

^aaUniversity of California, San Francisco, San Francisco, California, USA

^abCalifornia Pacific Medical Center, San Francisco, California, USA

Email address for correspondence: [email protected]

Keywords: immune activation; monocyte, anticoagulant

Background: Persistently activated monocytes expressing increased levels of HLA-DR and CD16 co-expressed are present in blood from patients with ALS. The degree of blood monocyle activation is correlated with plasma levels of LPS, a well-known monocyte activator, and is directly related to rate of disease progression in ALS. Our recent studies of ALS patient peripheral blood mononuclear cell gene expression showed upregulation of both M1 interferon-induced genes and M2 alternative activation genes, both consistent with a pattern of LPS activation. Together these data have led to the speculation that systemic immune system may play a significant role in ALS disease pathogenesis. However, there are discrepancies in the reported levels of monocyte activation in ALS (1,2). In vitro immune assays that require macrophage function can only be performed on heparinized blood, as calcium chelation with acid-citrate-dextrose (ACD) or EDTA blocks macrophage function.

Objectives: The goal of the current study was to test whether ACD, a calcium chelator that interferes with macrophage function, would also effect activation antigen expression as compared to a parallel evaluation of heparinized blood a form of anticoagulation that conserves macrophage function, in ALS.

Methods: 10 paired blood specimens from ALS patients were collected into two vacationer tubes containing either sodium heparin or ACD. Flow cytometry was performed to evaluate both T-cell and monocyte activation antigens.

Results: CD14 + monocytes in heparinized blood expressed significantly higher levels of HLA-DR (873.1 ± 244.6 vs. 423.4 ± 97.6, p = 0.0020) and CD16 (42.0 ± 11.2 vs. 10.5 ± 5.8, p < 0.0001) than monocytes in ACD blood. Monocytes expressing elevated HLA-DR and CD16 in heparinized blood showed significant increases in CD14-associated side scatter as a measure of granularity as compared to monocytes from ACD blood (p = 0.0039). A different pattern of anticoagulant effect was observed in analysis of T-cell activation. There were no differences in proportional levels of CD4 and CD8 T-lymphocyte subsets between heparinized and ACD blood. T-cell activation levels as quantified by detection of CD38 antigens on the surface of CD4 and CD8 T-cells was also similar between two anticoagulants.

Discussion and conclusion: The current study showed that two commonly used anticoagulants exerted various effects on expression of monocyte-inflammation/activation-related antigens. Significantly different levels of monocyte activation were observed between two anticoagulants. Monocytes which showed significantly increased levels of activation in heparinized blood had all of those disease associated levels revert to background when ACD was employed as the anticoagulant. T-cell markers were not different in either anticoagulant. As an anticoagulant, ACD works by chelating Ca²⁺, which is essential for coagulation but also as a modulator of calcium-dependent monocyte activation. Our results suggest that special care must be taken in choosing an anticoagulant for a sample in which monocyte activation is to be evaluated.

P193 NEURONAL LOSS IN FUNCTIONAL ZONES OF THE CEREBELLUM IN ALS

Tan R¹

McGinley C²

Hassani M¹

Kril J²

Kiernan MC²

Hornberger M¹

Halliday G¹

^acNeuroscience Research Australia, NSW, Australia

^adThe University of Sydney, NSW, Australia

Email address for correspondence: [email protected]

Keywords: cerebellum, neurons, pathology

Introduction: The neuropathological hallmark of ALS is degeneration of upper and/or lower motor neurons. Depending on the pattern of disease onset, approximately equal numbers of patients have upper limb-,lower limb- or bulbar-onset (1).The cerebellum has traditionally been regarded as a crucial relay station for motor regulation, and has multiple reciprocal connections with the motor regions implicated in ALS (2).We previously demonstrated cerebellar atrophy in ALS, and the severity of atrophy in the inferior lobules and cerebellar vermis was found to correlate with measures of motor functional status (Tan 2014, submitted). However, the integrity of cerebellar neurons, particularly the large Purkinje cells (PC), which constitute the sole output neurons of the cerebellar cortex, has not been examined in ALS.

Objective: To assess PC in the lateral lobules and cerebellar vermis in ALS.

Methods: Following institutional approvals, paraffin-embedded tissue blocks from patients with pathologically-confirmed ALS (n = 30, 10 of each onset) and controls (n = 10) from the NSW Tissue Resource Centre were prepared for histological examination of neurons. The number of PC along two half lobules were quantified and normalized to umber of folia within each region-of-interest. ANOVA with posthoc Bonferoni compared PC densities/folia dichotomised by main clinical diagnosis or by type of onset.

Results: PC loss (∼22%) was identified in the cerebellar vermis of ALS cases with lower-limb onset disease (p < 0.05) and correlated with disease duration (Rho = -0.38, p = 0.04).This was significant in comparison to upper limb- and bulbar-onset cases (p = 0.001),which did not differ from controls (p > 0.05).An average of ∼10% loss of neurons occurred per year with no loss estimated at onset. No significant differences were identified with disease-onset in the lateral cerebellum.

Discussion: This is the first report of PC loss in the cerebellar vermis in ALS and we demonstrate a significant reduction in cases with lower limb-onset. The vermis is classically thought to receive somatic sensory input from ascending spinal pathways and be involved in the kinematics of ongoing movement. However, recent evidence in animal models have challenged this theory and shown that some PC located in the vermis receive dense direct input from motor neurons in the motor cortex and innervate lower but not upper limb motor neurons in the spinal cord (3, 4).Our findings suggest that such vermal PC are preferentially vulnerable in lower limb-onset ALS, most likely as a consequence of early distal axonopathy. This is likely to impact more widely on vermal function in lower-limb ALS patients.

Acknowledgements: The authors are very grateful for the support of MNDRIA and NHMRC.

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P194 MORPHOLOGICAL EVIDENCES OF GLIAL INFLAMMATION THAT CONFER SPINAL WHITE MATTER DEGENERATION OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

Hayashi S¹

Yamasaki R²

Masaki K¹

Murai H¹

Okamoto K

Kira J-I¹

^aeDepartment of Neurology

^afDepartment of Neurological Therapeutics, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

^agGeriatrics Research Institute and Hospital, Maebashi, Japan

Email address for correspondence: [email protected]

Keywords: glia, inflammation, MCP1

Background: We previously reported that glial inflammation (GI) markers were elevated in the CSF of sporadic amyotrophic lateral sclerosis (1). However, morphological profiles regarding GI are poorly understood. In 70% of ALS cases infiltrations of massive microglia/macrophage (Mi/MΦ) with foamy appearance were observed not only in the corticospinal tracts (CST), but also in the anterolateral funicles outside the CST (ALFoc), corresponding to the distribution of myelin pallor (2). Classical antibodies against CD 68 or Iba1, however, could not discriminate whether Mi/MΦ in the CNS were derived from peripheral monocytes or resident microglia. Regarding this issue, Yamasaki et al. demonstrated that chemokine receptor, CX3CR1, was expressed on resident microglia, while CCR2, on infiltrating monocytes (3). These findings might offer morphologically novel evidence of GI in sALS tissues.

Objectives: To characterize glial reactions and their related immune cells in sALS spinal white matter.

Methods: Ten-percent buffered formaline-fixed, paraffin-embedded 5-μm-thick transverse thoracic cord sections of sALS cases (n = 7) and non-ALS cases (controls, n = 5) were examined by immunohistochemical methods. The antibodies used were against: Iba-1 (Mi/MΦ); CD68 (Mi/MΦ); CD11c (dendritic cells); CCR2 (monocyte); CX3CR1 (resident microglia); MCP-1 (ligand of CCR2); fractalkine (ligand of CX3CR1); CD45RO (T cells); CD20 (B cells); iNOS; and GDNF. Degree and distribution of infiltrating cells in both CST and ALFoc were examined, and immunoreactive cells in each region were quantified for statistical comparison. For correlation analysis, data were obtained from 30 spinal cord sections of five sALS patients.

Results: Compared to controls, sALS spinal cords showed CD68-, Iba1-, CCR2-, and CD11c-immunopositive cells in the ALFoc as many as those in the CST (p > 0.05), while the numbers of T cells in the CST were significantly increased than those in the ALFoc (p < 0.01). Many MCP-1-immunopositive granules were detected not only in the entire (CST+ ALFoc) anterolateral funicles (ALF), but also in the posterior funicles (PF). There were positive correlations between the numbers of MCP-1- vs. CD11c- (r = 0.737, p < 0.001), MCP-1- vs. CD68- (r = 0.439, p < 0.05), and MCP-1- vs. Iba1-immunopositive cells (r = 0.450, p < 0.05), encompassed in the entire ALF. There were intense iNOS-immunopositive foamy cells in the entire ALF, but scarce on GDNF-immunoreactivities. No pathological findings were observed regarding CX3CR1-, fractalkine-, and CD20-immunostainings in the CST, ALFoc, and PF of all specimens.

Discussion and conclusion: Mi/MΦ in the entire ALF of sALS spinal cords might originate from peripheral monocytes attracted by MCP-1, and have neurotoxic nature, suggesting that MCP1 could be one of the therapeutic targets to ameliorate white matter degenerations of this disorder.

References:

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P195 CEREBRAL LESIONS OF AMYOTROPHIC LATERAL SCLEROSIS (ALS) PATIENTS IN COMMUNICATION ABILITY STAGE V: TOTALLY LOCKED-IN STATE

Hayashi K¹

Mochizuki Y^1,2

Nakayama Y³

Nagao M¹

Shimizu T¹

Komori T¹

Arai N³

Nakano I¹

^ahTokyo Metropolitan Neurological Hospital, Tokyo, Japan

^aiTokyo Metropolitan Kita Medical and Rehabilitation Center for the Disabled, Tokyo, Japan

^ajTokyo Metropolitan Institute of Medical Science, Tokyo, Japan

Email address for correspondence: [email protected]

Keywords: totally locked-in state, communication neuropathological findings

Background: We determined stage classifications based on communication ability and analyzed the characteristics of ALS patients. About 13% of patients (1) with ALS on long-term mechanical ventilation (LTMV) develop into a totally locked-in state (2), where all voluntary movements have been lost and communication is impossible by any means (communication stage V (3)). Neuropathological features of stage V ALS patients include severe degeneration of motor neurons and other systems. However, findings of cerebral lesions have not been reported.

Objective: To determine the characteristics of cerebral lesions in stage V patients.

Method: In four autopsied patients with ALS at stage V, we investigated the radiological profiles at the beginning of stage V as well as pathological features.

Results: Means and ranges of disease onset, disease duration, duration from onset to LTMV, and onset to stage V were 40 years (13–57), 14.8 years (9–26), 18.5 months (8–24), and 6.5 years (3–10), respectively. Patient 1’s symptoms were sporadic, and displayed an accumulation of transactivation response DNA-binding protein of 43 kDa (TDP-43). Patients 2 and 3 had copper/zinc superoxide dismutase (SOD1) gene mutations (p.V118L, p.C146R) respectively. Patient 4 had a p.P525L mutation in the fused in sarcoma (FUS) gene. They had no cognitive disturbances from onset to stage V or history of brain ischemia. Radiologically, all patients displayed mild frontal and temporal lobe atrophy at the beginning of stage V.

Neuropathological findings: All patients exhibited common elements including severe degeneration of both upper and lower motor neurons and severe fiber loss in the tegmentum of the brainstem and the anterolateral funiculus of the spinal cord. The globus pallidus and substantia nigra showed mild neuronal loss and gliosis. The patients also exhibited certain variations. Patient 1 displayed neuronal loss in the frontal and temporal cortex and severe fiber loss in the cerebral white matter. Patients 2 and 3 exhibited relatively preserved frontal/temporal lobes, but displayed severe fiber loss in the posterior column of the spinal cord. Patient 4 showed neuronal loss in the frontal cortex and fiber loss in the cerebral white matter.

Discussion and conclusion: All patients radiologically displayed mild frontal and temporal atrophy at the beginning of stage V. However, pathological findings in terms of cerebral lesion indicated variations. Degeneration of the cerebrum of ALS patients progresses even after stage V, while disease progression may be distinct in each patient.

Acknowledgements: Study Supported by: JSPS KAKENHI (Grant-in-Aid for Scientific Research (B)) Grant Number 25293449 and by the Joint Program for ALS Research at the Tokyo Metropolitan Institute of Medical Science.

References:

• Kawata A. Rinsho Shinkeigaku. 2008; 48:476–480.
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P196 RESPIRATORY-AIDED, LONG-TERM SURVIVAL CASES OF AMYOTROPHIC LATERAL SCLEROSIS (ALS) WITH COMMUNICATION ABILITIES, MOTONEURON SYSTEM-CONFINED DEGENERATION, AND SCANTY TDP-43 AGGREGATION-A SUBGROUP OF ALS?

Mochizuki Y^1,2

Hayashi K¹

Nakayama Y³

Shimizu T¹

Kamide M⁴

Ogino M⁵

Komori T¹

Isozaki E¹

Nakano I¹

^akTokyo Metropolitan Neurological Hospital, Tokyo, Japan

^alTokyo Metropolitan Kita Medical and Rehabilitation Center for the Disabled, Tokyo, Japan

^amTokyo Metropolitan Institute of Medical Science, Tokyo, Japan

^anAtsugi City Hospital, Kanaagaa, Japan

^aoKitasato University School of Medicine, Kanagwa, Japan

Email address for correspondence: [email protected]

Keywords: totally locked-in state, long-term mechanical ventilation (LTMV), neuropathological findings

Background: About 13% of patients (1) with ALS surviving on long-term mechanical ventilation (LTMV) develop into a totally locked-in state (TLS) (2), which refers to a state in which all voluntary movements are lost and communication by any means is impossible. However, it is known that some respirator-aided, long-term survival patients maintain communication abilities. The position that the latter group occupies along the spectrum of ALS cases has not been described.

Objective: To clarify, along the ALS cases, the position of the long-surviving ALS subgroup with good communication abilities.

Methods: Three autopsied ALS cases with an approximately 30-year survival and good communication abilities were clinicopathologically investigated in direct comparison with previously reported ALS cases suffering from a TLS (2, 3).

Case reports: Clinical course: Age of onset and disease duration of cases 1, 2, and 3 were 48, 55, and 31, and 28, 29, and 33 years, respectively. Case 1 underwent a tracheostomy 8 years after disease onset, and required mechanical ventilation regularly 26 years after onset. Case 2 and case 3 underwent mechanical ventilation 14 and 6 years after disease onset, respectively. Case 1 and 3 were in a state of complete tetraplegia and case 2 was able to move only his knee joint a little, and the three cases were in a state of bulbar palsy. However they could communicate well via eye movements.

Neuropathological findings: In all the cases, both upper and lower motor neurons were markedly degenerated while the brainstem tegmentum was preserved. Multiple system degeneration, a characteristic pathology of ALS in the TLS (2), was not seen. A few normal-looking motor neurons remained in the anterior horn of the spinal cord. Neither hypertrophic astrocytes nor macrophages were observed. There were no TDP-43-immunoreactive inclusions in the lower motor neurons of any case and only occasional ones in the cerebral cortex of case 3. Bunina bodies were not seen.

Discussion and conclusion: Each of the present cases displayed a much slower disease progression into required respiratory assistance compared with typical ALS patients and also maintained good communication in spite of survival on LTMV. Neuropathological findings of remaining good-shaped motor neurons, motoneuron system-confined degeneration, and a few TDP-43-immunoreactive inclusions indicate a significant difference from findings of ALS in the TLS. Thus, our clinicopathological investigations of these three cases give rise to the possibility that there is a distinct subgroup characterized by the above-mentioned features.

Acknowledgements: Study Supported by: JSPS KAKENHI (Grant-in-Aid for Scientific Research (B)) Number 25293449 and the Joint Program for ALS Research at the Tokyo Metropolitan Institute of Medical Science.

References:

• Kawata A. et al. Rinsho Shikeigaku. 2008; 48:476–480.
   PubMedGoogle Scholar
• Hayashi H. Kato S. J Neurol Sci. 1989; 93:19–35.
   PubMed Web of Science ®Google Scholar
• Hayashi K. et al. Rinsho Shinkeigaku. 2013; 53:98–103.
   PubMedGoogle Scholar