THEME 8 IN VITRO EXPERIMENTAL MODELS

P127 DEVELOPMENT OF AN IN VITRO MODEL OF HUMAN MOTOR NEURONS

LEE JM¹, CHUNG RS², GUILLEMIN GJ^{1 ,3}

¹University of New South Wales, Department of Pharmacology, Sydney, Australia, ²NeuroRepair Group, Menzies Research Institute, University of Tasmania, Hobart Tasmania, Australia, ³St Vincent's Centre for Applied Medical Research, Sydney, Australia

Email address for correspondence: [email protected]

Keywords: primary cultures, human motor neurons

Background: Primary cultures of human primary motor neuron (MN) represent a unique tool to study cell characteristics and properties, neurotoxicity, and neuroprotection. This model can also be used to assess molecules of clinical relevance on MN. We have developed a new technique using a combination of centrifugation and gradient density separation process from foetal spinal cord to obtain primary cultures of highly purified human foetal MN.

Methods: We have isolated motor neurons from the human foetal spinal cord (16-20 weeks of gestation). At day 3 in vitro (DIV), 1-β-D arabino furanosylcytosine (Ara-C) was used to reduce non-neuronal cell growth. The purity of MN cultures was then assessed by immunocytochemistry for p75^NTR, SMI-32 markers and cell counting. Following dissociation of spinal cords and separation by gradient centrifugation, cells were analyzed 7 days after plating by immunocytochemistry using a labeling with SMI-32 antibody and DAPI staining for the nuclei. This high purity was confirmed by flow cytometry quantification of MN purity following labeling with anti-p75^NTR.

Results: We have obtained highly purified (> 85%) primary culture of human MN. The percentage of SMI-32 + and p75^NTR + MN relative to the number of cell nuclei was 86.17 ± 2.78 and 84.74 ± 3.26 when treated with Ara-C.

Conclusions: We have successfully optimized a new method to obtain highly purified primary cultures of human fetal MN. This tool will be very important to study neurotoxicity and neuroprotection and to assess therapeutic strategies for MND.

P128 ESTABLISHMENT OF HUMAN IMMORTAL MESENCHYMAL STEM CELLS SECRETING MULTIPLE TROPHIC FACTORS USING A HUMAN ARTIFICIAL CHROMOSOME

WATANABE Y, KAWASE S, YASUI K, KITAYAMA M, NAKANO T, NAKASHIMA K

Division of Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago, Japan

Email address for correspondence: [email protected]

Keywords: mesenchymal stem cell, human artificial chromosome, gene therapy

Purpose: The administration of neurotrophic factors or growth factors is a hopeful strategy for the treatment of amyotrophic lateral sclerosis (ALS). Glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor (IGF-1), and hepatocyte growth factor (HGF) show encouraging outcomes in animal experiments. None, however, has been efficacious in human clinical trials. It is possible that a cocktail of several trophic factors could exert a mutually potentiating effect to alleviate the progression of ALS. Meanwhile cell transplantation, e.g., mesenchymal stem cell (MSC) transfer, is also promising, but it appears not to be potent enough for clinical application. It would therefore be of great interest, if technically possible, to obtain stem cells which expressed multiple trophic factors known to be beneficial for ALS. Here, we pursued that concept using a human artificial chromosome (HAC) system.

Method: A P1-derived artificial chromosome (PAC) vector was constructed with GDNF, IGF-1, and HGF genes, as well as green fluorescent protein (GFP) and luciferase genes (a total of 41,247 base pairs). It was transferred by microcell-mediated chromosome transfer (MMCT) into Chinese Hamster Ovary (CHO) hybrid cells containing the HAC with a loxP system. Trophic factors and markers genes were transferred into the HAC vector in CHO cells using Cre-loxP mediated chromosome translocation. From the CHO hybrids, the HAC vector was further transferred by MMCT to human immortalized MSCs (hiMSCs). The resultant clones were analyzed for the expression of trophic factors by ELISA studies of cultured media, luciferase assays of the lysates of hiMSC clones, and the signal intensities of GFP by fluorescence microscopy.

Results: Of 41 chromosomes transferred and CHO clones analyzed, 13 clones retained the entire DNA sequence of PAC origin (31.7%). Among them, two clones retained the genes of interest on the HAC, not on CHO chromosomes. During MMCT from the CHO hybrids to hiMSCs, 30 clones out of 46 retained the entire PAC-origin DNA sequence (65.2%). RT-PCR revealed two clones that highly expressed mRNAs of the introduced genes. ELISAs, luciferase assays, and microscopic observations for GFP revealed a single clone named hiMSC 4pacG3-31, which was suitable for transplantation in the ALS mouse model.

Conclusions: We obtained a hiMSC clone that stably expressed high levels of GDNF, IGF-1 and HGF. This clone also enables us to histologically track cell dynamics after transplantation by GFP and in vivo by luciferase. Using this clone, we are now undertaking experimental treatment of SOD1^G93A transgenic ALS model mice.

P129 INDUCE DIFFERENTIATION OF MOTOR NEURON-LIKE CELLS FROM HUMAN ADIPOSE-DERIVED STEM CELLS WITH RETINOIC ACID AND SONIC HEDGEHOG IN VITRO

ZHANG C, YANG L, GENG J, CAO J, GU R, CHEN F, KONG J, WANG Y

Department of Neurology, First Affiliated Hospital, Sun Sen-Yat University, Guangzhou, Guangdong, China

Email address for correspondence: [email protected]

Keywords: motor neuron cell-like cells, human adipose-derived stem cells, retinoic acid, sonic hedgehog

Background: The adipose-derived stem cell (ADSC) might possess the ability to differentiate into neuronal-like cells, however, there was no way to differentiate ADSCs into more specialized subtypes of neurons in vitro and in vivo. The nonspecific cells are often the source of aberrant tissue formation in transplant therapy.

Objective: To test whether retinoic acid (RA) and sonic hedgehog (Shh) have the ability to induce human adipose-derived stem cell (hADSCs) differentiation into the characteristics of motor neurons in the central nervous system.

Methods: The hADSCs were plated on plastic culture dishes at 1-104cells/cm2 and incubated for 2 days. At preconfluence, culture medium was replaced with preinduction medium composed of DF12, 20% of fetal bovine serum, 10 ng/ml of fibroblast growth factor 2 , 2% B27, 250 mM of isobutylmethylxanthine, and 100 mM of 2-mercaptoethanol. Then, the dishes were incubated for 6 h. After that, induction medium composed of DF12, 0.2% B27, 0.01 mM of all-trans RA, and 100 ng/ml of Shh was used. After 1 week, the induced medium was replaced with another one, which contained DF12, 0.2% B27, 200 ng/ml of vitamin C, 100 ng/ml of brain-derived neurotrophic factor, and 100 ng/ml of glial cell-derived neurotrophic factor. For immunocytochemistry test, the cell nuclei were labeled with Hochest. Anti-glial fibrillary acidic protein (GFAP), anti-nestin, anti-myosin heavy chain, anti- b-III-tubulin and anti-choline acetyltransferase were added. Cells were examined with a fluorescence microscope. RT-PCR was performed to compare the expression levels of Olig2, Nkx2.2, Pax6, Hb9, HoxC8, and Otx2.

Results: As early as 6 h after induction, hADSCs were changed toward neuronal morphology. After induction, hADSCs showed positive immunocytochemical staining for b- III-tubulin, choline acetyltransferase, and neuron-specific enolase. Reverse-transcriptase polymerase chain reaction characterization indicated that cells differentiated from hADSCs were restricted to the ventral spinal fate (Nkx2.2, Pax6, Hb9, and Olig2).

Conclusion: When induced by RA and Shh, hADSCs could be differentiated into motor neuron-like cells.

P130 MURINE EOC13 MICROGLIA EXPRESS FUNCTIONAL P2X7 RECEPTORS: POTENTIAL MODEL FOR AMYOTROPHIC LATERAL SCLEROSIS

BARTLETT R^1,2, SLUYTER R^1,2, YERBURY J^1,2

¹University of Wollongong, Wollongong, NSW, Australia, ²Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia

Email address for correspondence: [email protected]

Keywords: P2X7, microglia, inflammation

Background: The P2X7 purinergic receptor is a trimeric ATP-gated cation channel expressed on leukocytes including microglia. Stimulation of this receptor results in the uptake of organic ions including ethidium and the release of pro-inflammatory mediators (1). P2X7 has been implicated in a number of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P2X7 is up-regulated in microglia of human ALS spinal cords (2) and rodents carrying G93A mutant superoxide dismutase 1 (mSOD1) (3,4).

Objectives: The aim of the current study was to determine whether murine EOC13 microglia express functional P2X7 receptors for use as a model to study the role of this receptor in ALS.

Methods: Murine J774 macrophages were used as a positive control for P2X7. P2X7 mRNA and protein expression were determined by RT-PCR and immunoblotting respectively. P2X7 function was measured using a fixed-time flow cytometric ethidium uptake assay.

Results: P2X7 mRNA and protein was present in murine EOC13 microglia. ATP induced ethidium uptake into EOC13 cells in a concentration-dependent manner and with an EC50 of 130 μM, typical of murine recombinant P2X7. The most potent P2X7 agonist, BzATP, also induced ethidium uptake. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120 and AZ11645373 impaired ATP-induced ethidium uptake by 90-100%.

Discussion and conclusions: These results demonstrate that EOC13 microglia express functional P2X7 receptors. Thus, this cell line may represent a potential in vitro model for further study into the potential roles of P2X7 in ALS. Studies investigating the up-regulation of P2X7 by mSOD1 in EOC microglia are planned.

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P131 THE ALTERNATIVE ACTIVATION OF MICROGLIA FROM ALS TRANSGENIC MICE IS ATTENUATED BY THE EXPRESSION OF MUTANT SOD1

BUTTGEREIT A, REIMER V, CAMPANELLI L, TRITSCHLER H, MERDIAN I, WEYDT P, WITTING A

Ulm University, Ulm, Germany

Email address for correspondence: [email protected]

Keywords: microglia, mitochondria, PPAR

Background: Microglia are the resident macrophages in the central nervous system and screen their environment for insults. Activation of microglial cells is an early marker of disease in ALS and other neurodegenerative diseases. One activation status of microglia, the alternative activation, is characterized by a resolution of inflammation. We have recently shown that the alternative activation of microglia is inhibited by mitochondrial dysfunctions associated with neurodegenerative diseases, such as Parkinson's disease and Huntington's disease.

Objective: We hypothesize that mitochondrial dysfunctions in neurodegenerative diseases are directly linked to the inflammatory activation state of the microglia and that targeting the metabolic status of microglial cells might be a potential therapeutic approach for these diseases. We aimed to characterize the effect of the SOD1 mutation on the inflammatory profile of microglia and investigate potential pharmacological interventions.

Methods: We treated primary mouse microglia cells with agonists and antagonists of PPAR-receptors in combination with LPS and/or the alternative activation inducing cytokine IL-4 and quantified the production of IL-6. PPAR receptors play an essential part in the regulation of metabolism. In addition we isolated primary mouse microglial cells from the G93A SOD1 mouse model of ALS and investigated the alternative activation.

Results: We found that PPAR-alpha has a stimulatory effect on the alternative activation of microglial cells. We found that the alternative activation was reduced in microglial cells from transgenic mice with the mutated form of the SOD-1 gene in comparison to corresponding controls.

Conclusions: Targeting the metabolic system might therefore be a promising therapeutic approach for neurodegenerative diseases. This could be achieved by modulating the nature of the inflammatory response towards the alternative activation pathway.

P132 BCL2-A1 INTERACTS WITH PRO-CASPASE-3: IMPLICATIONS FOR AMYOTROPHIC LATERAL SCLEROSIS

CROSIO C^1,2, MURA ME¹, ESPOSITO S¹, SANNA G¹, CARRÍ MT², IACCARINO C^1,2

¹Department of Physiological, Biochemical and Cell Science, University of Sassari, Sassari, Italy, ²Fondazione Santa Lucia IRCCS, Rome, Italy

Email address for correspondence: [email protected]

Keywords: Bcl2-A1, pro-caspase-3, AP-1

Background: Expression of mutant SOD1 typical of ALS induces the expression of Bcl2-A1 (also known as Bcl2-related protein A1, BFL1; A1; Bfl-1/A1), a member of the Bcl2 family of proteins, specifically in motor neurons of transgenic mice already at the asymptomatic stage. Bcl2-A1 is protective against death of neuronal cells induced by expression of G93A-SOD1, but is detrimental upon stimulation of those cells with TNFalpha.

Objectives: We have investigated the molecular pathways leading to Bcl2-A1 transcriptional activation upon mutant SOD1 expression and the molecular mechanisms underlying the anti-apoptotic action of Bcl2-A1 in ALS cellular models.

Methods: Transfection of immortalized motor neurons (NSC-34), Western Blot GST-pulldown followed by Mass Spectrometry (MS) analysis and co-immununoprecipitation were used to study BCl2-A1 promoter and its interaction with pro-caspase-3.

Results: We report that up-regulation of Bcl2-A1 in ALS mouse model is limited to spinal cord and it is directly linked to mutant SOD1 expression, with a pattern that mirrors the tissue specificity of the disease. Although Bcl2-A1 is mainly expressed in lymphocytes, the present study indicates that constitutive mutant SOD1 overexpression does not modify either the expression level or the relative isoforms abundance in this cell type. Moreover, in immortalized motoneurons Bcl2-A1 is transcriptionally regulated by the redox sensitive transcription factor AP1, most likely contributing to lineage- and stimulus-dependent cell specificity of Bcl2-A1 transcription.

Using a GST-pull down approach combined to MS we were able to identify pro-caspase 3 as a binding partner for Bcl2-A1. This interaction is highly specific both in vivo and in vitro and it depends on Bcl2-A1 helix a9. Furthermore, Bcl2-A1 inhibits pro-caspase-3 activation in immortalized motor neurons expressing mutant SOD1 and thus induction of Bcl2-A1 in ALS mice represents a pro-survival strategy aimed at counteracting the toxic effects of mutant SOD1.

Discussion and conclusions: These data provide significant new insights on how molecular signalling, driven by expression of the ALS-causative gene SOD1, affects regulation of apoptosis in motor neurons. We also provide evidence of a new anti-apoptotic Bcl2-A1 mechanism of action. Bcl2-A1 can physically interact in vitro and in vivo, via helix a9, with pro-caspase-3, preventing its activation. To our knowledge, Bcl2-A1 is the only protein inhibitor of caspase-3 able to bind the zymogen precursor and to prevent its caspase-8 mediated activation, since both viral (serpin CrmA and p35) and cellular (IAP, inhibitor of apoptosis protein) inhibitors bind the activated form of this caspase.

P133 ULTRASTRUCTURAL STUDY OF SPINAL CORD MOTOR NEURONS IN ADAR2-DEFICIENT MICE

SASAKI S¹, YAMASHITA T², HIDEYAMA T², KWAK S²

¹Tokyo Women's Medical University, Tokyo, Japan, ²University of Tokyo, Tokyo, Japan

Email address for correspondence: [email protected]

Keywords: ADAR2-knockout mice, motor neuron, ultrastructure

Background: There have been some reports indicating that AMPA receptor-mediated excitotoxic mechanism plays a pathogenic role in ALS and SOD1-associated familial ALS model animals. Conditional ADAR2-knockout mice demonstrate that the loss of ADAR2 activity induces the slow death of motor neurons and are considered to be useful to research on sporadic ALS. However, motor neurons of spinal cords have not been studied ultrastructurally in ADAR2-knockout mice.

Objectives: To examine ultrastructural alterations of motor neurons and to clarify whether the pathological changes are similar to those reported in sporadic ALS.

Methods: We electron-microscopically studied the motor neurons of cervical spinal cords in ADAR2^flox/flox homozygous mice (15 weeks, n = 2), ADAR2^flox/+ heterozygous mice (74 wks., n = 2) and control mice (12 wks., 16 wks., 20 wks. and 24 wks., n = 2, respectively).

Results: Light-microscopically, on the plastic section of the cervical spinal cord of ADAR2^flox/flox VAChT-Cre mice stained by toluidine blue, some large anterior horn neurons with simple atrophy were scattered in the anterior horn, accompanied by astrogliosis. Anterior horn neurons showed no vacuoles. There were no myelin ovoids in the white matter, anterior root or posterior root. In ADAR2^flox/+ VAChT-Cre mice, vacuoles of various sizes were observed in the cytoplasm and nuclei of posterior horn neurons as well as in anterior horn neurons. Myelin ovoids and the swelling of myelin were frequently found in the anterior column and, to a lesser extent, in the lateral and posterior columns, and anterior and posterior roots. Electron-microscopically, in ADAR2^flox/flox VAChT-Cre mice, autophagosomes surrounded by a double-membrane and autolysosomes isolated by a single membrane were observed in the somata of anterior horn neurons. They contained sequestered cytoplasmic organelles such as mitochondria and ribosome-like structures. The cytoplasm and dendrites of motor neurons frequently contained electron-dense membranous structures. The cistern of ER was often dilated without accumulation of electron-dense material. Abnormal mitochondria containing electron-dense changes in the outer and inner membranes and cristae were frequently observed in the somata, dendrites and axons. In ADAR2^flox/+ VAChT-Cre mice, vacuolar changes were frequently demonstrated in the nuclei of motor neurons and astrocytes and, to a lesser extent, in the somata of motor neurons. The somata of anterior horn neurons often contained autophagy-related structures.

In controls, no vacuolar change was observed in the nuclei of motor neurons or astrocytes, while mitochondria occasionally showed electron-dense changes in the outer and inner membranes and cristae in the cytoplasm of motor neurons and the axons.

Conclusions: Pathological changes preferentially observed in the cytoplasm of spinal cord motor neurons in ADAR2-deficient mice are similar to those reported in sporadic ALS, but inclusions characteristic of ALS such as Bunina bodies, round bodies and skein-like inclusions have not been recognized in these mice.

P134 Functional analysis of FUS/TLS mutations involved in ALS

BIBA A¹, BAÜMER D^1,2, GUBBIN J¹, ANSORGE O², TALBOT K^1,2

¹Department of Physiology, Anatomy and Genetics, MRC Functional Genomics Unit, University of Oxford, Oxford, UK, ²Department of Clinical Neurology, John Radcliffe Hospital, University of Oxford, Oxford, UK

Email address for correspondence: [email protected]

Keywords: FUS/TLS, SMN, stress granules

Background: Onset of sporadic ALS before the age of 25 is rare, and has been hypothesised to constitute a distinct form of ALS on morphological grounds. We have recently reported a number of patients with a young onset form of ALS who presented with motor symptoms beginning between the age 17 and 22, rapid disease progression without dementia and no family history of ALS. At post-mortem, degenerating motor neurons showed intracellular basophilic inclusions staining positive for FUS protein and in three out of four of these patients, a mutation in the FUS gene was identified, in two cases a P525L change. The condition is therefore proposed to be a pathologically and genetically distinct form of sporadic ALS, juvenile ALS-FUS.

Objectives: The aim of this study was to provide in vitro correlation of the results obtained from post mortem tissue, looking for FUS co-localisation and interactions with RNA/DNA binding proteins involved in motor neuron disease.

Methods: HeLa cells and primary spinal motor neurons were transiently transfected with myc-tagged FUS containing the P525L or R521C mutation. Immunofluoresence was used to visualize the expression of FUS. Immunoprecipitation and Western blotting were performed to determine interactions of FUS.

Results: Immunofluorescence staining with confocal microscopy confirmed previous reports that WT FUS predominantly localizes to the nucleus whilst P525L-FUS, and to a lesser degree R521C-FUS, show a striking accumulation in the cytoplasm. Under conditions of oxidative stress, mutant FUS in the cytoplasm formed aggregates which also stained for the stress granule markers TiAL-1 and PABP. Double staining also revealed colocalization of mutant FUS and SMN, mutations in which cause spinal muscular atrophy. SMN from nuclear and cytoplasmic fractions was co-immunoprecipitated with anti-myc antibody indicating that SMN and myc-tagged FUS interact. The presence of mutations does not abrogate the SMN-FUS interaction.

Discussion and conclusions: In cells transfected with either the P525L or R521C FUS mutation we confirmed recent reports of mutant FUS cytoplasmic mislocalisation and accumulation in stress granules, suggesting that the insoluble inclusions seen at post-mortem originate from these stress granules. The transfected cells represent an in vitro system in which these preliminary results can be further investigated. Moreover we have shown, for the first time, that that there is co-localisation of SMN and FUS in these inclusions, and that SMN may interact with both WT and mutant FUS. Further investigation is required to explore the nature of the interaction of FUS and SMN within stress granules.

P135 STRESS KINASE ACTIVATION MODULATES ACCUMULATION OF TDP-43

MEYEROWITZ J¹, PARKER S¹, KANNINEN K¹, LIDDELL J¹, LIM N¹, SOON C¹, MASTERS C^1,2, LI Q-X^1,2, BARNHAM K^1,2, DONNELLY P¹, CROUCH P^1,2, WHITE A^1,2

¹The University of Melbourne, Parkville, Australia, ²Mental Health Research Institute, Parkville, Australia

Email address for correspondence: [email protected]

Keywords: TDP-43, JNK, metals

TDP-43 proteinopathies (FTLD-U and ALS) are characterized by loss of nuclear TDP-43 expression and C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses. This could be associated with subsequent formation of aggregates. However, the pathway of endogenous TDP-43 accumulation in SGs during chronic disease is not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in neurons exposed to chronic oxidative stress. Neuronal and non-neuronal cultures were treated with the oxidative stress inducers paraquat or sodium arsenite and examined for TDP-43 and SG processing. We found that mild oxidative stress caused a loss of nuclear TDP-43, increased cytoplasmic accumulation of the 35 kDa C-terminal TDP-43 fragment and led to formation of TDP-43 and human antigen R (HuR)-positive SGs, a proportion of which were ubiquitinated. Our studies revealed that TDP-43 accumulation induced by chronic oxidative stress can form irreversible protein aggregates that remain present after removal of stress. In contrast, HuR rapidly dissociates from SGs upon removal of stress. The co-localization of TDP-43 with SGs could be blocked by inhibition of the stress kinase, c-Jun N-terminal kinase (JNK), without effect on the localization of HuR to SGs. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. The control of TDP-43 accumulation by kinases may be mediated through phophorylation of hnRNPs that co-localize with TDP-43 in SGs. To investigate therapeutic approaches to control TDP-43 accumulation, we co-treated neurons with a potentially therapeutic metallo-complex (CuII(atsm)). This was found to inhibit kinase activation and block TDP-43 accumulation. CuII(atsm) also increased expression of key cell survival proteins and reduced oxidative stress and neurotoxicity. In vivo studies revealed that CuII(atsm) inhibited abnormal TDP-43 processing, delayed disease onset and extended lifespan in G93A and G37R SOD1 murine models of ALS. Our studies are the first to demonstrate a critical role for kinase activation in TDP-43 accumulation and describe a potential therapeutic approach based on modulation of stress kinase activity. These findings may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

P136 THE EFFECT OF EXTRACELLULAR STRESSES ON PATHOLOGY AND SURVIVAL/DEATH REGULATION IN TDP-43-FALS LCLS

WARRAICH S^1,2, YANG S¹, NICHOLSON G^1,3, BLAIR I^1,2

¹Northcott Neuroscience Lab, ANZAC Research Institute, Sydney, NSW, Australia, ²Sydney Medical School, University of Sydney, Sydney, NSW, Australia, ³Molecular Medicine Laboratory, Concord Hospital, Sydney, NSW, Australia

Email address for correspondence: [email protected]

Keywords: TDP-43, LCLs

Background: Transactive response DNA binding protein 43 kDa (TDP-43) is a major component of the ubiquitin-positive inclusions that are a pathological hallmark of affected cells in ALS. TDP-43 is an RNA/DNA binding protein that is predominantly nuclear but undergoes subcellular redistribution in ALS. It mislocalises to the cytoplasm and forms sequestered aggregates. Biochemical analyses of TDP-43 in brains and spinal cords of FTLD and ALS cases reveal that TDP-43 is pathologically modified. Since 2008, we and other groups have found missense mutations in the TARDBP gene encoding TDP-43 in familial and sporadic ALS cases, supporting a causative link between TDP-43 dysfunction and neurodegeneration.

Objectives: 1. Determine whether patient lymphocyte cell lines (LCLs) carrying ALS mutations can be used as a model for ALS; 2. To evaluate cell viability and proliferation activity of immortalized lymphocytes as a potential model of neuronal death in mutant TDP-43 familial ALS (TDP-43-FALS) cases.

Methods: Patient and control LCLs were treated with various stresses (apoptosis inducer; staurosporine, proteasome inhibitor; Mg132, and endoplasmic reticulum stress; thapsigargin) and were investigated using immunohistochemisty, immunofluoresence and Western blotting techniques. Growth curves of patient and control LCLs were obtained with trypan blue and MTS assay. Effect of extracellular stresses (staurosporine, Mg132, Thapsigargin) on patient and control LCLs was evaluated by using MTS and LDH assays.

Results: In treated LCLs carrying ALS mutations, TDP-43 was found to be pathologically modified: redistributed, ubiquinated, cleaved and hyperphosphorylated. There was no significant difference in the growth pattern and doubling time between patient and control LCLs. No difference in the endogenous level of apoptosis was found and also mutations did not seem to be rendering patient LCLs more susceptible to extracellular stresses.

Discussion and conclusion: Untreated lymphocytes did not show ALS pathology but lymphocytes treated with cellular stresses showed similar pathology to that of ALS patient cells, such as redistribution, aggregate formation, ubiquitination and phosphorylation, suggesting that these cells may be used to study disease mechanisms. The apoptotic pathophysiology in TDP-43-FALS was not reflected in the form of alteration in cell viability and proliferation activity under basal or stress stimulated conditions.

P137 THE RELATIONSHIP BETWEEN TDP-43 TRANSLOCATION AND MOTOR NEURON TOXICITY

YANG S¹, WARRAICH S^1,2, SOLSKI J¹, NICHOLSON G^1,2, BLAIR I^1,2

¹ANZAC Research Institute, Sydney, Australia, ²Sydney Medical School, University of Sydney, Sydney, Australia, ³Molecular Medicine Laboratory, Concord Hospital, Sydney, Australia

Email address for correspondence: [email protected]

Keywords: TDP-43, apoptosis, caspase

Background: The disease mechanism underlying ALS is poorly understood. Increasing evidence suggests that apoptosis and mitochondria play key roles in the death of motor neurons associated with gene mutations. This motor neuron degeneration includes features that resemble apoptosis. DNA fragmentation and increased caspase 3 activity have been found in selective vulnerable central nerve system regions (1). It has also been suggested that SOD1 mutations lead to activation of several apoptotic regulators (2). However, the relationship between mutant TDP-43 and neuro-toxicity remains poorly understood. Better knowledge of the death mechanism will also facilitate development of potential therapeutic targets.

Objective: 1) To identify the death signalling pathway of mutant TDP-43 induced toxicity; 2) To study the relationship between TDP-43 mislocation and neuron-toxicity.

Methods: Transfected NSC34 cells or fibroblasts were treated with a pan-caspase inhibitor Z-DEVD and/or a proteasome inhibitor MG132. MTT assay and LDH assay were performed to determine cell toxicity. Cells were visualised using confocal microscopy and the cells with TDP-43 mislocation were counted. Western blotting was performed to study the activation of caspase 3. JC-1 assay was used to study the effect of mutant TDP-43 on mitochondria.

Results: In our experimental systems, both NSC34 cells and stressed fibroblasts showed TDP-43 translocation from nucleus to cytoplasm. Overexpression of mutant TDP-43 caused a 4-fold increase in cells with cytoplasmic TDP-43 and caused a higher toxicity (68% ± 3.6 viability in mutant vs 100% ± 6.2 in control). We found TDP-43 cytoplasmic inclusions in mutant fibroblast cells, but not in control cell, even before treatment. The mutation carrying fibroblast cells also had a higher percentage of TDP-43 mislocation (34% ± 0.4, compared to control 13% ± 0.4) and showed a higher toxicity by LDH assay after treatment. We observed caspase 3 activation and mitochondrial membrane permeabilisation in transfected NSC34 cells. The level of activated caspase 3 was higher in mutant TDP-43 transfected cells than wild type. In an attempt to reduce the toxicity, a pan-caspase inhibitor Z-DVED was used. However, this was unable to reduce cytotoxicity in either wild type or mutant TDP-43 expressing NSC34 cells, regardless of the presence or absence of MG132.

Discussion and conclusions: In this study, we found that mutant TDP-43 caused greater cytotoxicity than overexpressing wild type TDP-43. TDP-43 mutation also rendered fibroblasts more susceptible to cellular stresses. Both caspase 3 and mitochondria were involved in TDP-43 mediated cytotoxicity, suggesting this toxicity was due to apoptotic mechanisms. There was a difference in caspase 3 activation between wild type and mutant TDP-43 transfected cells, suggesting cells carrying TDP-43 mutations may be more likely to undergo apoptosis. The inefficiency of a pan-caspase inhibitor suggested that the toxicity induced by TDP-43 may be regulated by a range of molecules, not only caspases. Work is currently underway to better understand these mechanisms.

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P138 EXPRESSION OF MUTANT TDP-43 ENHANCES CELLULAR SUSCEPTIBILITY TO IMPAIRED ENERGY METABOLISM

KANNINEN K¹, SAVVA M¹, TURNER B², ATKIN J³, HORNE M³, WHITE A^1,4, CROUCH P^1,4

¹University of Melbourne, Melbourne, Victoria, Australia, ²Florey Neuroscience Institutes, Melbourne, Victoria, Australia, ³LaTrobe University, Melbourne, Victoria, Australia, ⁴Mental Health Research Institute, Melbourne, Victoria, Australia

Email address for correspondence: [email protected]

Keywords: TDP-43, energy metabolism, cell culture model

Background: The TAR DNA-binding protein (TPD-43) is a common component of cytoplasmic inclusions present in the majority of ALS cases and other neurodegenerative diseases including frontotemporal lobar dementia with ubiquitinated inclusions, Alzheimer's disease and dementia with Lewy bodies. The fundamental causes of aberrant TDP-43 metabolism and cellular localisation are not completely understood but the association of TDP-43 mutations with some forms of ALS indicate mutations to TDP-43 induce and/or accelerate neuronal dysfunction and degeneration.

Objectives: The objective of this study was to directly compare cells expressing normal or mutated TDP-43 and to examine how mutations to TDP-43 affect cell growth and TDP-43 metabolism.

Methods: NSC-34 cells were transfected to express non-mutated human TDP-43 (WT-TDP-43) or human TDP-43 harbouring the A315T mutation present in familial forms of ALS (A315T-TDP-43). Growth of the cells was measured in real time over several days using the xCELLigence cell analyser. As a model to examine the effects of impaired energy metabolism, the cells were grown in normal media with adequate glucose levels or in low glucose media where glucose levels become limiting. Cellular distribution of TDP-43 was determined by fluorescence microscopy and expression of TDP-43 and other cellular proteins determined by Western blot.

Results: Real time cell growth analysis revealed the expression of A315T-TDP-43 impairs growth of NSC-34 cells under normal growing conditions. Further to this, decreasing the availability of glucose in the culture media demonstrated that while both cell lines were sensitive to conditions that limit cellular energy metabolism, these sub-optimal growing conditions have a greater impact on cells expressing the A315T TDP-43 mutation. After 6 days in culture under normal growing conditions the cellular location of both WT-TDP-43 and A315T-TDP-43 is almost exclusively nuclear. Under conditions of low glucose however the A315T-TDP-43 is predominantly cytoplasmic, compared to the WT-TDP-43 which remains nuclear. Western analyses revealed the low glucose growing conditions did not significantly alter overall expression levels of WT- or A315T-TDP-43. By contrast, expression of the survival of motor neuron protein (SMN) was significantly decreased under conditions of limited glucose availability, but only in cells expressing the A315T-TDP-43.

Discussion and conclusions: These cell culture studies indicate that motor neurons expressing mutated forms of TDP-43 are more susceptible to conditions in which energy metabolism is impaired. Under these conditions mutated TDP-43, but not WT-TDP-43, translocates to the cytoplasm. This abnormal redistribution of TDP-43 may contribute to the decline in motor neuron function and viability by disrupting homeostasis of proteins such as SMN.

P139 FAMILIAL ALS-LINKED TDP-43 MUTATIONS CAUSE THEIR PROTEIN STABILIZATION AND DYSREGULATE MRNA METABOLISM

WATANABE S^1,2, KANEKO K^1,2, YAMANAKA K^1,2

¹RIKEN, BSI, Saitama, Japan, ²JST, CREST, Saitama, Japan

Email address for correspondence: [email protected]

Keywords: TDP-43, protein stability, mRNA stability

Background and objective: Abnormal accumulation of TDP-43 was identified as a pathological hallmark of ALS and FTLD. More than 30 missense mutations in TDP-43 gene, most of which reside at C-terminus of the protein, have been identified in both sporadic and familial ALS patients. Hyperphosphorylated 25 kDa C-terminus TDP-43 fragments are accumulated and aggregated in the motor neurons of ALS patients (1, 2). Since C-terminal fragments actually behave as seeds to facilitate cytosolic aggregation of full-length TDP-43 in the culture cell model, these fragments may be relevant to motor neuron degeneration in ALS (3). However, the detailed cytotoxic mechanisms of ALS-linked TDP-43 mutant protein remain to be unclear. Therefore, the aim of our study is to elucidate the cytotoxic mechanisms of mutant TDP-43 by biochemical approaches.

Methods: Our experiments were carried out using mouse neuroblastoma Neuro2a cells as an in vitro model. We constructed a non-tagged TDP-43 expressing vector in order to eliminate the effect of artificial tag. First, wild-type and all familial ALS-linked (fALS) mutant TDP-43 proteins were transiently expressed in differentiated Neuro2a cells with dibutyl cyclic AMP, the localization and solubility of TDP-43 proteins were analyzed by differential centrifugation and solubilization, respectively. Next, to measure the stability of TDP-43 proteins in cells, half-lives of several fALS-linked mutant TDP-43 proteins were determined by pulse-chase experiment. To measure the exon-skipping activity and the mRNA destabilization activity, the monitoring plasmids containing CFTR exon 9 mini-gene and GFP fused TDP-43 3'UTR were constructed, respectively. The activity of TDP-43 was determined by co-transfection with these monitoring vectors.

Results and discussion: The localization and solubility of TDP-43 were not altered by the introduction of mutation under normal conditions. Mutant TDP-43 proteins, however, had longer half-lives than the wild-type. Intriguingly, half-lives of mutant TDP-43 proteins were negatively correlated with the onset age of ALS, but not with the duration. In addition, the mRNA destabilization activity of fALS-linked mutant TDP-43 proteins was approximately 6 fold lower than that of wild-type in the differentiated neuroblastoma cells. On the other hand, the exon-skipping activities were not lost in the mutant TDP-43 proteins.

Among these results, significant differences have been observed in both the protein stability and the mRNA destabilization activity. We therefore speculate that they are the key phenomena to understand the cytotoxic mechanism of mutant TDP-43 proteins. We are in process of investigating why fALS-linked mutant TDP-43 proteins impair mRNA regulation through protein stabilization. The detailed results will be reported and discussed.

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P140 MUTANT TDP-43 AND ASTROCYTES

MALMEVIK J¹, ATKIN J², WALKER A², MUYDERMAN H¹

¹Flinders University, Adelaide, SA, Australia, ²La Trobe University, Bundoora, Vic., Australia

Email address for correspondence: [email protected]

Keywords: astrocytes, gtpases, TDP-43

Background: The RNA-binding protein TDP-43 has recently been demonstrated as an aetiological factor in motor neuron disease (MND). TDP-43 is a DNA and RNA binding protein regulating transcription and splicing. TDP-43 is also involved in transport and local post-transcriptional modification of mRNAs. The protein is abundantly expressed in motor neurons and astrocytes. TDP-43 pathology is triggered by abnormal processing and cytosolic aggregation of the protein or by mutations in the TDP-43 gene. The pathology is similar and the outcome is directly linked to cell death. Mutant TDP-43 causes familial forms of human MND, MND-like disease in transgenic animals and kills motor neurons in primary culture.

TDP-43 pathology is also found in astrocytes: a cell type that plays critical roles in the pathology of MND. Astrocytes are the major contributor to defence against oxidative damage and express transporters that clear excess glutamate from the extracellular space. Loss of these transporters leads to excitotoxic damage. In MND, changes in astrocyte physiology occur prior to those of motor neurons and their severity can be correlated to disease progression. Thus, the extent of preservation of key astrocytic properties and the ability of these cells to mount appropriate defensive responses are important determinants of tissue viability in many neurological diseases.

Methods: We have established cellular models of TDP-43 proteinopathies by expressing fluorescently-tagged TDP-43 (wild-type and mutants) in astrocytes in primary cultures. We have also silenced TDP-43 expression in these cells. We have used these models to investigate the role of TDP-43 and its mutants on normal cell function and on the response of these cells to injury.

Results: Presence of TDP-43 mutations, caused reorganisation of the actin cytoskeleton and lead to impaired wound healing in an in vitro injury model (n = 5). Moreover, astrocytes transfected with the Q133k TDP-43 mutation had decreased expression of GLT-1 glutamate transporters (n = 3) and displayed impaired mitochondrial function as evaluated by the mitochondrial membrane potential (n = 3). These cells also had an impaired ability to withstand oxidative stress (n = 3). Finally, the presence of mutant TDP-43 increased the activists of the Rho family GTPases Rho A and Rac-1 while significantly reduced Cdc42 activity suggesting a direct role for TDP-43 in the regulation of the Rho-family GTPases.

Discussion and conclusions: These results demonstrate severe effects on normal astrocytic function by mutant TDP-43 and suggest that astrocytes could play a role in motor neuron demise in TDP-43 proteinopathies.

P141 CONTACT WITH AGGREGATED SOD1 CAUSES EFFICIENT ACTIVATION OF MICROGLIA AND ASTROCYTES IN VITRO

ROBERTS K^1,2, YERBURY J^1,2, CAMPBELL I³

¹University of Wollongong, Wollongong, NSW, Australia, ²Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia, ³University of Sydney, Sydney, Australia

Email address for correspondence: [email protected]

Keywords: microglia, astrocytes, SOD1

Background: A large body of literature suggests that amyotrophic lateral sclerosis pathology is intimately linked with neuroinflammation, specifically activation and recruitment of microglia and astrocytes. The actual cause of gliosis is unclear. However, extracellular Cu/Zn superoxide dismutase (SOD1) has recently been shown to activate microglia providing one potential mechanism by which glial cells activated (1).

Objectives: As protein inclusions are thought to be an important part of ALS pathology and are associated with all forms of ALS we sought to determine if aggregated SOD1 would activate microglia and/or astrocytes.

Methods: Recombinant SOD1 was expressed in E.coli in conjunction with its copper chaperone to ensure correct metal loading. The resulting enzyme showed correct secondary structure and enzyme activity. To promote aggregation, SOD1 and a small panel of mutants were incubated with 30 mM Dithiothreitol (DTT) and 5 mM Ethylenediaminetetraacetic acid (EDTA), (to break disulphides and remove metals respectively). The samples were extensively dialyzed against PBS after aggregation to remove DTT and EDTA. Aggregated and monomeric forms of SOD1 were then added to either microglia or astrocyte cells in culture. To ensure that the effects we observed were not due to LPS contamination we also used the EOC-13 microglial cell line (LPS non responsive) and or compared the response to treating the cells with LPS. Cell lysates were collected for Western blot analysis and supernatants were analysed for cytokine secretion via ELISA kits.

Results: Both monomeric and aggregated SOD1 bound to the surface of glial cells and was internalized. Although monomeric mSOD1 has been shown to promote microglial activation in the past we found that aggregated SOD1 was able to much more efficiently activate microglia and astrocytes in culture when compared to the monomeric form of mSOD1.

Conclusion: We have for the first time shown that aggregated mSOD1 potently activates both microglia and astrocytes. These results suggest that aggregated SOD1 is potentially more neurotoxic than monomeric SOD1 and may play a key role in disease progression in ALS.

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P142 ROLE OF EXTRACELLULAR SOD1 IN THE PATHOGENESIS OF ALS

SUNDARAMOORTHY V, WALKER A, FARG M, ATKIN J, YERBURY J

Latrobe University, Melbourne, Australia

Email address for correspondence: [email protected]

Keywords: extracellular SOD1, uptake, ER stress

Background: Mutations in the superoxide dismutase 1 (SOD1) gene account for about 20% of familial ALS and the presence of SOD1 inclusions is also a common pathological feature in sporadic ALS. SOD1 has been previously shown to be secreted by neuronal cell lines and is found in the CSF of familial and sporadic ALS patients. The exact role of this extracellular SOD1 remains unclear. This study shows that extracellular SOD1 is taken up by motor neuron like NSC34 cells and the uptake of mutant SOD1 results in the activation of ER stress. Our laboratory has shown that activation of ER stress occurs early in the pathogenesis of both familial and sporadic ALS. Thus the uptake of extracellular SOD1 could be a possible mechanism for the spread of neurotoxicity among motor neurons in ALS.

Objectives: The aims of this study were to investigate whether motor neurons could uptake extracellular SOD1, and whether the uptake of SOD1 could induce ER stress in these neurons.

Methods: Motor neuron-like NSC-34 cell lines were cultured and treated with recombinant SOD1 protein. Immunocytochemistry, epifluorescence microscopy and immunoblotting were performed to detect SOD1 uptake in these cells. The cells were also examined for ER stress markers.

Results: Immunocytochemistry and immunoblotting results showed that extracellular SOD1 is taken up by NSC-34 cells and the uptake of mutant G93A SOD1 resulted in marked increase of ER stress markers such as CHOP. The addition of mutant SOD1 also induced morphological changes indicative of cell death in NSC-34 cells.

Discussion: The mechanism of neurodegeneration in ALS is non-cell autonomous, a process by which an affected cell (motor neurons or glial cells) induces the disease phenotype in other neighbouring cells. This study has shown the uptake of extracellular SOD1 by motor neuron NSC-34 cells, indicating that cell to cell transmission of SOD1 could be involved in ALS. Hence SOD1 could be a possible toxic factor responsible for the non-cell autonomous mechanism in ALS. These results also suggest that targeting the pathways activated by extracellular mutant SOD1 could be a possible therapeutic approach for ALS.

P143 EXOGENOUS MUTANT SOD1 CAN BE INTERNALIZED BY MOTOR NEURONAL CELLS AND PROMOTES FORMATION OF INTRACELLULAR PROTEIN INCLUSIONS

PUNDAVELA JF, YERBURY J

Illawarra Health and Medical Institute, University of Wollongong, Wollongong, NSW, Australia

Email address for correspondence: [email protected]

Keywords: protein aggregation, NSC-34, green fluorescent protein

Background: Clinical evidence shows that motor neurone degeneration in Amyotrophic Lateral Sclerosis (ALS) is a process which starts focally and spreads progressively through the nervous system (1). Like many other neurodegenerative diseases, protein inclusions are closely linked to ALS pathology. Inclusions positive for Cu/Zn superoxide dismutase 1 (SOD1) have been found in familial ALS with mutations in SOD1, and studies have also found inclusions of misfolded wild-type SOD1 in sporadic ALS patients (2). Recent evidence suggests that a transfer of SOD1 aggregates emulate a prion-like mechanism (3). Collectively, these data suggest that aggregate propagation may play a role in ALS pathology.

Objectives: The aim of this study is to demonstrate whether exogenously applied aggregates and monomeric mutant or wild-type SOD1 can seed the formation of intracellular inclusions in both wild-type or mutant SOD1 expressing cells.

Methods: Recombinant mutant or wild-type SOD1 was co-expressed with the copper chaperone to ensure correct metal loading in E.coli, then purified through size-exclusion and anion exchange chromatography. Recombinant SOD1 was incubated with 50 mM dithiothreitol (DTT) and 5 mM ethylenediaminetetraacetic acid (EDTA) for 70 hours with agitation to promote aggregation. The samples were then dialysed in Phosphate buffer saline to remove DTT and EDTA. NSC-34 cells were transiently transfected with human SOD1-EGFP-SOD1 expression constructs (wild-type, G93A, or EGFP alone). Aggregated and monomeric G93A or wild-type recombinant protein was added on to culture of transfected NSC-34 cells. The proteasome inhibitor MG132 acted as a positive control for inclusion formation promotion. At early time points, cell lysates were collected for Western blot analysis while at later time points cells were analysed by confocal microscopy.

Results: Western blot analysis demonstrated insoluble protein aggregates were taken up by NSC-34 cells, this occurred as early as 2 h. Confocal imaging showed that protein inclusions were promoted by incubation with either monomeric or aggregated mutant SOD1. This was the case in both WTSOD1-EGFP and G93ASOD1-EGFP expressing cells.

Discussion and conclusions: This study suggests that exogenous SOD1 aggregates can be internalized by motor neurone like cells and initiate the formation of intracellular protein inclusions. We propose that these inclusions are toxic to cells leading eventually to further release of aggregated proteins and subsequently take up by neighbouring cells. This mechanism may explain the progressive pathology of ALS.

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P144 A NOVEL MECHANISM OF SET/I2PP2A SHUTTLE BETWEEN NUCLEUS AND CYTOPLASM

YU G

Tongji Medical College, Wuhan, China

Email address for correspondence: [email protected]

Keywords: Alzheimer's Disease, SET/I2PP2A, nuclear localization signal (NLS)

Background and objectives: SET/I₂^PP2A acts as a key impetus on neurodegenerative disease, especially Alzheimer's Disease (AD) onset. Until now, little has been known about the detailed regulatory mechanism by which SET was detained in cytoplasm and the consequent events in mammalian cells. Phosphorylation is the best-understood mechanism of regulation of nuclear transport. SET sequence (6-11aa) AKVSKK almost coincided with the consensus nuclear localization signal (NLS) sequence KKXXKX or XKXXKK. Therefore, the present study is to explore whether the sequence ⁶AKVSKK¹¹ of SET is a potential NLS, and plays an essential role on SET nuclear import.

Methods: Site-directed mutagenesis; HEK293/tau cells culture and transfection; confocal microscopy; cytoplasmic and nuclear extraction; Western blot; immune-precipitation.

Results: 1) Pseudophosphorylation of SET at serine 9 was observed mostly in cytoplasm, while nuclear accumulation of pseudounphosphorylated SET and wt SET was not significantly affected. 2) Phosphorylation of SET at Serine 9 significantly reduced SET/karyopherin complex formation compared to npSET and wtSET. 3) Lysine11 mutation to alanine induced SET exclusively localizing in cytoplasm while K7A and K10A SET still mainly stayed in nucleus. 4) PP2A activities in either pSET or wtSET in treated HEK293/tau cells were significantly decreased as compared with that of control group, but npSET didn't influence PP2A activity. Interestingly, when directly comparing between pSET and wtSET, we also found that pSET inhibited PP2A activity more efficiently than wtSET by 25.2% (p = 0.006).

Conclusion: Phosphorylation of SET at Ser 9 inhibits SET nuclear import and promotes an inhibition of PP2A activity.

P145 ENDOCYTIC TRANSPORT ABNORMALITIES AS A DETERMINANT OF MOTOR NEURON VULNERABILITY IN ALS

TURNER B¹, HILL A², AUMANN T¹, SAWBRIDGE J-L¹, SHEEAN R¹, ATKIN J³, HORNE M¹

¹Florey Neuroscience Institutes, Melbourne, Australia, ²University of Melbourne, Melbourne, Australia, ³La Trobe University, Melbourne, Australia

Email address for correspondence: [email protected]

Keywords: endosome, exosome, Rab

Background: ALS is characterised by accumulation of pathological misfolded proteins in vulnerable motor neurons by unclear mechanisms. Increasing evidence suggests a key role for membrane trafficking defects in ALS neurodegeneration. Many ALS genes encode molecular machinery or enzymes directly mediating intracellular transport; these gene products are often cargoes of transport vesicles, and defective transport features early and prominently in ALS models. The endosome-lysosome system (ELS) is a major intersection for intracellular traffic and responsible for autophagic clearance and exosomal secretion of misfolded proteins. Both autophagic activation and exosome abnormalities are reported in ALS models, suggesting a disruption to the ELS.

Objectives: We hypothesised that abnormal endosome transport is a common pathogenic process engaged by leading ALS-linked proteins such as SOD1, TDP-43 and FUS. ELS morphology and transport was therefore examined in ALS models and patients.

Methods: NSC-34 cells stably expressing normal or mutant SOD1, TDP-43 and FUS were studied for endocytic Rab and autophagic markers by Western blotting and immunocytochemistry. Cells were treated with endocytic tracers including fluorophore-tagged transferrin and dextran to measure endosome transport rate. Cells were separately co-transfected with endocytic Rab-GFP or ubiquitin-RFP to modulate endosome transport. Lastly, endosome pathology was examined in post-mortem spinal cord tissues from sporadic ALS patients (n = 10) and non-neurological disease controls (n = 5).

Results: We first demonstrated that SOD1, TDP-43 and FUS were secreted by cell-derived exosomes and that ALS-linked forms of these proteins were depleted in exosomes, preceding cytoplasmic inclusion formation, ER stress and cell death activation. Exosome deficits correlated with early endosome defects determined by abnormal Rab5 upregulation, enlarged early endosomes and endocytosis of tracers. Rab5 induction was confirmed in spinal cords of presymptomatic transgenic SOD1^G93A mice and sporadic ALS patients, unlike controls.

Discussion and conclusions: Based on these findings, we propose that early endosome transport defects leading to impaired exosomal secretion and increased cytoplasmic protein burden may be an early determinant of motor neuron loss and common denominator of key pathological ALS-linked proteins. Abnormal endocytic transport is likely to point to more fundamental mechanisms of vesicle trafficking defects implicated in ALS and innovative potential therapeutic approaches.

P146 DEFECTIVE RELOCALIZATION OF ALS2/ALSIN TO RAC1-INDUCED MACROPINOSOMES ACCOUNTS FOR LOSS OF THEIR CELLULAR FUNCTION AND LEADS TO DISTURBED AMPHISOME FORMATION

OTOMO A^1,2, KUNITA R², SUZUKI-UTSUNOMIYA K², IKEDA J-E², HADANO S^2,3

¹Department of Bioscience, School of Science, Kitasato University, Sagamihara, Kanagawa, Japan, ²Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan, ³Research Center of Brain and Nervous Diseases, Tokai University Graduate School of Medicine, Isehara, Kanagawa, Japan

Email address for correspondence: [email protected]

Keywords: ALS2/Alsin, autophagy, amphisome

Background: ALS2, the causative gene product for juvenile recessive motor neuron diseases, regulates macropinocytosis and following endosome fusion. Recently, we reported that loss of ALS2 in SOD1^H46R mice resulted in an earlier death and accelerated accumulation of abnormal autophagosomes in the spinal axons. These findings suggest that ALS2 acts as a positive regulator for autophagolysosome-mediated protein degradation, and loss of its function leads to malfunction of autophagolysosome-mediated protein degradation in neurons. Consistently, growing evidence supports the notion that impairment of autophagy (autophagosome formation, maturation and degradation) is indeed associated with the formation of protein aggregates and neurodegeneration. Thus, autophagy serves an adaptive role to protect neurons from degeneration. However, the molecular mechanisms by which loss of ALS2 results in the accelerated accumulation of abnormal autophagosomes remain elusive.

Objectives: To clarify the molecular basis for phenotypic modification of autophagosome trafficking and maturation by loss of the ALS2 function.

Methods: We investigated Rac1-induced relocalization of ALS2^WT or its pathogenic mutants (ALS2^C157Y and ALS2^G540E) using HeLa cells. We also performed indirect immunofluorescent analysis to observe the colocalization of ALS2 with EEA1 (endosome marker) and microtubule-associated protein 1A/1B-light chain 3 (LC3) (autophagosome marker). The association of ALS2 with specific lipid molecules was examined by PIPStrip overlay assay using purified FLAG-ALS2.

Result: First, we observed the localization of ALS2^WT and its mutants in Rac1^Q61L expressing cells. Interestingly, unlike ALS2^WT, both ALS2^C157Y and ALS2^G540E failed to be localized to both Rac1^Q61L-induced macropinosomes and macropinosome-derived early endosomes (EEs), and yet sequestered in the cytoplasm. Next, to clarify the mechanisms for the mislocalization of the ALS2 mutants, we examined whether the ALS2 mutants could directly bind to phosphoinositide phosphates (PIPs), membrane-compartment specific lipid molecules. Both FLAG-ALS2^C157Y and FLAG-ALS2^G540E exhibited lower affinities to PI(3)P and PI(4)P compared to FLAG-ALS2^WT. Lastly, we investigated the effect of ALS2 mislocalization in autophagosome trafficking and maturation. Expression of ALS2^WT induced enlarged EEA1-positive vesicles that were colocalized with LC3, indicating an enhanced formation of amphisomes. Notably, expression of neither ALS2^C157Y nor ALS2^G540E enhanced the formation and/or enlargement of amphisomes.

Discussion and conclusions: We showed that loss of the affinities to specific lipid molecules accounted for the mislocalization of pathogenic ALS2 mutants in cells. Since defective relocalization of ALS2 leads to loss of the ALS2 function as a Rab5 activator on macropinosomes/endosomes, resulting in disturbance of the autophagosome/endosome maturation, malformation of amphisomes might underlie the pathogenesis of the ALS2-linked MNDs. Future studies on the molecular basis of ALS2 will uncover the roles of macropinocytosis and amphisome formation in selective cargo sorting and degradation, which brings new insights into the pathogenesis for ALS2-linked MNDs and other neurodegenerative diseases.

P147 DISTINCTIVE HIGH-MOLECULAR WEIGHT OLIGOMERIC COMPLEXES OF ALS2/ALSIN ARE ENRICHED IN THE BRAIN SYNAPTOSOMAL COMPARTMENTS

SUZUKI-UTSUNOMIYA K¹, OTOMO A², KUNITA R¹, IKEDA J-E¹, HADANO S¹

¹Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan, ²Department of Bioscience, School of Science, Kitasato University, Sagamihara, Kanagawa, Japan

Email address for correspondence: [email protected]

Keywords: ALS2, synaptosome, protein complex

Background: The loss-of-functional mutations in the ALS2 gene account for a number of recessive motor neuron diseases (MNDs). Thus, the ALS2 gene product, ALS2/alsin, plays an important role in maintenance and/or survival of motor neurons. Previously, we have demonstrated that ALS2 acts as a guanine nucleotide exchange factor (GEF) for a small GTPase Rab5, and forms a homophilic oligomer. This homo-oligomerization is crucial for the Rab5-GEF activity in vitro and for the ALS2-mediated endosome enlargement in cultured cells. Moreover, the intracellular distribution of ALS2 is drastically shifted from cytosol to the membrane/vesicle compartments by the activation of an ALS2-upstream factor Rac1, thereby inducing endosome enlargement in its Rab5-GEF activity-dependent manner. These findings prompted us to hypothesize that the conformational changes and/or differences in the ALS2 complex might be associated not only with its intracellular distribution but also with the tissue-specific ALS2 function in vivo. However, it remains unclear whether there are differences in the conformation of the ALS2 complex in different cellular compartments among different tissues, let alone whether the conformation-activity relationship of the ALS2 complex is implicated in its physiological function.

Objectives: To delineate the conformation-activity relationship of the ALS2 complex in vivo.

Methods: Whole brain tissue from C57BL/6N mouse was lysed, and subjected to immunoprecipitation using the anti-ALS2 antibody, followed by MALDI-TOF/MS, to detect possible ALS2 interactors in vivo. Whole brain and liver from mice were homogenized and fractionated by a series of centrifugations, resulting in subcellular fractions; P1, P2, P3, and S3. Synaptosomes were enriched from P2 by the gradient-centrifugation, and soluble synaptosomal fraction (P2-S) was obtained from synaptosomes by the treatment with non-ionic detergent. P2-S and S3 fractions were subjected to gel-filtration analysis with the use of the Superose 6 HR 10/30 column. By Western blot analysis, the subcellular distribution of ALS2 and molecular weight of the ALS2 complex were determined.

Results: In the brain, no major ALS2 interactors other than ALS2 itself were detected. In the liver, ALS2 was almost exclusively distributed in S3 (cytosol). By contrast, ALS2 was mostly enriched in P2 in the brain. Gel-filtration analysis revealed that cytosolic (S3) ALS2 from either the liver or brain tissue was eluted at an apparent peak molecular masses of ∼750 kDa, indicating that ALS2 forms a tetramer. Remarkably, ALS2 in P2-S showed a distinctive high-molecular weight distribution (presumably octamer) in addition to their tetrameric complex.

Discussion and conclusions: Our interim results suggest that the subcellular distributions of ALS2 in the CNS and peripheral tissues are different, and that the synaptosomal high-molecular weight ALS2 complex may be associated with neuron-specific ALS2 function in vivo. Currently, to delineate the conformation-activity relationship of the ALS2 complex, biochemical analyses of the wild-type and mutant ALS2 complexes are underway.

P148 THE FAILURE OF ER-GOLGI TRANSPORT IN MOTOR NEURON-LIKE CELLS EXPRESSING MUTANT SOD1 PROTEINS

SOO KY¹, FARG M¹, WALKER A¹, ATKIN J^1,2

¹Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia, ²Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, Parville, Australia

Email address for correspondence: [email protected]

Keywords: SOD1, ER-Golgi transport, VSVG

Background: Increased evidence shows that axonal transport and endoplasmic reticulum (ER) stress are the pathogenic mechanisms of ALS. We previously showed that ER stress is triggered early in the disease process, suggesting a significant role in pathophysiology. Motor neurons are large cells with long axons and are particularly susceptible to the failure of axonal and cellular transport. However, how ER stress is triggered in cells expressing mutant SOD1 proteins has not been established. Transfection of a gene encoding an abundant membrane glycoprotein (G protein) from vesicular stomatitis virus (VSV) in culture cells rapidly synthesize the VSVG protein on the ER like normal secretory protein. Use of mutant encoding a temperature sensitive VSVG (VSVGtsO45) allows the subsequent transport of this protein from ER to Golgi.

Objective: In this study, we investigated whether the transport of VSVGtsO45 from ER to Golgi is inhibited in cells expressing mutant SOD1 proteins and whether the failure of ER-Golgi transport occurs before ER stress in cells expressing mutant SOD1. Furthermore, we also examined whether overexpression of Sar1 (a COPII subunit) or protein disulphide isomerase (PDI) rescues ER-Golgi transport in cells expressing mutant SOD1.

Methods: NSC-34 cells were co-transfected with SOD1-EGFP vectors (either WT or mutants) and VSVGtsO45-mCherry. For overexpression of Sar1 or PDI, cells were triple transfected with SOD1-EGFP vectors (either WT or mutants), VSVGtsO45-mCherry and Sar1-Flag or pCMV-PDI vectors. After transfection, cells were then stained with ER or Golgi markers to determine whether VSVGtsO45 proteins are localized in the ER or Golgi.

Results: After 72 h transfection, the inhibition of VSVGtsO45 transport from ER to Golgi is significant in cells expressing mutant SOD1 proteins. This inhibition begins at 16 h transfection while ER stress occurs at 18 h transfection, and inclusions are only seen at 24 h transfection time points. Overexpression of Sar1 or PDI rescues transport of VSVGtsO45 from ER to Golgi in cells expressing mutant SOD1 proteins. In addition, mutant TDP-43 proteins also block the ER-Golgi transport of VSVGtsO45 in NSC-34 cells.

Discussion and conclusion: These data provide the evidence that ER-Golgi transport of proteins is restrained in cells expressing mutant SOD1 proteins. The failure of ER-Golgi transport occurs early and before ER stress, indicating that it triggers ER stress in cells expressing mutant proteins. Overexpression of Sar1 or PDI rescues ER-Golgi transport, suggesting a possible neuroprotective role in motor neuron death. Mutant TDP-43 may act similarly to mutant SOD1 in ALS pathogenesis.

P149 G93A HSOD1 IMPAIRS MITOCHONDRIAL CALCIUM HANDLING AND CAUSES ER STRESS IN EMBRYONIC MOTOR NEURONE CULTURES

LAUTENSCHLAEGER J, PRELL T, RUHMER J, WEIDEMANN L, WITTE OW, GROSSKREUTZ J

Hans-Berger Department Of Neurology, Friedrich-Schiller-University Hospital Jena, Jena, Germany

Email address for correspondence: [email protected]

Keywords: mitochondrial dysfunction, calcium dynamics, UPR

Background: Motor neurones vulnerable to amyotrophic lateral sclerosis (ALS) express low amounts of calcium binding proteins (1,2) so intracellular calcium passing through the ER mitochondria calcium cycle (ERMCC) must be buffered primarily in mitochondria (3). Isolated G93A hSOD1 mitochondria show reduced calcium buffering capacity (4). FCCP releases less calcium from G93A hSOD1 mitochondria (5,6), but studies on ERMCC dynamics in mutant hSOD1 motor neurones with near physiological stimuli are missing.

Objectives: To analyse cytosolic and mitochondrial calcium transients after very brief AMPA receptor activation comparing non-transgenic and G93A hSOD1 motor neurones. Further to evaluate if impaired mitochondrial calcium buffering leads to ER stress and activation of the unfolded protein response (UPR).

Methods: Mixed motor neurone cultures were prepared from E13 ventral spinal cord of non-transgenic and G93A hSOD1 mice. AMPAR were stimulated with kainate for 2 s. Cytosolic calcium transients were measured using fura-2 AM and mitochondrial calcium dynamics with rhod-2 AM. CGP 37157 was applied to inhibit mitochondrial calcium export through the mitochondrial Na/Ca exchanger thereby reducing the calcium shuttling back to the ER. Markers of the unfolded protein response were visualised using immunofluorescence.

Results: Non-transgenic motor neurones (n = 18) had a slightly faster decay of cytosolic calcium transients than non-motor neurones (n = 18). Calcium decay was decelerated in G93A hSOD1 motor neurones (n = 12) compared to the non-transgenic motor neurones. Whereas G93A hSOD1 non-motor neurones (n = 17) showed a cytosolic calcium decay comparable to the non-transgenic non-motor neurones. Mitochondrial calcium export was accelerated in G93A hSOD1 motor neurones compared to non-transgenic motor neurones, possible causing higher cytosolic calcium levels. The treatment with CGP 37157 activated the UPR in non transgenic motor neurones, demonstrating that reduced mitochondrial calcium release can introduce ER stress.

Discussion and conclusions: The results demonstrate that G93A hSOD1 induces disturbance of the cytosolic calcium decay after a near physiological activation of AMPA receptors. This is caused by a reduced calcium retention capacity in mitochondria of G93A hSOD1 motor neurones, and triggers ER stress. The study provides evidence that mitochondrial calcium handling is crucial for maintaining ER function, and that motor neurones are particularly vulnerable to ERMCC dysfunction. EMRCC stabilization may therefore provide a new therapeutic principle in motor neurone disease.

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P150 MODELING RETROGRADE DEGENERATION OF MOTOR NEURON AXONS AS A CONSEQUENCE OF MITOCHONDRIAL DYSFUNCTION

LE MASSON G¹, HENDERSON C², PRZEDBORSKI S², ABBOTT LF³

¹Neurocentre Magendie, INSERM U862, University of Bordeaux 2, Bordeaux, France, ²Center for Motor Neuron Biology and Disease, Columbia University, New York, USA, ³Center for Theoretical Neuroscience, Columbia University, New York, USA

Email address for correspondence: [email protected]

Keywords: energy metabolism, electrophysiology, computer model

Background: Mitochondrial dysfunction is now recognized as a fundamental player in many neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In ALS, anomalous mitochondria are found early in the disease in humans and in the SOD1 mouse model. The role of mitochondrial dysfunction in triggering the selective degeneration of fast motorneurons is still unknown since mitochondria have many functions. However, one of the most essential is to produce the ATP needed for homeostatic processes such as pumping ions across the cell membrane and so we decided to model the consequences of reduced ATP for motorneuron function and survival.

Methods: To better assess the consequences of mitochondrial perturbations and specifically of reduced ATP production, we built a realistic computer model of a spinal motorneuron. This model is based on motor neuron morphology, reproduces realistic firing properties and includes biochemical pathways involved in the production, distribution and consumption of ATP. The model emphasizes the role of ATP in running Na + /K + pumps and maintaining motorneuron homeostasis. We use this model to study the requirements for ATP production and distribution in maintaining the electrical properties of the neuron and holding the intracellular calcium concentration in a normal range.

Our results show: 1) When ATP levels reach a critical lower bound, an electrical instability develops, leading to a maintained depolarized state, and causing massive calcium influx. The instability leads to high energy consumption, further worsening the energy deficit. This pathological state is not spontaneously reversible and is likely to lead to neuronal degeneration and death. 2) If the ATP deficit is localized to the distal end of the axon, the instability depolarizes the terminals, triggering ectopic action potentials reminiscent of the hyperexcitability associated with fasciculation potentials. More importantly, this instability can spread in a retrograde fashion until it reaches the soma. 3) Finally, the vulnerability of each neuron to this instability strongly depends on its specific morphology and firing properties. We tested the hypothesis that selective motor neuron vulnerability in ALS might be linked to specific firing and energy profiles. We find that motor neurons have a high metabolic demand and that different subtypes of motor neurons (FF or S) have different vulnerability to energetic stress.

Conclusions: Using a realistic computer model, we studied the relationship between functional activity (firing properties) and metabolic energy requirements. The model is unique in that it includes ionic channels together with intracellular metabolic pathways linked to mitochondrial function, ATP synthesis and consumption, and pumps and buffers. We describe an instability triggered by mitochondrial failure that leads to a persistent energy deficit through a positive feedback loop, and that may be involved in the selective degeneration of fast motor neurons in ALS.

P151 A SMALL MOLECULAR COMPOUND CPN-9 SELECTIVELY PROTECTS AGAINST OXIDATIVE STRESS-INDUCED CELL DEATH BY ACTIVATING THE NRF2-ARE PATHWAY

KANNO T^1,2, TANAKA K¹, YANAGISAWA Y^1,2, YASUTAKE K², HADANO S², HIRAYAMA N², IKEDA J-E^1,2

¹Neugen Pharma Inc., Isehara, Kanagawa, Japan, ²Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan

Email address for correspondence: [email protected]

Keywords: neuroprotectant, Nrf2, oxidative stress

Background: Several lines of evidence have indicated that oxidative stress is implicated in the pathogenesis of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Thus, anti-oxidative stress therapy is thought to be one of the promising remedies for those diseases. Recently, we have shown that a small-molecule neuroprotectant CPN-9 selectively protects oxidative stress-induced cell death in vitro, and suppresses disease progression in an ALS mouse model (SOD1-H46R). However, the molecular mechanism by which CPN-9 selectively protects cells from oxidative stress remains unclear.

Objectives: To define the molecular signaling pathway for the CPN-9-mediated anti-oxidative stress activity.

Methods: We used human neuroblastoma SH-SY5Y cells differentiated by retinoic acid. To evaluate the effects of CPN-9 on the expression of the antioxidant stress genes and their products as well as on the cell viabilities, we performed RT-PCR, Western blotting, RNAi-mediated gene silencing, and in vitro cell viability assay.

Results: We first examined whether CPN-9 activates the transcription factor Nrf2 that promotes several antioxidant and detoxifying enzyme gene expression. CPN-9-treated SH-SY5Y cells revealed an increase in the nuclear Nrf2 levels, indicating the activation of Nrf2 in cells. Several antioxidant and detoxifying enzymes including heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase modifier subunit (GCLM), whose expressions were regulated by Nrf2, were indeed significantly upregulated in SH-SY5Y cells by CPN-9. Consistently, the cytoprotective effect of CPN-9 was suppressed either by HO-1, NQO1, or GCL inhibitor. N-acetylcysteine prevented the CPN-9-induced nuclear translocation of Nrf2 with a concomitant attenuation of the CPN-9-mediated protection against oxidative stress. Further, knockdown of Nrf2 expression also resulted in decreased levels of HO-1, NQO1 and GCLM induced by CPN-9 and abolished its anti-oxidative stress activity, indicating that the Nrf2-antioxidant response element (ARE) pathway is essential for the CPN-9 mediated cytoprotectivity.

Discussion and conclusion: The present study showed that CPN-9 suppressed oxidative stress selective cell death via the activation of the Nrf2-ARE pathway with a concomitant induction of the antioxidant and detoxifying enzymes, suggesting that the Nrf2-ARE pathway serves as a potential neuroprotective drug target. Thus, CPN-9, the Nrf2-ARE activating compound, might represent a promising new therapeutic agent for ALS and other neurodegenerative diseases.

Acknowledgements: This work was supported by the National Institute of Biomedical Innovation (NIBIO).

P152 DEFECTIVE REGENERATION OF OXIDATIVELY-INACTIVATED 2-CYS PEROXIREDOXINS IN SOD1-RELATED AMYOTROPHIC LATERAL SCLEROSIS

RAJAN SS, WOOD-ALLUM C, SHAW P

Sheffield Institute for Translational Neuroscience, University of Sheffield, UK

Email address for correspondence: [email protected]

Keywords: peroxiredoxins, oxidative stress, SOD1

Background: Peroxiredoxins (Prxs) are abundant redox-sensitive anti-oxidant enzymes that reduce hydrogen peroxide (H₂O₂) to water, becoming oxidized themselves. Oxidized 2-Cys Prxs may either be returned to their reduced state by thioredoxin or become overoxidized. Overoxidized 2-Cys Prxs (PrxSO_2/3) lose their hydroperoxidase activity but may then form multimers with a molecular chaperone function. Overoxidation of 2-Cys Prxs may be reversed by Sulfiredoxin-1. Previous studies demonstrated altered Prx levels in SOD1-ALS. This and the relevance of Prx anti-oxidant and protein chaperone roles to extant pathogenetic hypotheses led us to ask whether the oxidation state of the 2-Cys Prxs might be altered in ALS.

Objectives: We hypothesized that 2-Cys Prxs might spend longer in a more oxidized state in ALS and that this could have implications for disease pathogenesis and/or progression. We aimed to test this hypothesis in models of SOD1 ALS.

Methods: Fibroblasts were obtained from patients with I113T SOD1-related familial ALS and age and sex-matched controls. Western blotting for total 2-Cys Prxs, PrxSO_2/3, Sufiredoxin-1 and Sestrin 2 (previously thought to reduce PrxSO_2/3) was performed on cells grown under basal conditions and after exposure to 15 min 300mM H₂O₂. Stress recovery experiments (15 min 300mM H₂O₂, washout, 26 hours recovery) were performed with and without cycloheximide. Whole brain and spinal cord homogenates from G93A- overexpressing transgenic mice were also examined for PrxSO_2/3.

Results: No difference in total 2-Cys Prxs was found between patient and control fibroblasts and minimal PrxSO_2/3 was detected in either under basal conditions. Exposure to 300mM H₂O₂ for 15 min overoxidized 2-Cys Prxs to saturation with no detectable difference between patient and control cells. PrxSO_2/3 recovery was, however, significantly delayed in patient fibroblasts. Sulfiredoxin-1 induction after H₂O₂-treatment was both delayed and reduced in patient fibroblasts compared to controls. Both Sulfiredoxin-1 induction and PrxSO_2/3 recovery were abolished by cycloheximide pre-treatment. PrxSO_2/3 was almost undetectable by Western blotting in whole cord or brain preparations from G93A or non-transgenic littermate mice.

Discussion and conclusions: Delayed PrxSO_2/3 recovery after oxidative stress in I113T-FALS fibroblasts implies that 2-Cys Prxs spend longer in an oxidatively-inactivated state after oxidative challenge. Defence against H₂O₂ will be impaired for longer after each challenge suggesting the possibility of an above-normal rate of accumulation of oxidative damage with repeated exposures. Our results thus far suggest that the delay in PrxSO_2/3 regeneration may be due to both reduced and delayed Sulfiredoxin-1 induction after oxidative stress, an induction dependent upon protein translation. The underlying cause and functional effect of this deficit are currently under investigation. We further plan to address whether this effect can be seen in other forms of familial and sporadic ALS and, more importantly, whether it is also a feature of motor neuronal cells.

P153 EXCITOTOXICITY IN CULTURED MOTOR NEURONS

HOSIE K^1,2, KING A^1,2, BLIZZARD C^1,2, VICKERS J^1,2, DICKSON T^1,2

¹Menzies Research Institute, Hobart, Tasmania, Australia, ²University of Tasmania, Hobart, Tasmania, Australia

Email address for correspondence: [email protected]

Keywords: excitotoxicity, axon degeneneration, neuromuscular junction

Background: ALS is characterised by degeneration of both motor neurons and the neuromuscular junction. Although the causes of this degeneration remain largely unknown, evidence has strongly implicated glutamate excitotoxicity as a primary cause of motor neuron degeneration. However, it is currently unclear whether this toxicity is directed to the neuronal soma or via the neuromuscular junction.

Objectives: We aim to investigate the hypothesis that “somatodendritic excitotoxin exposure can result in primary degeneration of the distal axon”. We are investigating this hypothesis by utilising a variety of motor neuron cell culture models, including established and novel techniques.

Methods: Primary cultures of spinal motor neurons were derived from E15 embryonic Sprague Dawley rats. Additionally, primary spinal glia and skeletal muscle cultures were obtained from neonates. Motor neurons were cultured onto either glial or muscle monolayers, or into compartmented chambers (Xona Microfluidics) containing proximal spinal glia and distal skeletal muscle. Cultures were fixed and characterised using standard immunocy to chemical techniques.

Results: Firstly, established culture techniques were evaluated, indicating spinal motor neurons grown on muscle feeder layers exhibit different growth characteristics from motor neurons cultured on glial cells. At 7 days in vitro (DIV) motor neurons demonstrated a significant (p < 0.05) increase in mean axon length (n = 5, 1363 μm ± 104 μm) when compared to glial cultured neurons (n = 5, 759 μm ± 1054 μm), and significantly (p < 0.05) reduced mean number of neurites when cultured on muscle monolayers (n = 5, 2.6 ± 0.3) compared with glial cultures (n = 4, 4.5 ± 0.8). By 21 DIV motor neurons cultured on both skeletal muscle and glial feeder layers were > 85% immunopositive for dephosphorylated neurofilaments, indicating relative culture maturity. Putative neuromuscular junctions were present in motor neuron-skeletal muscle co-cultures, indicated by clustering of acetylcholine receptors. Mean neuron survival was diminished when cultured directly onto skeletal muscle monolayers, evident from 3 DIV when compared with motor neurons on glial cells. Excitotoxicity was induced in both culture systems (25 μM kainic acid for 24 hours), resulting in significant (p < 0.05) and severe loss of motor neurons co-cultured with astrocytes (n = 3, 85% ± 1.8%) compared with motor neurons on a skeletal muscle monolayer (n = 3, 53% ± 6.3%). Currently, we are developing techniques to culture compartmented motor neurons with proximal glial cells and distal skeletal muscle.

Discussion and conclusions: These data indicate that a compartmented culture strategy may facilitate normal motor neuron development, and simultaneously allow focal excitotoxin exposure. This will enable investigation into the primary site of excitotoxic degeneration, providing insights into the mechanisms of degeneration in ALS.

P154 THE ROLE OF THE PHOSPHATIDYLINOSITIDE-3-KINASE PATHWAY AND INTRACELLULAR PH IN TDP-43 MODELS OF ALS

HALLORAN M¹, CREWTHER S¹, FARG M², WALKER A², WALLACE R³, ATKIN J²

¹Department of Neuroscience in the School of Psychological Science, La Trobe University, Bundoora, Victoria, Australia, ²Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia, ³School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia

Email address for correspondence: [email protected]

Keywords: HIF-1α, SGK1, NHE1, PI3-kinase, TDP-43

Background: Hypoxia and chronic respiratory deficiency are associated with the clinical pathology of ALS, and have been shown to occur early in the disease process. Hypoxia-inducible factor 1 alpha (HIF-1α) is involved in activating various factors which play an integral role in hypoxia, with previous research indicating that the selective inhibition of HIF-1α may occur in ALS. HIF-1α is activated via the phosphatidylinositide-3-kinase (PI3-kinase) pathway, which also activates serum and glucocorticoid-inducible-kinase-1 (SGK1) in response to cell stress. SGK1 has been shown to be involved in the activation of various enzymes and transcription factors, and has also been found to be upregulated in a transgenic model of ALS. Research also indicates that SGK1 and the Na + /H + exchanger 1 (NHE1) genes are activated in response to hypertensive stress. NHE1's role is to maintain neutral intracellular (i) pH, as inhibition of NHE1 is associated with the acidification of pHi and neurodegenerative disease. ALS is also characterised by the presence of intracellular inclusions comprised of aggregated proteins, such as TAR DNA binding protein 43 (TDP-43). However, the relationship between HIF-1α, SGK1, NHE1 and TDP-43 has not been explored.

Objectives: To investigate the association between HIF-1α, SGK1, NHE1 and TDP-43 using Pathway Studio.

Methods: Pathway Studio 8.0 (Ariadne Inc. Rockville MD) will be used to analyse the pathways between HIF-1α, SGK1, NHE1 and TDP-43.

Results: Mutant TDP-43 was found to be associated with HIF-1α through a mutual binding to glutathione-s-transferase. SGK1 expression was found to be associated with COMMD1 which in turn binds with the glutathione-s-transferase/ ubiquitin pathway. NHE1 function was associated with TDP-43 through Zinc (Zn²⁺) and the caspase pathway.

Discussion and conclusions: Results indicate that HIF-1α binds to the factor inhibiting HIF-1α (FIH-1) region on glutathione-s-transferase leading to a downregulation of HIF-1α, while TDP-43G37V was also shown to bind to glutathione-s-transferase. SGK1 was associated with COMMD1, with a knockdown of COMMD1 leading to an increased expression of SGK1. COMMD1 may be of significance in relation to TDP-43 as it binds to the glutathione-s-transferase/ ubiquitin proteasomal system and is stated to be involved in ensuring protein stability. NHE1 was shown to be regulated by Zn^{2 +} with an increase in extracellular Zn^{2 +} associated with the aggregation of endogenous TDP-43. NHE1 and TDP-43 were also associated with the caspase pathway, with an increase in caspase activation linked to TDP-43, and with caspase inhibition being related to the rescue of NHE1 expression. NHE1 may be involved in cell survival via a decrease in caspase activity and the activation of the PI3-kinase pathway. Therefore, factors in the PI3-kinase pathway, such as HIF-1α and SGK1, as well as the regulation of pHi may play a significant role in the development of TDP-43 related pathologies.

P155 ANALYSIS OF THE VASCULAR ENDOTHELIAL GROWTH FACTOR SYNTHETIC PATHWAY AND NUCLEOCYTOPLASMIC TRANSPORT IN SPINAL ANTERIOR HORN CELLS IN ALS AND MSOD1G93A TRANSGENIC MICE MODELS

NAGARA Y, TATEISHI T, KAWAMURA M, YAMASAKI R, KIKUCHI H, KIRA J-I

Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Department of Neurology, Fukuoka, Japan

Email address for correspondence: [email protected]

Keywords: VEGF, HIF1-α, karyopherin β1, nucleocytoplasmic transport

Background: The vascular endothelial growth factor (VEGF) exerts neuroprotective effects on motor neurons. However, the role of VEGF expression and nucleocytoplasmic transport in the pathogenesis of amyotrophic lateral sclerosis (ALS) remains to be clarified.

Object: To investigate the role of the VEGF synthetic pathway and nucleocytoplasmic transport in spinal anterior horn cells in the pathogenesis of ALS by using ALS and G93A mutant superoxide dismutase 1 (mSOD1) transgenic (Tg) ALS model mice.

Method: We performed immunohistochemical analysis of molecules involved in VEGF pathway and nucleocytoplasmic transport in mSOD1^G93A Tg mice, together with six postmortem ALS cases and six non-neurological disease controls.

Result: The immunoreactivity of VEGF receptors—VEGFR1 and 2—in the spinal anterior horn cells was significantly weaker in ALS cases than in the controls. In contrast, immunoreactivity of hypoxia-inducible factor (HIF)-1a in the anterior horn cells was significantly stronger in ALS cases than in the 9 controls. Nuclear staining of HIF-1a was observed only in several shrinking neurons. VEGF immunoreactivity in the anterior horn cells was also weaker in mSOD1^G93A Tg mice than in non-transgenic mice aged 12 (presymptomatic stage) to 18 weeks (terminal stage), despite of the stronger HIF-1a immunoreactivity in mSOD1^G93A Tg mice than in non-Tg mice. Nuclear staining of HIF-1a was detected in anterior horn cells after 16 weeks (early symptomatic stage); nonetheless, VEGF immunoreactivity in motor neurons continued to be weaken with age, whereas that in reactive astrocytes was more enhanced in mSOD1^G93A Tg mice than in non-transgenic mice. HIF-1a transports to nucleus by NLS system, such as Karyopherin b1. Double-fluorescence staining showed that HIF-1a co-localized with Karyopherin b1 in the cytoplasm of anterior horn cells in mSOD1^G93A Tg mice. Furthermore, nuclear staining in anterior horn cells was also weaker in mSOD1^G93A Tg mice than in non-transgenic mice aged 12 weeks (presymptomatic stage). Also as the disease progressed, discontinuous staining of nuclear membrne with Karyoperinb1 was increased. These intend to dysfunction nucleocytoplasmic transport.

Conclusion: These results indicate that pathogenesis of sporadic ALS ,as well as mSOD1^G93A Tg model mice, involves down-modulation of VEGF receptors and dysfunction of nucleocytoplasmic transport from the presymptomatic stage.

P156 GABA-INDUCED CURRENTS ARE UP-MODULATED BY MONOCYTE CHEMOATTRACTANT PROTEIN-1 IN CORTICAL NEURONS FROM THE G93A MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

ZONA C^1,2, CAIOLI S^1,2, PIERI M^1,2, ANTONINI A^1,2

¹Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy, ²I.R.C.C.S. Fondazione S. Lucia, Rome, Italy

Email address for correspondence: [email protected]

Keywords: GABAa receptor, MCP-1, cortex

Background: The inflammatory response in Amyotrophic Lateral Sclerosis (ALS) is well documented. Pathological analysis shows that the neurodegenerative process is accompanied by microglial activation and astrogliosis. Microglia, as the main immunocompetent cell type of the Central Nervous System (CNS), have the potential to secrete most of the inflammatory mediators that could contribute to disease progression. Some of these have been shown to be elevated in the cerebrospinal fluid (CSF) and CNS tissues of ALS patients, like the monocyte chemoattractant protein-1 (MCP-1). Interestingly, it has been described that the chemokine MCP-1 is able to alter the electrical activity of nociceptive neurons, indicating a modulatory effect on ionic channels involved in neuronal excitability.

Objectives: Since it has been reported that Cl^- influx through the ionotropic GABA_A receptors could be harmful to neurons and that in G93A spinal motor neurons an altered GABA_A receptor subunit composition has been observed, the aim of this work was to investigate whether MCP-1 was able to modulate GABA-induced currents both in control and G93A cultured cortical neurons.

Methods: Electrophysiological recordings were performed on 8-10 day-old in vitro cortical neurons from control and G93A mice, using the patch-clamp technique in whole cell configuration. Tetrodotoxin (1μM), cadmium (100 μM) and D- 2-amino-5-phosphonovaleric acid (10 μM) were added to the external solution to block voltage-gated Na⁺ and Ca²⁺ channels and N-methyl-D-aspartic acid receptors, respectively. GABA and MCP-1 were dissolved in extracellular solution and applied with a fast multibarrel pipette perfusion system controlled by electronic valves. The currents induced by GABA perfusion (5 μM) were recorded in control condition and during MCP-1 (100 ng/ml) perfusion, both in neurons from Control and G93A mice.

Results: GABA-induced currents were induced by bath application of GABA and the current amplitudes resulted significantly higher following MCP-1 perfusion (100 ng/ml), both in Control (n = 22; p < 0.01) and G93A (n = 27; p < 0.001) neurons. Moreover, the increase of the GABA-induced current was significantly higher in G93A neurons than Control (p < 0.05). The MCP-1 effect was not mediated by its G-protein coupled receptor, CCR2, because GABA currents were still higher both in Control (n = 8) and G93A neurons (n = 9) when the BMS-CCR2-22 (1 μM), a CCR2 antagonist, or when GDP-βS (2 mM), the G-proteins’ inhibitor, were added to the external and internal solutions, respectively.

Discussion and conclusions: These results suggest that: i) MCP-1 is able to modulate the GABA-induced currents both in control and G93A cortical neurons by increasing the amount of cloride influx; ii) in G93A neurons the increase of the cloride influx is significantly higher compared to controls and iii) GABA-induced current modulation is not mediated by CCR2 receptor activation. Further studies will be necessary to ascertain the MCP-1 mechanism of action on GABA_A receptors and the role of MCP-1 in ALS pathology.

P157 LOW-DOSE THAPSIGARGIN AND WILD-TYPE SOD-1 SYNERGISTICALLY ENHANCES NEURITE GROWTH IN MOUSE MOTONEURONAL CELLS

KIM J-S, CHOI J, LIM J-H, MOON J-H, KIM B-J, YOON B-N, SUNG J-J, LEE K-W

Seoul National University Hospital, Seoul, Republic of Korea

Email address for correspondence: [email protected]

Keywords: thapsigargin, NSC-34 cell, SOD1

Background: Thapsigargin (TG), an inhibitor of the sarco-endoplasmic reticulum Ca²⁺ -ATPase, modulates intracellular concentration of Ca²⁺ in a time- and compartment-dependent manner. TG at micromolar concentrations has been used as an apoptosis-inducing agent in cellular models of amyotrophic lateral sclerosis (ALS). However, cellular effects of TG at nanomolar concentrations have not been evaluated in models. We observed that nanomolar levels of TG enhanced neurite growth in mouse motoneuronal NSC-34 cells without inducing apoptosis. This finding led us to test whether TG-enhanced neurite growth is affected by overexpressed SOD-1, considering that SOD-1 is involved in intracellular regulation of Ca²⁺ concentration. After confirming overexpressed SOD-1 affects TG-enhanced neurite growth, we are currently investigating whether spatiotemporal change of intracellular Ca²⁺ concentration is a molecular mechanism underlying synergistical enhancement of neurite growth by low-dose thapsigargain and overexpressed SOD-1.

Objectives: To determine whether overexpressed SOD-1 affects low-dose TG-enhanced neurite growth in NSC-34 cells, and to understand the underlying molecular mechanism

Methods: NSC-34 cells were transfected with GFP, or GFP-tagged wild-type SOD1 (WT) or GFP-tagged mutant SOD1 with G93A constructs, and then differentiated with serum withdrawal. After 1 day, TG at different concentrations (3, 30, 300 nanomole) was treated to cells for 20 minutes. After 1 day, cells were fixed and stained with neurite-specific antibodies. Neurite growth of each cell was determined by measuring total lengths of neurites, the sum of the lengths of all neurites from each cell, using Image J software with Neurite Tracer. For in vivo Ca²⁺ imaging, Rhod-3 and Rhod-2 were used for the visualization of intracellular Ca²⁺ . Data were analyzed by Mann-Whitney test or by one-way ANOVA. Differences were considered significant at the p < 0.05 level.

Results: Quantitative analysis of neurites growth demonstrated that 30 nanomolar TG treatment increased total neurite length, whereas the other TG treatments failed to increase the length (GFP 0nM: 154.15 ± 6.2; 3nM: 170.52 ± 16.31; 30nM: 218.19 ± 23.23; 300nM: 126.51 ± 8.04, G93A 0nM: 143.55 ± 15.7; 3nM: 168.35 ± 25.80; 30nM: 190.23 ± 12.4; 300nM: 119.02 ± 4.83, WT 0nM: 151.46 ± 11.84; 3nM: 173.31 ± 16.84; 30nM: 235.4 ± 25.68; 300nM: 141.27 ± 10.09 (unit = micrometer, n = 10, p < 0.05)). The analysis also demonstrated that in the cells treated with 30 nanomolar TG, the cell overexpressing WT contain increased level of neurite growth compared with the cells overexpressing G93A or GFP (GFP 30nM: 218.19 ± 23.23, G93A 30nM: 190.23 ± 12.4, WT 30nM: 235.4 ± 25.68 (unit = micrometer, n = 10, p < 0.05)).

Discussion and conclusion: We found that low-dose, 30 nanomolar, TG increased neurite growth in NSC-34 cells, and that this effect was accelerated by WT, but not G93A. These findings suggest that WT and G93A are differentially involved in the intracellular Ca²⁺ regulation, and thus the identification of how WT and G93A affect the low-dose TG-mediated spatiotemporal change of intracellular Ca²⁺ concentrations would shed light on the pathogenic mechanism of ALS.

P158 NEUROTOXIC PATHWAYS IN VITRO – NEURORESCUE EFFECT OF RILUZOLE ON THAPSIGARGIN(THAPS), BUT NOT STAUROSPORINE(STS), HYDROGEN PEROXIDE(H₂O₂) AND HOMOCYSTEINE(HCY) TOXICITY IN DIFFERENTIATED MOUSE MOTOR NEURON-NEUROBLASTOMA HYBRID(NSC-34D) CELLS

HEMENDINGER R¹, ARMSTRONG III E¹, RADIO N³, BROOKS BR^1,2

¹Carolinas Medical Center, Charlotte, NC, USA, ²Univeristy of North Carolina School of Medicine, Charlotte, NC, USA, ³ThermoScientific, Pittsburgh, PA, USA

Email address for correspondence: [email protected]

Keywords: riluzole, drug development, pathogenetic pathways

Background: An in vitro model was developed to examine the pathways underlying ALS pathogenesis using a panel of neurotoxins and the differentiated form of the NSC-34 cell line, NSC-34D. Each of these compounds has a mechanism of action which is potentially involved in the molecular pathogenetic pathways found in ALS. We have previously shown neuroprotection in this model system (1,2). Other groups have shown neuroprotection in vitro by riluzole against excitotoxins (3) and STS (4). As ALS patients begin their treatment when some pathways are already activated, we have moved to a new paradigm, neurorescue, where neurotoxicity occurs simultaneous with the therapeutic agent being examined. Our goal is to examine the new neurorescue paradigm with each neurotoxin with respect to mechanism to analyze treatment effect size as well as potentiation of this treatment effect size in vitro.

Objective: We utilized the NSC-34D cells to examine the effects of riluzole on cell death induction by STS, Thaps, H₂O₂ and HCy (5).

Methods: Nuclear morphology, caspase-3/7 activation and high content imaging were used to assess toxicity of these compounds with and without co-treatment with riluzole.

Results: STS was the most potent compound at killing NSC-34D cells with a toxic concentration at which 50% of maximal cell death is achieved(TC₅₀ = 10 nM), followed by Thaps(TC₅₀ = 0.9 μM) and H₂O₂(TC₅₀ = 15 μM). HCy required higher concentrations to kill at the same level(TC₅₀ = 2.2 mM)(5). Using the neurorescue paradigm in NSC-34D cells, we show that riluzole provides neurorescue against Thaps-induced cell death (by 20.3%; 42.7 + 2.7 vs. 63.0 + 3.0; p < 0.05) but had little to no effect on STS-, H₂O₂ - and HCy-induced cell death. This effect of riluzole on cell death induction was independent of caspase-3/7 activation. Further analysis in a pilot study utilizing high content analysis revealed that toxicity with STS could be observed continuously over 24 h in a dose-dependent manner with no early or late benefit of riluzole on STS toxicity.

Conclusions: Riluzole, in a neurorescue paradigm associated with co-administration of riluzole at the initiation of neurotoxin exposure, is capable of reducing cell death, independent of caspase-3/7 activation, only of Thaps neurotoxicity but not STS, H₂O₂, or HCy neurotoxicity.

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