THEME 9 IN VITRO EXPERIMENTAL MODELS

P197 THE ACADEMIC MEDICAL CENTRE ALS BRAIN TISSUE BANK AND DOIS: A FEASIBILITY STUDY

Casula M

Troost D

Academic Medical Centre, Amsterdam, The Netherlands

Email address for correspondence: [email protected]

Keywords: brain tissue bank, datacite, digital object identifiers (DOI’s)

Tissue banks serve a useful purpose insofar as they host a valuable scientific resource but their full potential is often not realized because samples are not easily discovered. Even when tissue samples are made available, the work associated with their collection and curation is often not acknowledged and financial support for the maintenance of these important collections is often lacking as well. With the emergence of data citation infrastructures, notably DataCite, there is the potential to address these issues while still respecting sample confidentiality.

With the aim of encouraging discovery and acknowledgement of tissue samples for ALS research, a feasibility study was undertaken at the Neuropathology Department at the AMC in Amsterdam to assign DataCite DOIs (Digital Object Identifiers) to the tissue samples of the ALS Brain Tissue Bank. This tissue bank consists of a unique collection of brain and spinal cord tissue of ∼220 ALS patients who donated their body to science for research on ALS. The feasibility study involved assigning DataCite DOIs to a subset of 50 spinal cord paraffin samples. Assigning DataCite DOIs is of benefit to both the custodian of the tissue samples and the end-user because the former is cited for their contribution and the latter benefits from access to a valuable resource. Thus, in the circumstance that the work is published, the contribution of all concerned is acknowledged, potentially also leading to further use of the tissue bank.

While the confidentiality of the samples is respected, there is always a limited amount of bibliographic information (metadata) about the associated data set that is in the public domain that allows the samples to be discovered. The result is that for the ALS Tissue Bank, approval is always required form the curator before research collaborations are initiated.

In conclusion, when used to identify individual tissue samples, DataCite DOIs have the potential to encourage a better use of the ALS Brain Tissue Bank and hence stimulate research of ALS pathogenesis.

P198 DEVELOPMENT OF IN VITRO MODELS OF ALS

Pugh V¹

Powell L²

Matthews T²

Rattray M³

Sweeney J²

^aUniversity of Reading, Reading, UK

^bUniversity of Huddersfield, Huddersfield, UK

^cUniversity of Bradford, Bradford, UK

Email address for correspondence: [email protected]

Keywords: in vitro model, neuroprotection, excitotoxicity

Background: Riluzole is the licensed neuroprotective drug for the treatment of amyotrophic lateral sclerosis (ALS). Riluzole is proposed to have various effects at clinically relevant concentrations including inhibition of Na+ currents (1), potentiation of calcium-dependent K+ currents (2) and inhibition of glutamate release (3). Although riluzole is clinically proven to extend lifespan in ALS patients, it has limited efficacy, extending lifespan by three months, on average (4). In order to find drugs with an improved potency, robust assays must first be developed in which to test novel compounds.

Objectives: We have developed a range of neurotoxicity assays against which to test a variety of compounds, including novel riluzole derivatives. The assays have been designed to mimic particular features of the pathological process observed in motor neurone disease.

Methods: Primary cultures of mouse cortical neurones and motor neurones were exposed to kainate, NMDA, tunicamycin and arsenite to induce cell damage or neuronal death. FM1-43 and Fura 2 AM fluorescent dyes were used to observe any loss of synaptic activity and an increase in intracellular calcium, respectively, as read outs of excitotoxicity. Morphological assessment of these cells was also conducted using MAP2 immunocytochemistry.

Results: Under all treatment paradigms we were able to induce degradation of neuronal processes in both cortical and motor neurones in a concentration dependent manner. We have observed an increase in Fura 2 AM fluorescence and a reduction in FM 1-43 fluorescence following exposure to toxins with respect to vehicle treated controls.

Discussion and conclusion: Using these assays we found that the toxins we used are capable of inducing excitotoxicity and ultimately cell death. The reduction in FM 1-43 fluorescence suggests a loss of synaptic vesicle recycling under excitotoxic treatment and ultimately a loss in synaptic activity. The increase in Fura 2 AM staining with lower concentrations of kainate and NMDA highlights an initial increase in intracellular calcium, an early event in the excitotoxicity in motor neurones seen in ALS. Upon developing these robust assays we will screen novel riluzole derivatives in these assays to find those which are capable of protecting neurones more than riluzole.

Acknowledgements: MND Association, ALSA and Research Endowment Trust Fund (RETF) of the University of Reading for funding.

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P199 IN VITRO HIGHLY DIFFERENTIATED PRIMARY MUSCLE FIBER: A MODEL RECAPITULATING MUSCLE DIFFERENTIATION IN SOD1G93A MOUSE

Vilmont V¹

Cadot B¹

Gomes ER^1,2

^dMyology Research Center, Sorbonne Universités, UPMC Université Paris, Paris, France

^eInstituto di Medicina Molecular, Lisbon, Portugal

Email address for correspondence: [email protected]

Keywords: muscle, in-vitro differentiation, SOD1^G93A

Background: Amyotrophic lateral sclerosis (ALS) is a highly debilitating fatal disease in humans with life expectancy ranging from two to five years after diagnosis. This pathology is characterized by weakness, muscle atrophy and spasticity leading to death in patients mostly due to respiratory failure caused by impaired diaphragm contraction. Muscle atrophy is believed to be triggered by the loss of upper motor neurons (UMNs) in the cortex and of lower motor neurons (LMNs) in brainstem and spinal cord. Genetic defects in the zinc/copper superoxide dismutase (SOD1) gene, has been linked to familial and sporadic ALS. While this model has been extensively used to study loss of MNs and neuromuscular junctions, less attention has been given to the potential role of impaired muscle development.

Objective: To investigate the possible pathophysiological link between muscle development and neuromuscular junction loss in the SOD1^G93A mice.

Methods: Primary myoblasts were isolated 6 to 7 days after birth from transgenic mice expressing the G93A mutant form of human SOD1 and from WT littermates. Myoblasts were directed to differentiate to advanced stages. Features of high differentiation like triad formation, movement of nuclei to the periphery, fiber thickness as well as neuromuscular junction dynamics were assessed.

Results: Myoblasts from SOD1^G93A could be differentiated in vitro like the WT littermates. Assessment of differentiation features revealed a decrease in the number of fibers with peripheral nuclei, impaired triad formation and reduced fiber thickness. Moreover, staining with bungarotoxin showed a reduced number of AchR clusters as well as a decrease in their length.

Discussion and conclusion: These preliminary data secure this protocol as a robust model to study highly differentiated fibers recapitulating muscle development. Our findings from SOD1^G93A mice revealed that muscle development is impaired in fibers taken from young mice which are still asymptomatic supporting the hypothesis that muscle development could be an independent trigger of neuromuscular junction loss and muscle atrophy.

P200 INVESTIGATING MISFOLDED SOD1 IN ALS USING PATIENT-DERIVED CELLS

Forsgren E¹

Lehmann M¹

Amaroso MW²

Garcia-Diaz A²

Sandoe J³

Kiskinis E³

Nordström U¹

Andersen P¹

Brännström T¹

Eggan K³

Wichterle H²

Henderson CE²

Marklund S¹

Gilthorpe J¹

^fUmeå University, Umeå, Sweden

^gColumbia University, New York, USA

^hHarvard University, Boston, USA

Email address for correspondence: [email protected]

Keywords: induced pluripotent stem cells (iPSCs), SOD1, disease model

Background: Mutations in the superoxide dismutase 1 (SOD1) gene were first found to be associated with ALS (1). SOD1 mutations have now been found in 12–23% of familial ALS cases and in up to 6% of all cases of ALS. The leading hypothesis as to how SOD1 mutations lead to ALS is by the induction of conformational changes within the protein, accumulation of unfolded SOD1 and the subsequent formation of cytotoxic aggregates. Significantly, the presence of aggregates of unfolded SOD1 in both familial and sporadic ALS suggests that unfolded SOD1 might represent part of a final common pathway leading to motor neuron death is ALS (2).

Numerous studies have alluded to different, potential mechanisms of unfolded SOD1 toxicity. However, the precise nature of the toxic SOD1 species is not yet clear. The pathogenesis of ALS is proposed to be due to both cell autonomous mechanisms acting in motor neurons, and non-cell autonomous ones, where other cells types such as astrocytes are involved (3). We have focused on the use of induced pluripotent stem (iPS) cells as a model system to investigate both cell and non-cell autonomous mechanisms of unfolded SOD1 toxicity in vitro, using patient-derived cells.

Methods: iPS-derived motor neurons and astrocytes have been generated from different ALS patients carrying SOD1 mutations, as well from non-diseased controls. The amounts of unfolded SOD1 in patient derived fibroblasts, iPS cells, iPS-motor neurons and astrocytes were analysed.

Results: Our results show that: misfolded SOD1 is present in patient-derived cell cultures; amounts of misfolded SOD1 vary between cell types and SOD1 mutations; high amounts of misfolded SOD1 in neurons carrying G85S-SOD1 but even more in iPS-astrocytes; iPSCs are a useful system to study misfolded SOD1 in ALS; the presence of high levels of misfolded SOD1 could explain the selective vulnerability of motor area cells in ALS

Acknowledgements: This project was supported by Knut and Alice Wallenberg Foundation, Kempe Foundation, NHR, Ulla-Carin Lindquist Foundation For ALS Research, StratNeuro, Umeå University

P201 THE EFFECTS OF METABOLIC DISTURBANCES ON THE LEVELS OF MISFOLDED SOD1 IN ALS PATIENT-DERIVED FIBROBLAST LINES

Keskin I¹

Forsgren E¹

Gilthorpe J¹

Tokuda E²

Weber M³

Birve A¹

Andersen PM¹

Marklund Sl²

ⁱDepartment of Pharmacology and Clinical Neuroscience

^jDepartment of Medical Biosciences, Umea University, Umeå, Sweden

^kNeuromuscular Diseases Unit/ALS Clinic, Kantonsspital St. Gallen, St. Gallen, Switzerland

Email address for correspondence: [email protected]

Keywords: SOD1, misfolded, patient-derived fibroblast lines

Background: The neurotoxicity of mutant SOD1 is believed to be exerted by misfolded SOD1 species. The patho-mechanism is poorly understood but has been suggested to involve perturbation of mitochondria, induction of ER-stress, reduction of proteasome and autophagy efficiency and aggregation. Another unresolved feature of ALS is why carriers of SOD1 mutations are apparently healthy until middle age, and then undergo rapid neurological decline. Perhaps age-related decline in proteostasis and energy metabolism contributes, amplified by vicious circles that increase the levels of misfolded SOD1 in the tissue.

Objectives: We have generated fibroblast lines from skin biopsies derived from 8 ALS patients carrying mutations in SOD1 (A4V, H46R, E78_R79insSI, N86S, D90A, L117V, D126fsX24, G127instggg); from 1 ALS and 1 FTD patient with C9ORF72 GGGGCC-hexanucleotide repeat expansion, from 2 healthy control subjects. These cells, which express the mutant SOD1 under the native promoter, offer opportunities for exploration which are not accessible in other model systems. We used the cells to gain information on the effects of various metabolic disturbances on the levels of misfolded SOD1.

Methods: We cultured the fibroblast cell lines under a variety of stress conditions and with/without inhibitors. Misfolded SOD1 from fibroblast extracts was measured with a specific ELISA (misELISA) as described (1).

Results: All fibroblast lines derived from the ALS patients contained more misfolded SOD1 than those from control individuals. The proteasome inhibitor bortezomib caused marked increases in misfolded SOD1 levels. Induction of ER stress with tunicamycin, inhibition of mitochondria with rotenone, and suppression of autophagy with 3-methyladenine, also decreased the levels of misfolded SOD1. Hyperoxia might conceivably stabilize SOD1 by artificially promoting the C57-C146 disulfide bond. There were, however, no differences between cells cultured in physiological oxygen tension (4% O₂) and hyperoxia (20% O₂). Under none of the conditions could any detergent-resistant aggregates be demonstrated in the cell extracts.

Conclusion: Proteasome inhibition caused large increases in misfolded SOD1 levels. Other cellular perturbations occurring in ALS and aging did not per se induce increases in misfolded SOD1 levels.

Reference:

• Zetterström P et al. J Neurochem. 2011; 117: 91.
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P202 NEUROPROTECTIVE POTENTIAL OF HUMAN MESENCHYMAL STROMAL CELLS IN ALS IN VITRO MODELS

Böselt S¹

Rath KJ¹

Klöss S²

Esser R²

Köhl U²

Petri S¹

^lDepartment of Neurology

^mInstitute of Cellular Therapeutics, Hannover Medical School, Hannover, Germany

Email address for correspondence: [email protected]

Keywords: cell therapy, mesenchymal stromal cells (MSC), apoptosis

Background: Mesenchymal stromal cells (MSC) are currently discussed as potential candidates for the therapy of neurodegenerative diseases such as ALS and are already investigated in clinical trials, mainly based on the assumption that non-neuronal cells can provide a more protective environment for degenerating motor neurons. We have previously shown protective effects of an MSC cell line in vitro, based on modulations of cellular expression of growth factors, cytokines and chemokines in neurons and non-neuronal cells (1).

Objectives: We intended to establish good manufacturing practice (GMP) isolation of human MSC from bone marrow available from healthy donors and to develop appropriate quality control. Protective effects and underlying mechanisms of action were studied in cellular models of ALS.

Methods: 10-colour flow cytometric protocols for quality control including detection of residual T and B cells were established and validated. Expression of MSC surface markers was studied by FACS analysis and laser-induced fluorescence microscopy. Differentiation potential into osteocytes, chondrocytes and lipocytes was confirmed. Mouse embryonic primary motor neurons (derived from both non-transgenic and mutant SOD1^G93A transgenic mice), NSC-34 cells and glial cells (astrocytes) (derived again from both non-transgenic and mutant SOD1^G93A ALS transgenic mice) were co-cultured with human MSC and the effects against staurosporine- and H₂O₂-induced cell death were determined by immunocytochemistry and MTT assay.

Results: Conditioned medium (MSC CM) as well as co-culture with MSC attenuated staurosporine - and H₂O₂-induced cell death in a concentration-dependent manner in primary motor neurons, NSC-34 cells and astrocytes. MSC-co-culture decreased neuronal mRNA expression of glutamate receptor 2 and of matrix metalloproteinase 9 (MMP-9). Ongoing analyses comprise MSC-mediated changes in gene- and protein expression of pro-and antiinflammatory mediators, neurotransmitters and corresponding receptors and growth factors.

Discussion and conclusion: Our data demonstrate that MSC have neuroprotective capacities, mediated by effects on apoptosis, oxidative stress and possibly neurotransmission and ER stress. Analysis of MSC-induced changes in motor neurons and glial cells, also regarding genotype and possibly disease-specific alterations can further clarify their mechanisms of action and therapeutic potential.

Reference:

• Sun H et al. PLOS ONE. 2013; 8: e72926.
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P203 INVESTIGATING OLIGODENDROCYTE DYSFUNCTION IN ALS USING HUMAN STEM CELLS

Magnani D¹

Livesey MR.¹

Burr K¹

Selvaraj BT¹

Vasistha N¹

Evans E¹

Rajan R¹

Story D¹

Zhao C¹

Borooah S¹

Shaw C³

Vallier L²

Wyllie DJA¹

Chandran S¹

ⁿEuan Macdonald Centre, University of Edinburgh, UK

^oCambridge stem cell institute, Cambridge, UK

^pUCL, London, UK

Email address for correspondence: [email protected]

Keywords: oligodendrocytes, induced pluripotent stem cells (iPSCs), TDP-43

Background: In ALS/MND nerve cells degenerate leading to muscle weakness and paralysis. Another key cell in the brain and spinal cord is the oligodendrocyte (OL). These cells provide metabolic support and insulation to neurons and until recently were not believed to be involved in MND. However, studies of ALS human post mortem samples and the SOD1^G93A mouse model of ALS, implicate OLs in the aetiopathogenesis of motor neuronal degeneration. Our aim is to investigate the cell autonomous defects of oligodendrocytes in ALS.

Methods: We have optimized a platform to efficiently convert human induced pluripotent stem cells (iPSCs) obtained from control and familial ALS patients, carrying the TDP43^M337V and TDP43^G298S mutation, into functional OL progenitors (OPCs) and OLs. Spinal cord patterned iPSC derived OPCs (OLIG2 + and PDGFRa+) can be expanded for several weeks in the presence of FGF and PDGFa. OPCs are further differentiated into OLs by removal of FGF and PDGFa.

Results: Upon differentiation very little overlap is observed (< 15%) between PDGFRa+ OPCs and O4 + OLs, while differentiated OLs begin to express myelin basic protein (MBP) (> 90%), becoming mature OLs within 10 days of differentiation. OL differentiation and maturation coincided with an increase in cell size and OL development was associated with an increase in the number and length of OL processes, as quantified by Sholl analysis. Furthermore, using pharmacological and biophysical approaches we have examined the profile of passive membrane properties of hPSC-derived OPCs through to their maturation into OLs. OL differentiation was associated with a loss of spiking activity and a reduced rectification of outward currents that indicated a change in the ion channel expression profile of OLs compared to those expressed in OPCs. Specifically, we observed decreases in tetrodotoxin-senstive voltage-gated Na⁺-channels (OPCs: 29.1 ± 1.1 pA/pF, n = 18; OLs: 0 pA/pF, n = 5), TEA-sensitive delayed-outwardly rectifying K⁺-channels (OPCs: 26.4 ± 0.4 pA/pF, n = 15; OLs: 0.5 ± 0.1 pA/pF, n = 9) and transient A-type K⁺-channels (OPCs: 105.6 ± 4.5 pA/pF, n = 12; OLs: 8.1 ± 0.8 pA/pF, n = 6). Conversely, immunohistochemistry revealed prominent expression of inwardly-rectifying K⁺-channel 4.1 subunits in OLs, whereas expression of this channel subunit was only negligibly detected in OPCs. This recapitulates ion channel expression profiles exhibited by native (rodent) OL-lineage.

Discussion and conclusion: Studying molecular and physiological differences between WT and mutant TDP oligodendrocyte lines is ongoing. Our work has established a platform to investigate cellular autonomy in MND with a focus on oligodendrocytes.

P204 CHARACTERISATION OF TDP-43 DYSREGULATION IN C9ORF72 IPSC-DERIVED MOTOR NEURONS

Scaber J¹

Mutihac R¹

Cowley S²

Turner M¹

Talbot K¹

^qNuffield Department of Clinical Neurosciences, John Radcliffe Hospital

^rOxford Stem Cell Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

Email address for correspondence: [email protected]

Keywords: C9ORF72, induced pluripotent stem cells (iPSCs), TDP-43

Background: An expanded hexanucleotide (GGGGCC) repeat in chromosome 9 open reading frame 72 (C9ORF72) has been identified as a major cause of familial amyotrophic lateral sclerosis (fALS) and frontotemporal lobar dementia (FTLD). At post-mortem, patients with the hexanucleotide repeat have the typical TDP-43 positive inclusions that are detected in over 90% of patients with ALS, but also have TDP-43 negative inclusions that contain repeat-associated non-ATG (RAN) dipeptides translated from the intronic repeats. The role of TDP-43 pathology in C9ORF72 disease remains unknown.

Methods: Fibroblasts were obtained from healthy subjects and three ALS patients carrying ∼500 and ∼1000 GGGGCC hexanucleotide repeats in the C9ORF72 gene. Pluripotency was induced by reprogramming the fibroblasts with Sendai viruses carrying Sox2, Oct3/4, Klf4 and c-myc. Embryoid bodies were generated and neuralization was induced by retinoic acid (RA) followed by ventralization, which was achieved by sonic hedgehog agonists. Motor neuron precursors were allowed to reach maturation for at least 4 weeks before assessment of TDP-43 pathology.

Results: We detected significantly elevated levels of full-length TDP-43 protein in C9ORF72 iPSC-derived motor neurons, as well as a 35 kDa isoform. At the mRNA level, quantitative analysis of TDP-43 transcripts showed that levels of TARDBP mRNA were similar in both control iPSC-derived motor neurons and C9ORF72 iPSC- derived motor neurons. Oxidative stress induced by sodium arsenite treatment resulted in a marked decrease of soluble TDP-43 in C9ORF72 motor neurons compared to healthy motor neurons. Analysis of TDP-43 subcellular localisation was performed in both stressed and unstressed conditions. In the absence of oxidative stress, no differences were detected in TDP-43 localization between C9ORF72 and healthy iPSC-derived motor neurons (p values to follow).

Discussion and conclusion: In our iPSC-derived neuronal culture enriched for motor neurons, there is evidence of derangement of TDP-43 at the protein level but not at the mRNA level. Given that TARDBP mRNA is tightly regulated by TDP-43, this may suggest a disturbance in the autoregulatory mechanism of TDP-43.

P205 G4C2 REPEAT-INDUCED TRANSLATIONAL REPRESSION IN A CELLULAR MODEL OF C9ORF72 ALS

Rossi S^1,2

Serrano A^1,2

Giorgi A⁵

Gerbino V³

Nencini M³

Di Francesco L⁵

Schininà ME⁵

Cestra G⁶

Achsel T⁴

Carrì MT^2,3

Cozzolino M¹

^sInstitute of Translational Pharmacology, CNR, Rome, Italy

^tDepartment of Biology, University of Rome Tor Vergata, Rome, Italy

^uFondazione Santa Lucia, Rome, Italy

^vCenter for the Biology of Diseases, KULeuven, Leuven, Belgium

^wDipartimento di Scienze Biochimiche A. Rossi Fanelli, Università Sapienza, Rome, Italy

^xIstituto di Biologia e Patologia Molecolari, CNR, Rome, Italy

Email address for correspondence: [email protected]

Keywords: C9ORF72, protein translation, Pur-alpha

Background: A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. By sequestering BRNA binding proteins, these RNA foci can be toxic. The reduced effective concentration of these factors can then affect various steps of post-transcriptional gene regulation, such as alternative mRNA splicing, translational regulation, mRNA transport, or mRNA stability. However, the precise step that is affected by C9ORF72 repeat expansions, the major genetic cause of amyotrophic lateral sclerosis, is still ill defined.

Objectives: We investigated the possible mechanisms whereby C9ORF72 repeats might affect cell viability by identifying proteins that bind to C9ORF72 repeats, and analysing their expression and subcellular distribution.

Methods: A 31 pure GGGGCC repeat (G4C2)31 was obtained by in vitro ligation of complementary nucleotides, and cloned into mammalian expression plasmids. Mouse motoneuronal NSC34 cells and human HeLa cells transfected with (G4C2)₃₁ repeats were used as cellular models. Immunofluorescence analysis, coupled to fluorescence in situ hybridisation (FISH), was used to assess RNA foci formation and localization of binding partners. Global protein synthesis was monitored with the SUnSET method based on puromycin incorporation.

Results: To get insights into the mechanisms whereby C9ORF72 might induce cell toxicity, we used an in vitro-transcribed biotinylated RNA containing the (G4C2)₃₁ repeats to identify binding proteins. Through mass spec analysis of bands excised from SDS PAGE, we were able to identify many different factors involved in post-transcriptional gene regulation. In particular, members of the hnRNP and SR family of proteins, both regulators of alternative splicing, as well as translational regulators, including initiation and elongation factors, Pur-alpha, Pur-beta and other translation regulatory proteins were also found. The expression of (G4C2)₃₁ repeats is sufficient to induce the formation of intra-nuclear RNA foci in NSC34 and HeLa cells. A significant, although not complete, sequestration of some of the above factors into RNA foci was observed. Most strikingly, (G4C2)₃₁ repeat widely affects the overall distribution of Pur-alpha and its binding partner FMRP, which accumulate into intra-cytosolic granules that are positive for the expression of stress granules markers. In these conditions, global protein synthesis turned out to be decreased, as measured by the reduction in puromycin incorporation into nascent peptide chains.

Discussion and conclusion: Our observations show that C9ORF72 repeats are able to activate a stress response that leads to a general reduction of translation, and suggest that this might be due to C9ORF72 ability to bind and sequester translational regulators.

P206 GENERATION INTEGRATION-FREE AND XENO-FREE INDUCED PLURIPOTENT STEM CELLS FROM BLOOD CELLS OF FALS PATIENT CARRYING FUS GENE MUTATION

Liu X¹

Gao S²

Yu C²

Deng M¹

^yPeking University Third Hospital, Beijing, China

^zTongji University, Shanghai, China

Email address for correspondence: [email protected]

Keywords: induced pluripotent stem cells (iPSCs), FUS gene mutation, motor neuron

Objective: To generate integration-free and Xeno-free iPSCs from ALS patients carrying FUS gene mutation by introducing episomal plasmids into peripheral blood mononuclear cells with nucleofection, and to provide safe and reliable resources for clinical application in the future. In addition, we aimed to differentiate ALS-iPS into motor neurons and provide cell-based disease model for ALS disease in Han Chinese.

Methods: Transfer non-integrated episomal plasmid into peripheral blood mononuclear cells from FALS with FUS P525L gene mutation by using Amaxa Nucleofector^® I and other related nucleofection reagents. Remove the effect of animal-derived feeder cells and other components on human iPS cells by using E8 culture medium. Karyotype analysis, immunofluorescence staining, quantitative RT-PCR, teratome formation assay and bisulfate genomic sequencing were used to identify the pluripotency of these iPS cell lines. In addition, motor neurons were derived from these iPS cells by inhibiting SMAD pathway.

Results: 6 integration-free iPS cell lines were established from ALS patients carrying the P525L mutation. Cell lines can be stably cultured in Xeno-free medium longterm, demonstrate pluripotency and are similar to human ES cells.Furthermore, motor neurons were successfully induced from these iPS cells. Dislocation and aggregates of mutant FUS protein were observed not only in motor neurons but also in iPS cells.

Discussion and conclusion: Gene mutations do not affect the reprogramming of blood cells and pluripotency of iPS cells, nor does it prevent differentiation of motor neurons. The non-integrated ALS-specific iPS cell lines we generated can avoid the integration of foreign genes into the genome, and remove the impact of animal-derived feeder cells and other components on human iPS cells, which is more suitable for clinical application in the future. Furthermore, the disease model we generated can recapitulate key aspects of ALS pathogenesis. They offer a cell-based disease model and indispensible resource for further elucidating ALS disease pathogenesis and screening appropriate drug candidates in Han Chinese.

Acknowledgements: We thank the ALS patients who participated in the study. This work was made possible with support from National Natural Science Foundation of China (81072374, 31171048, 30700906) and Beijing Municipal Natural Science Foundation (7112146), Beijing Nova Program (2009A04).

References:

• Meng X et al. Mol Ther. 2012; 20(2): 408–416.
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P207 NOT AS FUS-SY AS PREVIOUSLY THOUGHT; A STUDY OF THE CHANGING SUB-CELLULAR LOCALISATION OF TDP-43 AND FUS IN RESPONSE TO CELL STRESS

James J

Moujalled D

Duncan C

Liddell J

White A

The University of Melbourne, Parkville, Victoria, Australia

Email address for correspondence: [email protected]

Keywords: FUS, TDP-43, stress-granule

Background: Mutations in the RNA binding proteins TDP-43 and FUS have been associated with familial and sporadic forms of MND. Recent findings have implicated RNA binding proteins such as TDP-43 in stress response and have shown that both wild-type and mutant TDP-43 incorporate into stress granules. However, it is widely believed that wild-type FUS does not incorporate into stress granules (1), thus differing from TDP-43.

Objectives: To investigate changes in sub-cellular localisation of TDP-43, and FUS in response to various inducers of stress in cell culture; and to determine if wild-type FUS incorporates into stress granules.

Methods: Primary cortical neurons, and SH-SY5Y neuroblastoma cells, were grown in culture and treated with various inducers of sub-toxic cell stressors which are known to cause mitochondrial impairment or oxidative stress; sodium arsenite, paraquat, ammonium ferric (III) citrate (n ≥ 3). The level of cellular LDH release was measured to indicate cell death. Changes in cellular metabolic activity were measured using both the MTT assay and the bioluminescent ATP assay. Cells were fixed and immunocytochemistry was used to determine TDP-43 and FUS localisation, and incorporation into stress granules, using the markers TIAR and HuR. The software Imaris v7.1 was used on confocal images to determine changes in nuclear vs cytosolic distribution of proteins.

Results: Wild type FUS does incorporate into stress granules with 300μM sodium arsenite treatment for 3hr in culture, but not 1hr in culture. A significant number of stress granules were induced (n ≥ 3, p<0.05 increase of 36.7 granules per 100 cells). FUS translocated from the nucleus to the cytosol with this treatment (n ≥ 3, p = 0.0432). These changes occurred with minimal cell death (no significant LDH release, p = 0.696) but a significant decrease in metabolic activity was observed by reduction in MTT metabolism (n ≥ 6, p < 0.0001), and confirmed via the ATP assay (n ≥ 3, p = 0.0171).

Discussion and conclusion: It has become widely accepted that wild type FUS (1) does not incorporate into stress granules, however this study shows that during cell stress (ATP depletion in response to sodium arsenite treatment), wild-type FUS moves from the nucleus to the cytosol and incorporates into stress granules, yet this occurs several hours after TDP-43 incorporates into stress granules. This suggests different roles for TDP-43 and FUS in response to cell stress.

Conclusion: Wild type FUS incorporates into stress granules with prolonged stress treatments. This result suggests that the normal role of FUS during conditions of metabolic impairment is to translocate from the nucleus to the cytosol and to regulate cytosolic RNA processing.

Acknowledgements: BOMP microscopy platform, University of Melbourne, Rotary Health and The Rotary Club of Bendigo South, MND Research Institute of Australia.

Reference:

• Bosco DA et al. Human Mol Gene. 2010; 19(21): p. 4160–4175.
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P208 PUR-ALPHA INHIBITS MRNA TRANSLATION AND AFFECTS STRESS GRANULES MODULATING LOCALIZATION AND ACTIVITY OF FUS

Di Salvio M^1,2

Gerbino V³

Piccinni V^1,2

Camerini S⁴

Chellini L⁵

Carrì MT^3,5

Loreni F⁵

Cozzolino M^3,6

Cestra G^1,2

^aaIBPM, Istituto di Biologia e Patologia Molecolari, CNR, Rome, Italy

^abUniversity of Rome “Sapienza”, Dept. of Biology and Biotechnology Charles Darwin, Rome, Italy

^acLaboratory of Neurochemistry, Fondazione Santa Lucia, IRCCS, Rome, Italy

^adDept of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy

^aeDepartment of Biology, University of Rome “Tor Vergata”, Rome, Italy

^afIFT, Istituto di Farmacologia Traslazionale, CNR, Rome, Italy

Email address for correspondence: [email protected]

Keywords: Drosophila, retinal degeneration, Pur-alpha

Background: FUS protein carrying ALS causative mutations mislocalizes and coalesces in stress granules (SGs). We identified Pur-alpha as a protein that specifically binds to mutated FUS. Pur-alpha is a highly conserved protein that interacts, in a sequence-specific fashion, with single-stranded DNA and RNA (1). Interestingly, Pur-alpha was recently demonstrated to bind to GGGGCC expanded repeats of C9ORF72 gene, one of the most frequent mutations associated with ALS (2). In a Drosophila model of neurodegeneration caused by GGGGCC repeats expression, Pur-alpha ameliorates the phenotype (3).

Objectives: We investigated the protein-protein interaction network around the C-terminus of FUS and we identified Pur-alpha as a FUS binding protein. Thus we addressed the role of Pur-alpha in the regulation of stress granules and its effect on localization and toxicity of FUS.

Methods: We performed affinity purification from rat brain extracts and mass spectrometry to identify FUS interacting proteins. We verified FUS/Pur-alpha interaction in pull down and co-immunoprecipitation experiments. We addressed the role of Pur-alpha in the regulation of stress granules in mammalian cell lines and its role in FUS- mediated neurodegeneration in Drosophila melanogaster transgenic flies.

Results: We have found that Pur-alpha binds specifically to the C-terminal region of mutant FUS in an RNA-dependent fashion and that the two proteins colocalize in SGs. We also report that Pur-alpha associates with monomeric ribosomes and polyribosomes. Interestingly, phosphorylation of the alpha subunit of eIF2 initiation factor is upregulated by either Pur-alpha or mutated FUS overexpression. According to an inhibitory effect of Pur-alpha on mRNA translation, its expression suppresses SG coalescence and relocalizes mutated FUS in a cytosolic diffuse pool. RNAi-mediated ablation of Pur-alpha produces locomotion defects in Drosophila indicating a pivotal role for this protein in motoneuronal function. Surprisingly, the ectopic expression of Pur-alpha in different Drosophila tissues, although is sufficient to produce a diffused pool of mutated FUS, exacerbates neurodegeneration.

Discussion and conclusion: We propose that mutated FUS and Pur-alpha affect parallel pathways converging on the inhibition of protein translation, and that translation inhibition is involved in FUS-mediated ALS. Since Pur-alpha expression in Drosophila motoneurons, as well as in cultured cells, reduces mutant FUS aggregation and worsens the degenerative phenotype, we propose that soluble mutant FUS in the cytoplasm is more dangerous for the cell than clustered in SGs.

In conclusion, we unveil a specific physical and genetic interaction between Pur-alpha and FUS and a novel functional role of Pur-alpha in the regulation of SGs and protein synthesis, which affects localization and toxicity of mutant FUS proteins.

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P209 MUTANT FUS/TLS EXPRESSION INHIBITS PML NUCLEAR BODY TURNOVER

Ko HK

Zhou H

Hayward L

Univ of Massachusetts Medical School, Worcester, MA, USA

Email address for correspondence: [email protected]

Keywords: FUS, PML, nuclear body

Background: More than 30 dominant mutations in the nucleic acid binding protein FUS/TLS (fused in sarcoma/translocated in liposarcoma) cause ∼5% of familial ALS cases. Multiple roles of FUS/TLS have been proposed in both the nucleus and the cytoplasm, but the mechanism(s) by which mutant FUS/TLS renders motor neurons vulnerable to degeneration in ALS have not been specifically defined. The promyelocytic leukemia (PML) protein is a major constituent of PML nuclear bodies (PML-NBs), which are dynamic structures implicated in nuclear homeostasis and stress responses that may influence motor neuron health.

Objectives: Because the functional roles of PML-NBs and associated components remain incompletely understood, we sought to test whether expression of human WT or mutant FUS/TLS variants may perturb the dynamics of PML-NB formation, localization, composition, or turnover.

Methods: We expressed human GFP-FUS/TLS variants (wild type or R495X, R521G, or P525L mutants) stably in HEK293 cells and performed immunostaining for GFP, PML, proteasome 11S subunit, and the PML-associated protein Daxx. Cells were exposed to stress for 1–24 h using arsenic trioxide (ATO), an agent known to induce PML-NBs acutely and to potentiate subsequent PML protein degradation by the proteasome.

Results: We observed that exposure to 1 μM arsenic trioxide (ATO) acutely induced the formation of large PML-NBs within 1–2h for both mutant and control HEK293 cells. After 12–24h of exposure to ATO, cells expressing wild type FUS/TLS showed a reduction in PML-NBs, as would be expected due to normal turnover of PML bodies by the proteasome. In cells expressing mutant FUS/TLS, however, large PML-NBs persisted and localized to perinucleolar regions. Increased PML protein accumulation was detected in the RIPA- insoluble fraction from cells expressing FUS/TLS mutants compared to control. Within 1h of ATO exposure, the 11S proteasome subunit colocalized with PML-NBs in both mutant and control cells, and this colocalization persisted in the large PML-NBs of mutant cells at 24h. Immunostaining for the PML-associated protein Daxx, which has been implicated in stress-induced cell death and a Fas-induced pathway relevant to motor neuron degeneration, revealed strong accumulation of Daxx in large PML-NBs of cells expressing mutant FUS/TLS.

Discussion and conclusion: These results suggest that mutant FUS/TLS expression in HEK293 cells inhibits the normal turnover of PML-NBs during prolonged stress despite effective recruitment of the 11S proteasome subunit to PML-NBs. Ongoing studies are directed toward understanding the mechanism of failure to degrade PML-NBs and determining whether the accumulation of PML-NBs, or associated proteins such as Daxx, may impair cell viability.

Acknowledgements: Supported by the ALS Therapy Alliance and the ALS Association

P210 INVESTIGATING THE ROLE OF THE NBAF COMPLEX IN ALS

Tibshirani M

Durham H

McGill University, Montreal, Quebec, Canada

Email address for correspondence: [email protected]

Keywords: chromatin remodeling, dendrites, histone acetylation

Background: The nBAF (neuronal Brg1-associated factors) complex is a chromatin remodeling complex important for neuronal differentiation, dendritic arborization and synaptic plasticity. Complexes include multiple BAF subunits, CREST and Brg1 (1). Dendritic attrition and cytoplasmic accumulation of the DNA/RNA binding proteins FUS and TDP-43 are associated with ALS (2, 3). In addition, FUS interacts with components of the nBAF complex, including Brg1 (4). Recently, CREST variants were discovered in ALS trios, suggesting a role for the nBAF complex in ALS (4).

Objective: In the context of identifying epigenetic changes in motor neurons resulting from altered nuclear/cytoplasmic distribution of wild type and mutant FUS/TDP-43 and their binding partners, we examined components of the nBAF complex and the relationship of their distribution to maintaining dendritic architecture.

Methods: Spinal cord-DRG cultures were prepared from E13 mouse embryos. Motor neurons in mature cultures were microinjected with plasmids encoding flag-tagged WT or mutant FUS or TDP-43 along with mCherry to visualize cell morphology. Dendritic architecture was quantified using the MATLAB script Bonfire (Firestein Lab).

Results: In motor neurons expressing mutant FUS or TDP-43, dendritic branching and length were decreased and terminal dendritic branches were lost compared to WT or empty vector controls. Furthermore, Brg1 was depleted from the nucleus in neurons with redistribution of WT or mutant proteins to the cytoplasm. Overexpression of Brg1 was sufficient to completely prevent the dendritic attrition caused by mutant FUS (p = 0.00055) or TDP-43 (p = 1.98e-6). Treatment with the Brg1 inhibitor, PFI-3, dramatically reduced dendritic arborization in neurons, replicating the effect of mutant FUS/TDP-43. Brg1 contains bromodomains, which have a high affinity for acetylated lysine in histones; treatment with the histone deacetylase inhibitor Vorinostat, preserved actetylation of H3K9/14 in motor neurons expressing mutant FUS or TDP-43 and prevented the nuclear depletion of Brg1 and dendritic attrition associated with these mutant proteins.

Discussion and conclusion: Attrition of upper and lower motor neuron dendrites is a prominent feature in ALS, which would contribute substantially to dysfunction (4). Our study links the cytoplasmic accumulation of FUS and TDP-43, which occurs in familial and sporadic forms of ALS, to epigenetic changes including disruption of nBAF complex activity, leading to retraction of the dendritic arbor. Brg1 appears to be a master regulator, and potential therapeutic target, since maintaining Brg1 in the nucleus was sufficient to prevent dendritic attrition, despite redistribution of FUS andTDP-43 to the cytoplasm. The influence of other nBAF components is being investigated.

Acknowledgments: Supported by MDA and ALSA.

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P211 GRASPS™: A NOVEL, SIMPLE AND ROBUST METHODOLOGY TO IDENTIFY ALS-ALTERED MRNA MOLECULES UNDERGOING PROTEIN SYNTHESIS

Dodd JE

Walsh MJ

Shaw PJ

Hautbergue GM

SITraN, University of Sheffield, Sheffield, UK

Email address for correspondence: [email protected]

Keywords: RNA dysregulation, translating mRNA molecules, pathophysiology

Background: Dysregulation of messenger RNA (mRNA) metabolism constitutes a major determinant in the pathogenesis of several ALS-subtypes (1, 2). Many research groups around the world have characterized hundreds to thousands of abnormal RNA-processing events in cell and animal models of ALS as well as in post-mortem CNS tissues from patients. However, these studies (i) did not allow discrimination of pathophysiological events from those which were consequent upon initial dysregulations, and (ii) did not investigate at genome-wide level whether altered mRNAs are translated into proteins.

Objectives: Since proteins are ultimately involved in cell fate, survival or neurodegeneration, we set out to develop a reliable method to identify the levels and sequences of actively translated mRNA molecules, in order to apply this methodology to TDP-43 ALS-inducible cell models that present widespread dysregulation of RNA metabolism.

Methods: GRASPS^™ is based on stalling protein synthesis using potent translation inhibitors such as cycloheximide (3) or the bacterial toxin BLF1 (4), as well as various cross-linking reagents. Once stalled the stable translation complexes are purified under stringent conditions that also dissociate peripheral ribosomal components and contaminating RNA-binding factors indirectly bound to ribosomes. The specific purification of stalled ribosomes enables the trapped translating mRNA molecules to be purified and then identified using next generation RNA sequencing.

Results: We have established proof-of-principle for this methodology and named it GRASPS for Genome-wide RNA Analysis of Stalled Protein Synthesis. High purity and integrity of isolated ribosomes and associated-mRNA molecules were validated using mass spectrometry and the Bioanalyser (Agilent). The method was also functionally validated using quantitative RT-PCR analysis of a Green Fluorescent Protein (GFP) mRNA reporter in GRASPS^™ fractions from stable GFP-inducible human HEK cell lines with or without a mutation of the initiating start codon. We next applied the novel methodology to the investigation of the functional consequences of RNA dysregulation in TDP-43 Q331K ALS- inducible non-neuronal (HEK) and motor-neuron-like (NSC-34) cell models. We are currently proceeding with the Next Generation RNA sequencing analysis.

Discussion and conclusion: We have developed a novel method, GRASPS^™, which allows isolation and identification of translating mRNA molecules at genome-wide levels. We will be identifying the first ALS translatomes in TDP-43 Q331K cell models. In addition, GRASPS^™ is not only directly transferable to other ALS subtypes and for use in tissues, but also to any other disease in which RNA dysregulation may be involved.

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P212 CHARACTERIZATION OF MATRIN 3, A NEW RNA BINDING PROTEIN LINKED TO ALS

Boehringer A¹

Pioro E²

Chiò A³

Traynor BJ⁴

Bowser R¹

^agBarrow Neurological Institute, Phoenix, AZ, USA

^ahDepartment of Neurology Cleveland Clinic, Cleveland, OH, USA

^aiDepartment of Neuroscience University of Turin, Turin, Italy

^ajLaboratory of Neurogenetics, National Institutes of Health, Bethesda, MD, USA

Email address for correspondence: [email protected]

Keywords: Matrin 3, TDP-43, proteomics

Background: We recently described four mutations in the MATR3 gene encoding the nuclear matrix protein Matrin 3 that are associated with ALS in four families around the world (1). Matrin 3 is a RNA binding protein and by immunohistochemistry is located to the nucleus of motor neurons and glia in the spinal cord. While cytoplasmic inclusions containing Matrin 3 are rare, we detect increased Matrin 3 immunoreactivity in motor neuron nuclei in ALS subjects and diffuse cytoplasmic immunostaining in some motor neurons. Patients that harbor mutations in MATR3 exhibited the most intense nuclear immunoreactivity.

Objectives: Our goal was to characterize the function of wild-type and mutant Matrin 3 protein to help define its role in ALS.

Methods: Cell lines expressing each of the four mutations and wild type MATR3 were created in HEK cells and Neuro2a neuroblastoma cells. These cell lines as well as tissue from ALS patients, controls and an ALS patient with a MATR3 mutation were used in our studies. Immunoprecipitation was performed and proteins separated by gel electrophoresis. Gel slices were digested with trypsin and proteins identified by liquid chromatography tandem mass spectroscopy (LC-MS/MS).

Results: We identified 192 proteins that specifically interact with wild-type Matrin 3 using immunoprecipitation coupled to mass spectroscopy (IP-MS). Our IP-MS results confirmed a prior report suggesting Matrin 3 binds to TDP-43(2). We further demonstrated that the binding between Matrin 3 and TDP-43 is affected by some of the mutations in MATR3. A number of other proteins that function in RNA metabolism and translation also interact with Matrin 3. The Matrin 3 protein interactome is altered by mutations within MATR3. Cells that stably express wild type or mutant MATR3 exhibit Matrin 3 immunostaining predominantly in the nucleus, similar to our findings in human tissue. We are also screening a panel of stressors to Matrin 3-expressing cells to determine changes in cell vulnerability caused by mutations in MATR3.

Discussion and conclusion: We recently identified mutations in the MATR3 gene linked to familial ALS. Our current study creates a model system to investigate the function of Matrin 3 and how disease associated mutations alter protein function. We performed IP-MS to define the protein interactome of Matrin 3 and confirmed direct interactions of Matrin 3 to TDP-43. We have also explored how disease associated mutations in Matrin 3 alter protein interactions. Our studies will lead to a better understanding of how Matrin 3 functions in RNA metabolism and its role in the pathophysiology of ALS.

Acknowledgments: This work was supported by NIH grant NS061867 to RB.

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P213 DEVELOPMENT OF AN ANTISENSE OLIGONUCLEOTIDE-BASED METHOD TO MANIPULATE RNA EDITING OF AMPAR SUBUNITS

Chaytow H

Popplewell L

Dickson G

Chen P

Royal Holloway University, London, UK

Email address for correspondence: [email protected]

Keywords: calcium signalling, neurodegeneration

Background: AMPA receptors (AMPARs) are a subset of glutamate receptors composed of one or more of four different subunits (GluA1–4). AMPARs are generally permeable to calcium, unless the GluA2 subunit is present. AMPAR subunits undergo RNA editing at specific sites in their sequence, a post-transcriptional modification caused by deamination of an adenosine subunit. This forms inosine, which is read by the cell’s translation machinery as guanosine therefore changing the codon, critical for the GluA2 subunit’s impermeability to calcium. Studies have shown that reduced editing is found in spinal cord motor neurons of MND patients, and this may lead to their cell death via increased calcium influx (1). Deamination is carried out by a family of enzymes called Adenosine Deaminases Acting on RNAs (ADARs). ADAR2 has multiple alternatively-spliced variants within mammalian cells: some have been shown to reduce the efficiency of ADAR2’s deamination. One such alternatively-spliced transcript contains the addition of an exon containing an AluJ sequence. We can interfere with endogenous exon splicing using Antisense oligonucleotides (ASOs); short complementary nucleotide sequences which can be used to prevent AluJ exon inclusion.

Objectives: We designed ASOs targeting the splice sites surrounding the AluJ-containing exon in ADAR2 and examined their effects on AluJ inclusion and the RNA editing process in mammalian cell lines.

Methods: HeLa, NSC34 and SHSY5Y cells were grown in culture and treated with ASOs (n = 3 per treatment group). HeLa cells do not endogenously express AMPARs, and so a plasmid containing a section of the GluA2 subunit was transfected into cells in order to measure RNA editing. Editing efficiencies were measured using a RT-PCR based assay on RNA extracts.

Results: Endogenous ADAR2 within HeLa cells exhibited an inclusion rate of the AluJ exon of 59.23% (± 0.62) within ADAR2 transcripts (ie, there is endogenous exon skipping of around 40%). When exposed to 2 μM of ASO, exon skipping was enhanced to 99.37% (± 0.41, p < 0.05). Exclusion of the AluJ exon in ADAR2 significantly increases editing of a minigene containing a section of GluA2 from 23.1 ± 1.07% to 29.5 ± 0.98% (p < 0.05). We are now examining the effects of our ASOs in neuronal-like cell lines (NSC34s and SHSY5Ys) known to endogenously express the GluA2 subunit.

Discussion and conclusion: This shows promising preliminary data for the use of ASOs to alter RNA editing of AMPARs. Ultimately this ASO will be tested in primary neuronal cultures, where this increased editing efficiency can be enhanced and may prove to be beneficial to neuronal viability.

Acknowledgements: This work was supported by an MND Association PhD studentship.

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P214 FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED PROTEINS FORM AGGREGATES VIA DISTINCT PATHWAYS IN CELLS

Yerbury J^1,2

Farrawell N^1,2

Lambert-Smith I^1,2

^akUniversity of Wollongong

^alIllawarra Health & Medical Institute, Wollongong, NSW, Australia

Email address for correspondence: [email protected]

Keywords: protein aggregation, protein homeostasis, cell biology

Background: ALS pathology is characterised by intraneuronal protein deposits including ubiquitylated round, conglomerate or skein-like inclusions. The composition of ubiquitylated inclusions varies considerably depending on whether the disease is sporadic or familial and the genetics of the familial forms. Both FUS and SOD1 mutations cause ALS with FUS (1, 2) and SOD1 positive inclusions (3), respectively. However, most other cases have inclusions positive for TDP-43 (3). Given that protein aggregation is linked to toxicity we sought to understand the types of aggregates formed by TDP-43, FUS and SOD1.

Objectives: In cells, protein aggregation is thought to procede via formation of juxtanuclear quality control compartments (JUNQ) or insoluble protein deposits (IPOD) (4, 5). This study aimed to characterize the aggregation pathways of mutant TDP-43, FUS and SOD1 in cells.

Methods: Cell models were created in NSC-34 cells using mutant TDP-43, FUS and SOD1 fused to GFP. Inclusion formation was followed by GFP foci, ubiquitin-RFP and with a Htt_ex1-46Q-RFP reporter of iPOD formation. Microtubules were destabilized by treatment with nocodazole. Inclusion mobility was measured using fluorescence recovery after photobleaching (FRAP) on a Leica SP5 scanning confocal microscope.

Results: SOD1 JUNQ-like inclusions are always positive for ubiquitin, do not co-localize with Htt, contain a mobile fraction and are dependant on microtubules as found previously (4, 5). FUS inclusions contain a mobile fraction, do not require microtubules and co-localize to Htt. In contrast TDP-43 inclusions are not dependant on microtubules, are immobile, but do not co-localize to Htt. Moreover, TDP-43 and FUS inclusions progressively co-localise with ubiquitin with time and are adjacent to LC3-positive foci.

Discussion and conclusion: These data suggest that the properties of TDP-43-, FUS- and SOD1-inclusions and the pathways through which they form are distinct. In addition, we propose that TDP-43 inclusions represent a newly identified mechanism of protein aggregation in the cell. This will have implications for potential therapeutics targeting protein aggregation pathways in ALS.

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P215 ALS-ASSOCIATED MUTANTS OF SOD1, TDP-43, FUS, C9ORF72 AND UBQLN2 DISRUPT PROTEOSTASIS IN NEURONAL CELL CULTURE

Lambert-Smith I^1,2

Saunders D²

Yerbury J¹

^amUniversity of Wollongong, Wollongong, NSW, Australia

^anGarvan Institute of Medical Research, Sydney, NSW, Australia

Email address for correspondence: [email protected]

Keywords: protein homeostasis, ubiquitin, genetic mutations

Background: The ability of motor neurones to maintain protein homeostasis (proteostasis) is significantly compromised in ALS. Proteostasis maintenance is necessary for proper cellular function, and clear evidence for its disruption lies in the common occurrence of ubiquitin-positive inclusions within affected motor neurones in the various genetic forms of ALS.

Objectives: This study aimed to investigate the proteostasis disruptions caused by familial ALS (fALS)-associated gene mutants in a neuronal cell culture model, using reporter substrates for the ubiquitin-proteasome system (UPS) and cellular chaperone activity.

Methods: NSC-34 cells were transfected with mutants of SOD1 (A4V), TDP-43 (M337V), FUS (R495X), C9ORF72 (38x and 72x expansions of the GGGGCC repeat), and UBQLN2 (P525H) fused to GFP. Inclusion formation was followed by either GFP foci presence in cells or by co-transfection with ubiquitin-RFP. NSC-34 and SH-SY5Y cells were also co-transfected with the GFP fusion proteins and a tomato-CL1 reporter of UPS activity. The CL1 degron sequence targets it for proteasome degradation (1); any blockade of this system results in accumulation of the tomato fluorescent protein signal. Additionally, SOD1 and TDP-43 fused to tomato were co-transfected with mutants of firefly luciferase fused to GFP (FLuc-GFP), which act as reporters of the proteostasis environment (2); the FLuc mutations destabilise the protein, therefore any perturbations in the chaperone-mediated folding environment will result in an inability of chaperones to monitor and refold the FLuc reporters.

Results: In cells expressing the fALS-associated mutants we observed differences in the timing of ubiquitin localisation to inclusions, potentially reflecting different mechanisms underlying inclusion formation. High level expression of SOD1, C9ORF72 and UBQLN2 caused tomato-CL1 accumulation. This accumulation was particularly striking in cells expressing mutant TDP-43 and FUS. In addition we found SOD1 expression caused an increase in mutant FLuc aggregation, indicating a deficiency in the availability of chaperones and therefore a reduction in global proteostasis capacity.

Discussion and conclusion: These data provide insight into the ways in which these fALS-associated genetic mutations lead to disruptions in motor neurone ability to maintain proteostasis. In particular we have discovered that these mutations compromise the UPS. Protein quality control systems involve many different proteins with defined roles. It will be important to closely examine the specific perturbations caused by the fALS-mutants and assess whether these changes in proteostasis result in the same motor neurone pathology that characterises this disease.

Acknowledgments: We would like to acknowledge the Australian Research Council and Australian Rotary Health/Rotary Club of Dural.

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P216 STRUCTURAL AND DYNAMIC CHARACTERISATION OF TDP-43 USING NMR SPECTROSCOPY

Weise A¹

Kirkpatrick JP¹

Robertson AL¹

Ng JSW²

Kumita JR²

Scotter EL³

Shaw CE³

Dobson CM²

Christodoulou J¹

Cabrita LD¹

^aoUniversity College London, Institute of Structural and Molecular Biology and Birkbeck College, School of Crystallography, London, UK

^apUniversity of Cambridge, Department of Chemistry, Cambridge, UK

^aqKing’s College London, Institute of Psychiatry, London, UK

Email address for correspondence: [email protected]

Keywords: TDP-43, structure, NMR

High-resolution structural information describing TDP-43 is scarce despite the clinical relevance and the powerful prospect of such data significantly aiding the understanding of this protein. Composed of an NLS-containing N-terminus and two RNA-binding domains (RRM1, RRM2) followed by a glycine-rich C-terminus (CTD_274–414), TDP-43 forms toxic aggregates as part of its disease-related pathology that lead to ALS and FTLD.

The CTD_274–414 harbours almost all of the ALS-associated mutations and is predicted to be an intrinsically disordered protein (IDP) domain. Disorder however does not preclude the existence of an overall architecture as has been shown to be particularly relevant in other disease relevant IDPs such as α-synuclein and tau. We have developed an expression and purification strategy using E. coli to produce isotopically-labelled and monomeric material and used NMR to provide a detailed structural and dynamic description of aggregation-prone CTD_274–414 both in isolation and as it exists within living cells. These data are discussed in the context of our biophysical observations (using EM and ThT-binding) of the amyloid-like aggregates formed by this aggregation-initiating region of TDP-43.

P217 PROFILIN 1 ASSOCIATES WITH STRESS GRANULES AND ALS-LINKED MUTATIONS ALTER STRESS GRANULE DYNAMICS

Figley M¹

Bieri G¹

Kolaitis R-M²

Taylor JP²

Gitler A¹

^arStanford University, Stanford, California, USA

^asSt. Jude Children’s Research Hospital, Memphis, Tennessee, USA

Email address for correspondence: [email protected]

Keywords: profilin, stress granules, ataxin-2(ATXN2)

Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial amyotrophic lateral sclerosis (ALS) (1). Profilin 1 is a well-studied actin-binding protein but it is unknown how PFN1 mutations cause ALS. The budding yeast, Saccharomyces cerevisiae, has one profilin ortholog.

We expressed the ALS-linked profilin 1 mutant protein in yeast, demonstrating a loss of protein stability, assayed by Western blot, and failure to restore temperature-sensitive growth to profilin mutant cells when compared to yeast expressing wildtype human profilin 1. Overexpression of wildtype profilin 1 or the ALS-linked mutants in a wildtype yeast background was tolerated, indicating a lack of gain-of-function toxicity in this model. This model provides for simple and rapid functional screening of novel ALS-linked profilin 1 variants.

To gain insight into potential novel roles for profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with yeast cells lacking profilin (pfy1Δ). Using Synthetic Genetic Array analysis, we queried ∼4800 nonessential yeast genes, identifying 127 genes which demonstrate a synthetic sick or lethal interaction with pfy1Δ yeast. Unexpectedly, deletion of several stress granule and processing body genes, including pbp1, were found to be synthetic lethal with deletion of pfy1.

Mutations in ATXN2, the human ortholog of PBP1, are a known ALS genetic risk factor, and ataxin 2 is a stress granule component in mammalian cells (2, 3). Given this genetic interaction and recent evidence linking stress granule dynamics to ALS pathogenesis (4), we hypothesized that profilin 1 might also associate with stress granules. Here we report that profilin 1 and related protein profilin 2 are novel stress granule-associated proteins as assessed by immunocytochemistry in mouse primary cortical neurons (cultured from E18 embryos) and in human cell lines in vitro (HeLa and U2OS) in response to various stressors (heat shock, oxidative stress induced by arsenite, and endoplasmic reticulum stress induced by DTT).

We also report that ALS-linked mutations in profilin 1 alter its stress granule dynamics. When overexpressed by transient transfection in U2OS cells, several of the mutant proteins are defective at localizing to arsenite-induced stress granules (C71G, M114T, G118V mutants, p = < 0.001) or being cleared from heat shock-induced stress granules after stress removal (E117G, p = < 0.01) when compared to wildtype profilin 1 (n = 100 transfected cells/condition and 3 experiments). This study provides further evidence for the potential role of stress granules in ALS pathogenesis.

Acknowledgements: This work was supported by NIH Director’s New Innovator Award 1DP2OD004417, NIH Grant NS065317, Muscular Dystrophy Association Grant MDA294366, the ALS Association, Target ALS, the Packard Center for ALS Research, the Stanford Genome Training Program (NIH/NHGRI T32 HG000044), and the Stanford Graduate Fellowship. We acknowledge Erfei Bi (University of Pennsylvania) for providing some yeast profilin mutant strains and for helpful suggestions.

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P218 HSPB8 IS RECRUITED INTO STRESS GRANULES UPON STRESS AND PROTECTS AGAINST TDP-43 MEDIATED TOXICITY IN VIVO IN DROSOPHILA MELANOGASTER

Seguin SJ¹

Morelli FF¹

Diacci C¹

Ganassi M¹

Poletti A²

Carra S¹

^atUniversity of Modena and Reggio Emilia, Modena, Italy

^auUniversity of Milan, Milan, Italy

Email address for correspondence: [email protected]

Keywords: protein quality control, stress granules, HSPB8

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease comprising clinically indistinguishable sporadic (s) and familial (f) forms, associated with a number of genes (SOD1, TDP-43, FUS, UBQLN2, VCP, FIG4, CHMP2B, SQSTM1, C9ORF72). Most ALS proteins are involved in protein degradation, ubiquitin proteasome pathway (UPP) and autophagolysosomal pathway (APLP), RNA processing or stress granule (SG) response. In ALS, motoneurons accumulate protein aggregates that contain RNA- binding proteins markers of SGs. Both aggregated proteins and persistent SGs are cleared by autophagy (1). Thus, proteostasis (mediated by the protein quality control, PQC) and ribostasis (involving SGs) may be interconnected and their imbalance may participate in ALS. Thus, upregulation of key players of the PQC, including molecular chaperones (heat shock proteins), which can assist the autophagy-mediated degradation of specific clients, thereby restoring proteostasis (and indirectly, ribostasis), may be beneficial in ALS.

Objectives: We previously published that overexpression of the small heat shock protein HSPB8 protects against TPD-43 mediated toxicity in immortalized motoneurons. In fact, HSPB8 facilitates the autophagy-mediated degradation of truncated TDP-43, thereby decreasing its cytosolic aggregation (2). Here we further investigated HSPB8 mode of action, focusing on its potential role both at SGs and protein aggregates. We also investigated the protective role of HSPB8 against TDP-43 mediated toxicity in vivo in Drosophila melanogaster.

Methods: For the in vitro work, HeLa cells were treated with SG inducing agents, including arsenite, MG132, heat shock. Cells were fixed and HSPB8 subcellular localization, as well as SG formation was investigated by immunofluorescence using specific antibodies. HSPB8 colocalization with mutated FUS containing SGs was studied in HeLa cells transfected with HA-tagged R518K FUS. For the in vivo work, the following transgenic flies were used: GMR-mut-NLS-TDP-43/CyO; UAS-HSP67Bc/TM3; UAS-HSPB8/CyO and UAS-HSPB8/TM3. Flies were grown and crossed at 25°C.

Results: We found that HSPB8 is recruited into SGs upon various stress. HSPB8 also colocalizes with mutated FUS at SGs, suggesting that it may act both at the level of protein aggregates (previous results) and SGs. Next, we tested HSPB8 protective role in vivo, using a Drosophila model expressing a NLS-TDP-43 mutant that accumulates in the cytosol, thereby causing eye degeneration (kindly provided by Prof. JP Taylor). We found that overexpression in vivo of both human HSPB8 and its Drosophila functional ortholog HSP67Bc protects against TDP-43 mediated toxicity. This is associated with a decrease in NLS-TDP-43 levels, supporting HSP67Bc/HSPB8 role in facilitating client degradation.

Discussion and conclusion: Our results support that HSPB8 may act both at the level of aggregate-prone proteins as well as SGs, facilitating the degradation of mutant ALS proteins (eg, TDP-43) and decreasing their cytosolic accumulation, thereby exerting protective functions.

Acknowledgements: AriSLA, MIUR (Programma Giovani Ricercatori Rita Levi Montalcini).

P219 TDP-43 REGULATES RNA GRANULE DYNAMICS AND INTERACTION: A NOVEL ELEMENT OF ALS PATHOGENESIS

Aulas A¹

Khalfallah Y¹

Mohamed N-V¹

Gkogkas C²

Caron G¹

Destroismaisons L¹

Sonenberg N²

Leclerc N¹

Parker JA¹

Vande Velde C¹

^avCentre de recherche CHUM, Universite de Montreal, Montreal, Canada

^awMcGill University, Montreal, Canada

Email address for correspondence: [email protected]

Keywords: TDP-43, RNA granules, translation

Background: Stress granules (SG) are an integral part of the cellular stress response and are a recent convergence point in studies related to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). RNA binding proteins, such as TDP-43, are now well linked to ALS, in both familial and sporadic contexts. We have previously reported TDP-43 to be an important regulator of the SG response. We hypothesize that motor neuron loss/dysfunction in ALS is due to a loss of normal TDP-43 function associated with SG dynamics.

Objective: To determine the role of TDP-43 in RNA granule dynamics.

Methods: Evaluate endogenous TDP-43 function via siRNA-mediated knockdown on RNA granules induced by oxidative stress as assessed by high resolution confocal microscopy and biochemical methods.

Results: TDP-43 depletion impairs secondary aggregation of SG such that SG size is significantly reduced. We have demonstrated this defect is via TDP-43 dependent expression of G3BP1. Here, we report that despite this defect, the recruitment of mRNA and several well-defined SG proteins is normal in TDP-43 depleted conditions. Moreover, the transient translational repression that characteristically accompanies SG formation also proceeds normally. However, TDP-43/G3BP1 depletion yields reduced basal translation despite elevated levels of mRNA and increased numbers of Processing Bodies (PB). The interaction of SG and PB during stress (docking) is thought to facilitate the bilateral exchange of mRNA and proteins. We find that docking is reduced by TDP-43 depletion and this correlates with poor mRNA protection. Both docking and mRNA protection can be rescued by exogenous expression of the TDP-43 target G3BP1.

Discussion and conclusion: We report a novel consequence of TDP-43 (via regulation of G3BP1) in the alteration of SG dynamics and define SG size as a determinant of SG-PB docking and mRNA preservation. This offers a new pathway for ALS pathogenesis.

Acknowledgements: ALS Society of Canada, Muscular Dystrophy Association, Natural Sciences and Engineering Research Council.

P220 EVIDENCE THAT β-METHYLAMINO- L-ALANINE (BMAA) CAN GENERATE ‘PRIONOID’ PROTEINS THAT CAUSE CHRONIC PROTEOTOXIC STRESS IN VITRO

Rodgers K¹

Dunlop R¹

Main B¹

Guillemin G²

^axUniversity of Technology, Sydney, Australia

^ayMacquarie University, Sydney, Australia

Email address for correspondence: [email protected]

Keywords: protein misfolding, environmental toxin, BMAA

Background: Gene-environment interactions are implicated in sporadic MND but the identities of the environmental triggers remain elusive. The high incidence of the ALS-Parkinsonism-Dementia complex reported on Guam has been linked to exposure to the cyanobacteria-derived amino acid β-methylamino-L-alanine (BMAA). Our recent observation that BMAA can be mistakenly incorporated into proteins in place of L-serine in vitro causing protein misfolding in cells (1) provides a mechanism whereby exposure to BMAA could increase the burden of protein aggregates in neurons over the lifetime of an individual; a process that might be further increased in individuals carrying mutations in aggregate-prone proteins.

Objectives: We test the hypothesis that incorporation of BMAA into protein can generate misfolded ‘prionoid’ proteins in cells capable of initiating further protein aggregation, establishing a chronic proteotoxic stress in cells, a decline in cell function and apoptosis.

Methods: Cells examined were MRC-5 (fibroblasts), SH-SY5Y (neuroblastoma cells) and human primary neurons (foetal brain). Proteomic analysis was by 2D-gel electrophoresis and ESI/MS/MS and/or MALDI/TOF. Protein quantification was by western blotting, mRNA by RT-qPCR and assessment of apoptosis and necrosis by caspase 3 and MTT/LDH activity, respectively.

Results: We compared the proteome of BMAA-treated cells to that of control cells. 10 proteins were identified that were significantly increased (p < 0.001) after BMAA exposure (300μM) including proteins involved in the ER stress response (Grp78 and calnexin) and the heat shock response (Hsc70 and HSP90). In support of these data, we demonstrated that the transcription of heat shock factor 1, a primary regulator of the heat shock response, was increased with BMAA but remained at basal levels on co-treatment with L-serine. Markers of ER stress were rapidly increased in response to BMAA by RT-qPCR (CHOP, ATF4). Changes were also evident in proteolytic activity after prolonged exposure to BMAA (500μM, 72 hours).

Discussion and conclusion: These studies show that exposure of a range of cells to BMAA produces changes consistent with the presence of misfolded proteins in the ER and activation of a global heat shock response in the cell. Since most neurodegenerative diseases are associated with protein misfolding, the presence of a non-protein amino acid, such as BMAA, in the polypeptide chain could increase the rate of misfolding and aggregation of vulnerable proteins, triggering ALS, Parkinsonism, and dementia in susceptible individuals, as was evident on Guam. The ability of the cell to handle misfolded proteins could decrease as the nervous system ages and the function of the defence systems and proteolytic machinery decline. This would provide a link between environmental factors, genetic susceptibility and ageing (gene-time-environment model).

Reference:

• Dunlop RA et al. PloS one 2013; 8(9): e75376.
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P221 INHIBITION OF DYNEIN ATPASE ACTIVITY REDUCES AGGREGATION OF MISFOLDED PROTEIN RESPONSIBLE FOR SBMA AND ALS

Cristofani R¹

Giorgetti E^1,2

Crippa V¹

Boncoraglio A^1,3

Cicardi ME¹

Rusmini P¹

Carra S⁴

Poletti A¹

^azDipartimento di Scienze Farmacologiche e Biomolecolari (DISFEB) e Centro di Eccellenza sulle Malattie Neurodegenerative (CEND), Università degli Studi di Milano, Milani, MI, Italy

^baDepartment of Pathology, University of Michigan, Ann Arbor, Michigan, USA

^bbDepartment of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

^bcDipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, Università degli Studi di Modena e Reggio Emilia, Modena, Italy

Email address for correspondence: [email protected]

Keywords: retrograde transport, autophagy, proteasome

Background: Spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are characterized by the presence of aggregates of aberrant proteins (androgen receptors (AR), superoxide dismutase 1 (SOD1), TAR DNA binding protein 43 (TDP-43), etc.) with abnormal conformations (misfolding). These accumulate into aggregates because the protein quality control system is not sufficiently active for their correct removal. Dynein motor complex seems to play a crucial role to maintain an efficient clearance of misfolded proteins by autophagy. Dynein mediated the transport of misfolded proteins/aggregates near microtubule organization center (MTOC). In addition dynein allows autophagosome nucleation by binding Vps34-Beclin1-Ambra1 complex, and assists the fusion of autophagosome to lysosome.

Objectives: In this study we aimed to identify the contribution of dynein motor complex in the removal of misfolded proteins which accumulate in motoneuron diseases.

Methods: We used NSC34 cells transfected with mutant SBMA-AR, G93A-SOD1 and ΔC-TDP-43. Dynein inhibition was obtained using the selective dynein ATPase inhibitor EHNA at 0,1mM for 48h. Protein accumulation was quantified by retained protein on cellulose acetate membrane in filter retardation assay (FRA). Autophagy activity was analyzed using the LC3 marker by western blotting. Misfolded proteins rerouting to proteasome was analyzed measuring the BAG1/BAG3 ratio in RT-qPCR.

Results: Inhibition of dynein, drastically reduce the LC3II/LC3I ratio when autophagy is induced by trehalose. Immunofluorescence on NSC34 transfected with SBMA-AR showed that dynein is sequestered into mutant SBMA-AR aggregates. Unexpectedly, EHNA reduces aggregates of mutant proteins in FTA also in presence of autophagy inhibitor (3-MA), but not in presence of proteasome inhibitor (MG132). In order to confirm these data we produced an inducible stable transfected GFP-SBMA-AR cell line. Also in this case EHNA treatment reduces aggregate retained in FRA. Moreover, EHNA treatment increases the levels of mutated AR in PBS and Triton-X100 fraction in progressive extraction. When we analyzed the mRNA levels of the co-chaperones BAG1 and BAG3, which route misfolding proteins to proteasome degradation or chaperone-mediated-autophagy (BAG1) and autophagy (BAG3), we found that BAG1/BAG3 ratio is increased after EHNA treatment.

Conclusion: These data suggest that dynein impairment, which results in autophagy blockage, also reduces aggregation of misfolding proteins involved in MNDs, by increasing their solubility and possibly through induction of alternative degradative pathways.

Acknowledgements: Telethon; Fondazione AriSLA; AFM, France; Regione Lombardia; Università degli Studi di Milano; Ministero della Salute.

P222 COORDINATED ACTIVATION OF AUTOPHAGY AND CATHEPSIN-MEDIATED PROTEOLYSIS INHIBITION IS ESSENTIAL FOR NEUROPROTECTION BY CYSTATIN C AGAINST MUTANT SOD1-MEDIATED TOXICITY

Watanabe S¹

Wakasugi K²

Yamanaka K¹

^bdDepartment of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, Japan

^beDepartment of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan

Email address for correspondence: [email protected]

Keywords: cystatin c (CysC), cathepsin b, AMP-activated protein kinase (AMPK)

Background: Recent studies have revealed that Cystatin C (CysC), an endogenous cysteine protease inhibitor and a major component of Bunina bodies in sporadic ALS, plays neuroprotective roles in various neurodegenerative diseases. We previously presented CysC reduces mutant SOD1- mediated toxicity through induction of autophagy, however, the precise neuroprotective mechanism of CysC was yet unidentified.

Objectives: The aim of this study is to reveal the mechanisms of neuroprotective activity of CysC.

Methods: We treated the neuro2a cells expressing wild-type or mutant SOD1 with several pharmacological agents or recombinant proteins as listed below: AICA-ribose (AICAR), an AMP-activated kinase (AMPK) agonist and an autophagy inducer; CA-074 methyl ester, a membrane-permeable cathepsin B (CatB) specific inhibitor; W106G CysC, CysC mutant lacking the CatB inhibitory activity. The cell viability was measured by MTS assay.

Results: Administration of CysC into neuro2a cells induced phosphorylation of AMPK and inhibited CatB aberrantly activated by mutant SOD1. Unexpectedly, AICAR treatment or W106G CysC did not rescue the cells regardless of autophagy induction. CA-074 also failed to protect the cells. Therefore, both autophagy induction and CatB inhibition were essential for the neuroprotection by CysC. In addition to this, CysC was leaked from lysosomes and formed intracellular aggregates dependent on oxidative stress, implying a relationship between the neuroprotective activity of CysC and Bunina body formation.

Discussion and conclusion: Our findings indicate that the neuroprotective property of CysC was dependent on the coordination of two distinct pathways: autophagy induction through AMPK activation and inhibition of CatB.

P223 THE INTERPLAY BETWEEN ENDOPLASMIC RETICULUM STRESS AND NF-KB IN ALS

Prell T

Jingyu L

Tadic V

Lautenschläger J

Gunkel A

Hammer N

Witte O

Grosskreutz J

Hans Berger Department of Neurology, Jena, Germany, Germany

Email address for correspondence: [email protected]

Keywords: ER stress, neuroinflammation, SOD1^G93A

Background: Recent studies have indicated that endoplasmic reticulum (ER) stress is involved in the pathogenesis of ALS (1, 3). ER stress occurs when misfolded proteins accumulate in the ER (2). New observations-particularly those stemming from cancer cell lines-suggest that ER stress and the subsequent engagement of the unfolded protein response (UPR) can activate the NF-κB pathway.

Objectives: We attempted to determine whether ER stress can activate the NF-κB pathway in murine motor neurons and vice versa.

Methods: We used an established motor neuron culture system derived from embryonic mouse ventral spinal cords and the mouse motor neuronal cell line NSC-34, both of which overexpress the G93A mutant of human SOD1 (SOD1^G93A). Cells were treated with an ER stressor (tunicamycin) and an inflammatory stimulus (lipopolysaccharide, LPS), and the subsequent activation of the UPR and the NF-κB pathway was measured by immunhistochemistry and western blotting (NF-κB, X-box binding protein 1, basic leucine-zipper transcription factor 6, and phosphorylated eukaryotic initiation factor-2α).

Results: We observed a constitutive expression of NF-κB in motor neurons and motor-neuron-like cells and the expression of NF-κB was enhanced in the presence of SOD1^G93A. The stimulation of NSC34 cells with LPS did not activate the UPR. Moreover, the stimulation of the glia-motor neuron culture with LPS did not activate the UPR. The chemical induction of the UPR by tunicamycin was accompanied by the activation of NF-κB in motor neurons and NSC34 cells.

Conclusion: Our data link two important pathogenic mechanisms of ALS, ER stress and NF-κB signalling, in motor neurons. The increased NF-κB activity that was observed here in the affected SOD1^G93A neurons may serve a neuroprotective function. Similar to the UPR, it can be an early protective response to ongoing disturbances in the ER-mitochondria calcium cycle in ALS.

Acknowledgment: This research is supported by a BMBF (the Bundesministerium fürBildung und Forschung) grant PYRAMID in the framework of the ERANET E-RARE program (http://www.e-rare.eu) and was undertaken in cooperation with the BMBF funded MND-NET.

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P224 FIBROBLASTS FROM SALS PATIENTS DO NOT SHOW AGE RELATED CHANGES IN BIOENERGETIC PROPERTIES AND MITOCHONDRIAL MORPHOLOGY

Allen S

Duffy L

Shaw PJ

Grierson A

University of Sheffield, Sheffield, UK

Email address for correspondence: [email protected]

Keywords: mitochondria, metabolism, aging

Background: Metabolic dysfunction plays a key role in amyotrophic lateral sclerosis (ALS) disease progression and has been observed in several cellular and animal models of the disease (1, 2). Evidence suggests that mitochondria play an important part in aging, which is a crucial risk factor in neurodegenerative disorders (3). Peripheral tissues such as fibroblasts have been observed to recapitulate pathophysiological abnormalities observed in the CNS (4, 5).

Objectives: To ascertain whether fibroblasts isolated from sporadic ALS patients show the defects in energy metabolism reported in the CNS and to ascertain how these patients respond to aging at a metabolic levels compared to controls.

Methods: Using mitochondrial morphology analysis, ATP assays and a XF24 bioanalyser, which simultaneously measures the two major energy producing pathways of the cell, mitochondrial respiration and glycolysis in real-time. We assessed the effect of metabolic dysfunction in SALS patient fibroblasts compared to controls and how ALS affects the metabolic response to aging.

Results: SALS fibroblasts show age independent reduced mitochondrial coupled respiration compared to controls (p = 0.043). Unlike SALS cases, control fibroblasts show increased mitochondrial spare respiratory capacity (SRC) with age (p = 0.025), decreased glycolytic flux (p = 0.025) and an increase in the OCR/ECAR ratio (p = 0.001) indicating an increased reliance on oxidative phosphorylation to meet energy needs. Unlike controls SALS fibroblasts show increased proton leak with age (p = 0.053) indicating damaged mitochondria. The oxphos: glycolysis ATP ratio tended to decrease with age in SALS cases but not controls. Mitochondrial network complexity increased with age in controls (p = 0.03) but not in SALS cases. The older SALS cases (70 years and above) had significantly reduced SRC and oxphos: glycolysis ATP ratio and also displayed increased glycolysis compared to controls.

Discussion and conclusion: Mitochondrial morphological and bioenergetic analysis, indicate that with age, control fibroblasts can rely more on mitochondrial function to meet energy demands. SALS fibroblasts are unable to do this as uncoupling and mitochondrial fragmentation increases with age in sporadic ALS. Older SALS cases display the greatest mitochondrial bioenergetic abnormalities and shift to a more glycolytic energy state. This highlights that from a mitochondrial functional perspective, older ALS patients are unable to cope with the aging process as well as controls and from a glycolytic metabolic perspective, SALS cells have an altered metabolic aging profile.

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P225 PGC-1ALPHA EXPRESSION AND STIMULATION IN ALS RELEVANT TISSUES AND CELLS

Bayer H

Barteczko L

Heinrich M

Merdian I

Weydt P

Witting A

Experimental Neurology, Ulm, Germany

Email address for correspondence: [email protected]

Keywords: PGC-1alpha, lactate-shuttle, metabolism

Background: The transcriptional co-activator PGC-1alpha is an important regulator of mitochondrial activity and biogenesis in many metabolically active tissues, including brain. Clinical and experimental evidence suggests that impaired function or activity of PGC-1α contributes to the pathogenesis of neurodegenerative disease spectrum disorders, including ALS. Of note, genetic variants of a brain-specific promoter modify age of onset in human ALS. We found that the brain-specific PGC-1α isoforms are differentially expressed in neurons vs. glial cells (1), suggesting that neurons and glia cells are metabolically differently regulated. According to the Lactate-Shuttle hypothesis lactate is released by glia cells to nourish neurons, whereas glutamate is released by neurons to stimulate lactate production by glia cells.

Objectives: Here we first examine the expression of brain-specific PGC-1α isoforms during disease progression in different brain regions and spinal cord of the ALS mouse model, B6SJL-Tg (SOD1^G93A)1Gur/J. Second, we analyzed the activation of PGC-1α in primary murine cell cultures of neurons and glia cells, including oligodendrocytes, astrocytes and microglia.

Methods: Tissues from SOD1 mice were harvested at 60 days (preclinical), at 100 days (onset) and at 130 days (end-stage). Primary murine cells were stimulated with lactate or glutamate. The expression of the different PGC-1α isoforms and enzymes involved in glycolysis and Krebs cycle was quantified by Real-time PCR.

Results: We showed that PGC-1α is significantly reduced in ALS end-stage mice in different brain regions and the spinal cord. PGC-1α is also differentially expressed in the different cell types, eg, neurons and oligodendrocytes, express much higher levels of the novel brain-specific isoforms of PGC-1α. Treatment of primary neurons with lactate leads to an increase in PGC-1α whereas treatment of oligodendrocytes with glutamate results in a decrease of PGC-1α and pyruvate dehydrogenase.

Discussion and conclusion: The different expression levels might reflect the specific metabolic needs of the different cell types and their different role in the contribution to the lactate shuttle. A reduced PGC-1α expression in ALS mice might therefore reflect an insufficient lactate shuttle, which has been shown to be of major interest to ALS.

Acknowledgements: The work is funded by a pilot grant from the Thierry-Latran-Foundation (to PW) and by the Helmholtz Virtual institute “RNA dysmetabolism in Amytrophic Lateral Sclerosis and Frontotemporal Dementia” (to AW and PW).

Reference:

• Soyal SM. et al. Hum Mol Gen 2012; 21(15): 3461–73.
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P226 MIRO1 EXPRESSION RESCUES ALS MUTANT SOD1-INDUCED INHIBITION OF ANTEROGRADE TRANSPORT OF MITOCHONDRIA

Moller A

De Vos KJ

Department of Neuroscience, SITRAN, University of Sheffield, Sheffield, UK

Email address for correspondence: [email protected]

Keywords: mitochondria, axonal transport, SOD1

Background: Damage to axonal transport is an early event in ALS that may contribute to motor neuron degeneration. We have shown previously that familial ALS mutations in SOD1 and VAPB selectively reduce anterograde transport of mitochondria (1, 2).

Anterograde transport of mitochondria is mediated by the molecular motor kinesin-1 that attaches to mitochondria via the adaptor protein TRAK and the atypical Rho GTPase Miro1 (3, 4). Miro1 plays a central role in the regulation of mitochondrial axonal transport in response to Ca²⁺ and mitochondrial damage. Firstly binding of Ca²⁺ to the Miro1 EF-hand motifs halts anterograde transport of mitochondria by regulating the interaction of kinesin-1 with Miro1 such that either kinesin-1 binding to microtubules or to Miro1 itself is disrupted (5, 6). Secondly, PINK1 phosphorylates Miro1 in response to mitochondrial damage. This activates proteasomal degradation of Miro1 in a Parkin-dependent manner and as a result detaches kinesin-1 from mitochondria and arrests anterograde mitochondrial movement (7).

Objectives: Here we investigated the mechanisms underlying defective anterograde transport of mitochondria in mutant SOD1-related ALS.

Methods: Rat cortical neurons were transfected with EGFP, EGFP-tagged wild-type SOD1 or SOD1^G93A and either empty vector, wild type Miro1 or the Ca²⁺ insensitive Miro1^E208K/E328K mutant. Axonal transport of mitochondria labeled with DsRed-Mito was quantified from time-lapse recordings. The effect of ALS mutant SOD1 on Miro1 protein levels was investigated in HEK293 cells co-transfected with myc-Miro1 and either wild type or SOD1^G93A.

Results: SOD1^G93A significantly decreased the total number of motile mitochondria by specific reduction of their anterograde transport; retrograde transport of mitochondria was not affected. This specific anterograde transport defect unbalanced axonal transport of mitochondria such that there was a significant shift toward net retrograde transport. Expression of either wild type Miro1 or Miro1^E208K/E328K rescued this defect and restored the transport balance, suggesting SOD1^G93A may affect Miro1 levels. In agreement, SOD1^G93A significantly decreased Miro1 levels compared to wild type SOD1 in HEK293 cells.

Discussion and conclusion: These data indicate that Miro1 degradation may play a role in ALS mutant SOD1-induced defects in axonal mitochondrial transport.

Acknowledgements: This work is supported by an MRC New Investigator Grant to KJDV (MR/K005146/1).

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P227 PERTURBATION OF CITRIC ACID CYCLE AND EXCITATORY NEUROTRANSMISSION IN AN IN VITRO MODEL OF AMYOTROPHIC LATERAL SCLEROSIS EXPOSED TO OXIDATIVE STRESS

Veyrat-Durebex C^1,2

Corcia P^1,3

Piver E⁴

Emond P^1,5

Dangoumau A¹

Vourc’h P^1,2

Devos D⁶

Andres CR^1,2

Blasco H^1,2

^bfUMR INSERM U930, Tours, France

^bgLaboratoire de Biochimie et Biologie moléculaire, CHRU de Tours, Tours, France

^bhService de Neurologie, CHRU de Tours, Tours, France

^biINSERM U966, Tours, France

^bjPPF-ASB, Université François Rabelais, Tours, France

^bkEA1046, Faculté de médecine de Lille, Lille, France

Email address for correspondence: [email protected]

Keywords: co-culture, oxidative stress, metabolism

Background: Although genetic and environmental factors have been associated with amyotrophic lateral sclerosis (ALS), pathophysiology of ALS remains unknown in the majority of cases. Preliminary results from our laboratory on CSF components of ALS patients indicate a potential modification of energetic substrates consumed by the central nervous system (1).

Objectives: In order to improve the knowledge of the pathophysiology of this disease and associated metabolic alterations, we developed a metabolomic approach on a cellular model reproducing genetic (mutation in SOD1 gene) and environmental (oxidative stress) conditions of ALS.

Methods: A co-culture model of mouse astrocytes (C8-D1A cell line) and motor neurons (NSC-34 cell line) expressing or not human SOD1 protein wild-type or G93C mutant were exposed to oxidative stress (menadione 10 μM). The two cell types were collected at 3 times post-stress (for 48h). Analyses of intracellular media were performed by gas chromatography coupled to mass spectrometry after extraction (methanol/water/chloroform) and derivation (oxymation and silylation) of intracellular metabolites. Data were analyzed using multivariate analysis (OPLS-DA) using Simca P+ software (Umetrics, Umea, Sweden).

Results: Twenty six metabolites observed in GC-MS were used in statistical models. Preliminary analysis of the results showed a difference of metabolism between the two cell types leading to a separate statistical analysis of the co-cultured astrocytes and motor neurons. We also noticed an influence of genetic and time on metabolism. We observed a different metabolism in the 48h of stress between stressed and unstressed cells, with different discriminant metabolites depending on cell type and genetic conditions. Thus we showed that stressed cells no over-expressing SOD1 have significant higher concentrations of glutamate and aspartate (astrocytes) and threonine, serine and aspartate (motor neurons). Meanwhile, stressed cells over-expressing mutant SOD1 have significant higher concentrations of glycine (astrocytes) and fumarate and valine (motor neurons).

Discussion and conclusion: Preliminary results from this study revealed the feasibility of a metabolomic approach on a cellular model of ALS. We brought out the potential disruption of citric acid cycle and excitatory transmission under oxidative stress in ALS, which joined the assumptions already raised in this disease. These data could open perspective of functional, genomic and transcriptomic approaches focused on metabolites identified in this study and help to better understand mechanisms of the disease.

Acknowledgements: We thank the ARSla for its financial support.

Reference:

• Blasco H. et al. PLoS One 2010; 5:e13223.
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P228 SYNAPTIC MECHANISMS UNDERLYING THE EXCESSIVE AND PRECOCIOUS GLUTAMATE RELEASE IN THE SPINAL CORD OF SOD1G93A EXPERIMENTAL MICE

Bonifacino T¹

Milanese M¹

Musazzi L²

Treccani G²

Giribaldi F¹

Usai C⁴

Onofri F³

Popoli M²

Bonanno G^1,4

^blDepartment of Pharmacy, Pharmacology and Toxicology Unit, University of Genoa, Genoa, Italy

^bmDepartment of Pharmacological and Biomolecular Sciences, Laboratory of Neuropsychopharmacology and Functional Neurogenomics, University of Milan, Milan, Italy

^bnDepartment of Experimental Medicine, Section of Physiology, University of Genoa, Genoa, Italy

^boInstitute of Biophysics, National Research Council, Genoa, Italy

^bpCenter of Excellence for Biomedical Research, Genoa, Italy

Email address for correspondence: [email protected]

Keywords: glutamate release, excitotoxicity, synaptic proteins, SOD1^G93A

Background: Glutamate (Glu)-mediated excitotoxicity plays a major role in the degeneration of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS) and reduced astrocytic uptake was suggested as a cause for the increased synaptic availability of Glu (1). On the basis of our studies, we have proposed that abnormal release may represent another source for excessive extracellular Glu levels (2, 3, 4). Acting at the altered Glu release mechanisms may represent a possible strategy for new therapeutic approaches to ALS (5).

Objectves: To investigate the molecular mechanisms supporting the excessive Glu exocytosis in experimental ALS.

Methods: 4 weeks or 17 weeks old mice were used. Nerve terminals (synaptosomes) were obtained from the spinal cord of control (SOD1) and pathologic (SOD1^G93A) mice. As a functional readout we measured glutamate release, by exploiting the superfusion technique. We also measured the intra-synaptic calcium concentration and the expression/activation state of synaptic proteins by confocal microscopy and western blot experiments.

Results: The spontaneous and the stimulus-evoked exocytotic Glu release are increased in SOD1^G93A mice, respect to controls, either at 4 weeks or 17 weeks of life. The expression of several synaptic proteins involved in neurotransmitter release does not show differences except for synaptotagmin which results over-expressed at the active zone in both 4 weeks and 17 weeks old ALS mice. Accumulation of high molecular weight SNARE complexes and disregulation of cytoskeletal proteins are also present in isolated nerve terminals from the spinal cord of SOD1^G93A mice. Increased pre-synaptic Ca²⁺ levels, over-activation of calcium/calmodulin-dependent kinase-II and ERK/MAP kinases, correlates with a hyper-phosphorylation of synapsin-I at both early and late clinical stages of the disease. In line with these findings, release experiments highlight the involvement of the readily releasable pool of vesicles for the excessive Glu exocytosis.

Discussion and conclusion: Our results indicate a dysregulation of glutamate exocytosis in the spinal cord of symptomatic SOD1^G93A mice. This event is accompanied by marked changes in specific pre-synaptic molecular mechanisms that lead to a significant augmentation of the readily releasable pool of vesicles and determine a higher probability of these vesicles to fuse. The same synaptic alterations are present also in 4 weeks old ALS mice and represent a key feature in the early phase of the disease, thus playing a pivotal role in the aetiopathogenesis of the disease.

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P229 ASTROCYTE-DERIVED TGF-β1 ACCELERATES DISEASE PROGRESSION IN ALS MICE BY INTERFERING WITH NEUROPROTECTIVE FUNCTIONS OF MICROGLIA AND T CELLS

Endo F¹

Komine O¹

Wyss-Coray T²

Yamanaka K¹

^bqNagoya University, Research Institute of Environmental Medicine, Nagoya, Japan

^brStanford University, Palo Alto, CA, USA

Email address for correspondence: [email protected]

Keywords: astrocyte, TGF-β1, SOD1

Background: Elevated level of transforming growth factor-β1 (TGF-β1), an anti-inflammatory cytokine, has been observed in the peripheral blood and the cerebrospinal fluid of ALS patients. However, the exact role of TGF-β1 in ALS pathomechanism has not been elucidated.

Objective: The aim of this study is to uncover the role of TGF-β1, anti-inflammatory cytokine in the pathomechanism of ALS.

Methods: Expression of TGF-β1 and its downstream signaling were examined in multiple lines of mutant SOD1 mice. SOD1^G93A mice were crossbred with GFAP-TGF-β1 mice, which moderately overexpressed TGF-β1 in astrocytes. The disease phenotype, the phenotypes of glia/immune cells, and the expression profiles of cytokines and neurotrophic factors were analyzed in SOD1^G93A/GFAP-TGF-β1 and SOD1^G93A mice. Finally, the role of astrocytic mutant SOD1 in the level of TGF-β1 was analyzed in LoxSOD1^G37R mice.

Results: We identify astrocytic TGF-β1 as determinant of disease progression through regulating the neuroprotective glia / immune response in ALS mice. We show that TGF-β1 level is elevated in astrocytes of symptomatic mutant SOD1 mice, and that astrocyte-specific overproduction of TGF-β1 in SOD1^G93A mice accelerates disease progression in a non-cell autonomous manner with reduced IGF-I production in deactivated microglia and fewer infiltrated T cells with a dysregulated IFN-γ / IL-4 balance. In addition, the level of TGF-β1 was decreased when mutant SOD1 is deleted specifically from astrocytes in LoxSOD1^G37R mice that are known to extend their life span. Moreover, expression levels of endogenous TGF-β1 in SOD1^G93A mice negatively correlate with survival time. On the other hand, we also show that canonical TGF-β signaling within motor neurons of SOD1^G93A mice is dysregulated regardless of exogenous TGF-β1.

Discussion and conclusion: These findings indicate that astrocyte-derived TGF-β1 accelerates disease through negatively regulating the neuroprotective inflammatory response by microglia and T cells. Furthermore, cell-type-specific dysregulation of TGF-β signaling is critical to the pathomechanism of ALS.