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MND Australia Research Meeting 2015 Working towards a world without MND 22 – 23 November 2015 www.mndresearch.asn.au Sponsors: PROGRAM SUNDAY 22 NOVEMBER 6.00– 8.00 pm Poster Reception and Welcome 6.15 pm Presentation MONDAY 23 NOVEMBER Professor Naomi Wray, Launch of MND Australia Ice Bucket Challenge Grant: Sporadic ALS Australia - Systems Genomics Consortium ORAL PRESENTATIONS 9.00 am Registration 9.30 am Welcome David Ali, President, MND Australia Opening address Introduction Professor Bruce Robinson, Dean, Sydney Medical School, The University of Sydney Professor Matthew Kiernan, Chair, MNDRIA Research Committee Session 1 Chair Professor Naomi Wray 10.00 am A/Prof Ian Blair, NSW Next-generation sequencing in Australian MND 10.20 am Prof Lars Ittner, NSW Novel MND mouse models 10.35 am A/Prof Greg Neely, NSW Genomic approach to find new MND disease genes and drug targets 10.50 am Dr Kelly Williams, NSW Investigating the molecular basis of ALS 11.05 am Prof Roger Chung, NSW Proteomic studies to identify the defects caused by mutations in a newly identified familial MND gene 11.20 am Morning Tea Session 2 Chair Associate Professor Tracey Dickson 11.40 am Dr Bradley Turner, VIC Androgen receptor abnormalities in MND 11.55 am A/Prof Julie Atkin, NSW 12.10 pm Dr Justin Yerbury, NSW 12.25 pm A/Prof Peter Noakes, QLD 12.40 pm Dr Jacqueline Leung, TAS Optimising the protective activity of protein disulphide isomerase in MND Monitoring accumulation of ubiquitin chains in ALS – Developing a potential imaging tool for monitoring preclinical disease progression Progress into the contributions made by motor neurons and muscle in the progression of MND: human neuromuscular and muscle analyses. Investigating the role of oligodendrocytes in ALS 12.55 pm Breaking news 1.10 pm Lunch Session 3 Chair Professor Glenda Halliday 2.15 pm Dr Rachel Tan, NSW 2.30 pm Dr Parvathi Menon, NSW 2.45 pm Dr James Howells, NSW Histopathological changes in the cerebellum of ALS cases with repeat expansions in the C9ORF72 and ATXN2 genes. Insights into ALS pathophysiology from patterns of disease progression Dysfunctional properties of single motor units in ALS 3.00 pm Dr Catherine Blizzard, TAS Synaptic dysfunction: an early mechanism of TDP-43 pathogenesis in ALS PROGRAM 3.15 pm 3.30 pm A/Prof Tracey Dickson, TAS Dr Shyuan Ngo, QLD 3.45pm Afternoon Tea Session 4 Chair Professor Matthew Kiernan 4.05 pm 4.35 pm A/Prof Trent Woodruff, QLD Prof Steve Vucic and Dr Fiona McKay, NSW Dr Robert Henderson, QLD 4.50 pm Dr Anne Hogden, NSW 5.05 pm Dr Sharpley Hsieh, NSW 5.20 pm Closing comments The innate immune system as a therapeutic target in motor neuron disease. Safety and biological efficacy of narrow-band UVB phototherapy in ALS Blood biomarkers in ALS: Translation into clinical practice of pNfH and search for additional biomarkers using proteomics Decision support tools for motor neurone disease multidisciplinary care My memories are important to me: autobiographical memory retrieval in MND Professor Matthew Kiernan 5.30 pm Close 4.20 pm Inhibitory regulation of motor neurons: A new target mechanism for ALS? Investigating the mechanisms underlying defective energy metabolism in MND POSTER PRESENTATIONS Name Institution Ms Monique Bax IHMRI, University of Wollongong Ms Jashelle Caga Brain & Mind Centre, University of Sydney Menzies IMR, University of Tasmania Menzies IMR, University of Tasmania MND Research Centre, Macquarie University Mr Jayden Clark Ms Rosie Clark Dr Nicholas Cole Ms Vandana Deora School of Biomedical Sciences, University of QLD Dr Emma Devenney Neuroscience Research Australia, University of NSW Dr Emily Don ASAM, Macquarie University Ms Kimberley Duncan Medicine & Health Sciences, Macquarie University Paediatrics, School of Women’s & Children’s Health, University of NSW Dr Michelle Farrar Title The impairment of the ubiquitin proteasome system in amyotrophic lateral sclerosis patient iPSC– derived neurons. Survival differences among ALS patients with different levels of apathy Targeting Microtubules to Improve Outcomes in ALS Intrinsic interneuronal vulnerabilities in the GAD67.SOD1 mouse cortex Fishing to Better Understand the Biology of Motor Neurone Disease/Frontotemporal Dementia Activation of the complement cascade and the NLRP3 inflammasome by hSOD1G93A protein Psychosis in the Frontotemporal Dementia – Motor Neurone Disease continuum – clinical features and neuroimaging correlates A Zebrafish Model of the MND Linked C9orf72 Hexanucleotide Repeat Expansion. Characterising the functional domains of an MND candidate protein Dysfunction of the motor cortex heralds onset of ataxia in Machado-Joseph Disease PROGRAM Ms Natalie Farrawell IHMRI, University of Wollongong Ms Jennifer Fifita Macquarie University Dr Nimeshan Geevasinga Western Clinical School, University of Sydney Western Clinical School, University of Sydney Menzies IMR, University of Tasmania Medicine & Health Sciences, Macquarie University Dr Nimeshan Geevasinga Ms Emily Handley Ms Alison Hogan Ms Janine James University of Melbourne Ms Tongcui Jiang Menzies IMR, University of Tasmania Dr Mark Laidlaw Applied Sciences, RMIT University Ms Isabella LambertSmith IHMRI, University of Wollongong Dr Linda Lau Florey Institute of Neuroscience & Mental Health, University of Melbourne School of Biomedical Sciences, University of QLD Dr John Lee Dr Jeffrey Liddell Mr Luke McAlary Ms Emily McCann Dr Diane Moujalled Dept of Pathology, University of Melbourne Biological Sciences, University of Wollongong Medicine & Health Sciences, Macquarie University Dept of Pathology, University of Melbourne Dr Jane Parkin Kullmann Brain & Mind Centre, University of Sydney Ms Nirma Perera Florey Institute of Neuroscience & Mental Health, University of Melbourne Bio21 Molecular Science and Biotechnology Institute, University of Melbourne Ms Mona Radwan Ubiquitin homeostasis and SOD1 aggregation in ALS Disease gene discovery in familial amyotrophic lateral sclerosis Revisiting early diagnosis in ALS Riluzole exerts short-term effects on cortical hyperexcitability in sporadic ALS Spine Loss is an Early Pathogenic Event in the TDP-43A315T Mouse Model of ALS Generation of novel zebrafish models of MND using traditional and emerging transgenic techniques What’s in a stress granule? Neurite outgrowth and spine formation in primary cortical neurons overexpressing human TDP-43A315T A Temporal Association between Accumulated Petrol (Gasoline) Lead Emissions and Motor Neuron Disease in Australia A collection of GFP fusion proteins reveals responses in the proteostasis network to TDP-43, FUS and SOD1 in a yeast model of ALS Establishing plutipotent stem cell-derived spinal motor neurons for screening CSF toxicity in ALS Opposing roles for complement C3aR and C5aR1 in the disease progression of hSOD1G93A mice Activation of Nrf2 is implicated in the disease-attenuating activity of CuII(atsm) Unfolding the Mystery: Common Misfolded Intermediates Captured with Mass Spectrometry Genetic analysis workflow in the Macquarie University MND Research Centre Phosphorylation of hnRNP K by cyclin-dependent kinase 2 controls cytosolic accumulation of TDP-43 ALS Quest: Initial Results from an Internationally Accessible Web-Based Questionnaire to Discover Risk Factors for Motor Neurone Disease Rilmenidine increases mTOR-independent autophagy enhancing disease progression in a mouse model of MND Determining the pathomechanism of C9ORF72 Repeat-Associated Non-ATG (RAN) translation products PROGRAM Dr Sarah Rea Sir Charles Gairdner Hospital, WA Dr Mary-Louise Rogers Centre for Neuroscience, Flinders University, SA Garvan IMR, University of NSW Dr Darren Saunders Dr Rebecca Sheean Dr Kazumoto Shibuya Dr Frederik Steyn Florey Institute of Neuroscience & Mental Health, University of Melbourne Brain & Mind Centre, University of Sydney University of Queensland Centre for Clinical Research Mr Jack Stoddart Medicine & Health Sciences, Macquarie University Mr Xin Lin Tan Royal Melbourne Hospital, University of Melbourne Brain & Mind Centre, University of Sydney Ms Hannah Timmins Dr Mehdi Van den Bos Dr Adam Walker Dr Robyn Wallace Department of Neurology, Westmead Hospital, NSW Macquarie University Mr Daniel Whiten Biomedical Sciences, Charles Sturt University, NSW IHMRI, University of Wollongong Ms Rafaa Zeineddine IHMRI, University of Wollongong Ms Katharine Zhang Medicine & Health Sciences, Macquarie University Spread the word! #MNDmeeting15 @MND_RIA SQSTM1 mutations associated with ALS, Frontotemporal dementia and Paget’s disease of bone lead to dysfunctional autophagy and altered cell signalling. Targeted gene delivery to neonatal p75NTR expressing motor neurons Defining the Neuronal Ubiquitome in ALS. Expansion of Tregs by interleukin-2/ antibody complexes slows disease progression in mutant SOD1 mice Motor neuronal hyperexcitability is a common in sporadic and familial ALS Metabolic dynamics in MND: Assessment of metabolic flux in patients reveal breakdown in energy supply and demand An In Vivo And Behavioural Study Of Als-Associated Protein, Tdp-43 In Transgenic Zebrafish Traumatic brain injury, Motor Neuron Disease, and TDP43 Identifying links between neurophysiological markers and patient reported outcomes in ALS. Cortical hyperexcitability in ALS: A heterogeneous process Clearance of pathological cytoplasmic TDP-43 allows functional recovery in new mouse models of sporadic ALS/FTLD Direct Conversion of Adult stem cells to induced neuronal and Muscle cells Clusterin ameliorates TDP-43 pathology SOD1 protein aggregates activate micropinocytosis in neurons to facilitate their entry Functional pipeline for determining pathogenicity of candidate gene mutations causing motor neuron disease ORAL PRESENTATIONS Associate Professor Ian Blair Next-generation sequencing in Australian MND Kelly Williams1, Jennifer Fifita1, Emily McCann1, Katharine Zhang1, Matthew Kiernan2, Roger Pamphlett2, Garth Nicholson3, Dominic Rowe1, Denis Bauer4, William Wilson4, Ian Blair1 1. Faculty of Medicine and Health Sciences, Macquarie University. 2 Brain and Mind Research Institute, University of Sydney. 3 ANZAC Research Institute, University of Sydney and Concord Hospital. 4 Translational Bioinformatics, CSIRO There have been dramatic advances in MND research over the past seven years that have been driven by gene discoveries. To date, the only proven causes of MND are gene mutations that lead to motor neuron death, typically in familial MND. There is strong evidence that sporadic MND is a multifactorial syndrome caused by combined effects and interaction of susceptibility alleles (genetic variations that confer risk to developing MND) and environmental exposure. These alleles may confer weak, modest, or strong risk for MND. The identity of most MND risk alleles, and their relative contribution to developing the disease, remain unknown. There is also strong evidence that unknown genetic factors contribute to the progression of MND, influencing the spread and speed of degeneration. Unknown genetic variation can also influence a patient’s response to treatment. Collectively, these genetic components are known as the genetic architecture of MND. With recent dramatic advances in DNA-sequencing technology (next-generation sequencing, NGS), we are working to unravel the genetic architecture of MND by 1). continuing to identify familial MND genes and mutations, and 2). sequencing the whole genomes of sporadic MND patients to identify MND risk alleles, as part of the largest multinational genetic study (called “Project MinE”) ever attempted in MND. Professor Lars Ittner Novel MND mouse models Yazi D Ke, Annika van Hummel, Claire Stevens and Lars M Ittner Dementia Research Unit, School of Medical Sciences, University of New South Wales The nuclear transactive response DNA-binding protein 43 (TDP-43) aberrantly localizes to the cytoplasm and forms cytoplasmic deposits in neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Disease-causative mutations in the TDP-43-encoding TARDBP gene in familial ALS and non- mutant human TDP-43 have been utilized to model FTD/ALS in cell culture and animals, including mice. Here, we report novel A315T mutant TDP-43 transgenic mice, iTDP-43A315T, with controlled neuronal over-expression. Constitutive expression of human TDP-43A315T resulted in pronounced early-onset and progressive neurodegeneration, which was associated with reduced motor performance and spatial memory, as well as disinhibition. Muscle atrophy resulted in reduced grip strength. Cortical degeneration presented with pronounced astrocyte activation. Using differential protein extraction from iTDP-43A315T brains, we found cytoplasmic localization, fragmentation, phosphorylation and ubiquitination and insolubility of TDP-43. Remarkably, suppression of human TDP-43A315T expression in mice with overt neurodegeneration for only one week was sufficient to significantly improve motor and behavioral deficits, and reduce astrogliosis. Our data suggest that functional deficits in iTDP-43A315T mice are at least in part a direct and transient effect of the presence of TDP-43A315T. Furthermore, it illustrates the compensatory capacity of compromised neurons once transgenic TDP-43 is removed, with implications for future treatments. A/Prof Greg Neely Genomic approach to find new MND disease genes and drug targets Thang Khuong, Willie Lin, and Greg Neely. Charles Perkins Centre, University of Sydney Recently large human genetics studies have been performed to identify MND susceptibility genes, however identifying strong genetic factors that predispose to MND have been difficult, suggesting complimentary approaches are required. Since defects in synaptic development and function have been linked to MND, we have combined human genetics or other genomic data with functional target validation in transgenic animals. To date, we have tested 302 candidate MND genes, and have successfully identified 99 genes that act presynaptically at the neuromuscular junction and are essential for proper motor neuron function. Reduced expression of these genes can cause reduced motor coordination and/or shortened lifespan in transgenic fruit flies, and resembles MND symptoms in human. We are continuing our gene discover efforts, and will begin to evaluate if any of these novel disease genes could be targeted to develop new treatments for MND. ORAL PRESENTATIONS Dr Kelly Williams Genome-wide and targeted analysis of DNA methylation in disease discordant MND/FTD cohorts Kelly L Williams1, Beben Benyamin2, Emily P. McCann1, Anjali Henders2, Sonia Shah2, Dominic B. Rowe1, Garth A. Nicholson1, Naomi Wray2, and Ian P. Blair1 1 Faculty of Medicine & Health Sciences, Macquarie University, Sydney 2 Queensland Brain Institute, University of Queensland, Brisbane Mutations in just two genes, C9ORF72 and SOD1, account for more than half of Australian hereditary MND cases. Substantial variation is seen in age of onset and progression of disease, including among patients with identical gene mutations. Comorbidity with frontotemporal dementia (FTD) is frequently observed, especially in patients with the C9ORF72 repeat expansion. The absence of a clear genotype-phenotype correlation provides strong evidence for the role of highly penetrant modifying factors, including epigenetic changes, in disease onset and progression. Discovery of modifiers will provide insight into the phenotypic discordance, highlight dysfunctional disease pathways, and offer potential targets to delay onset or progression of MND/FTD. We are examining both genome-wide and gene-specific DNA methylation in a large cohort of ALS and/or FTD families with C9ORF72 repeat expansions or SOD1 mutations. The discovery cohort comprises 315 individuals from 84 families, including patients with ALS and/or FTD, pre-symptomatic individuals (i.e. carry a disease-causing mutation but currently unaffected) and controls. gDNA samples have undergone genomewide methylation quantitation using the Illumina HumanMethylation450K BeadChip. An EpiTYPER assay determined quantitative methylation of the SOD1 and C9ORF72 CpG islands. Initial analysis shows a substantially higher level of C9ORF72-CpG island methylation in the single “pure FTD” patient. Genome-wide analysis has identified a highly significant differentially methylated CpG site (p=5x10-9) in C9ORF72 cases compared to controls. The CpG cite falls within the promotor region of a gene encoding an RNA-binding protein. Identical experiments in the replication cohort (n>150 samples from >20 families, including multiple “pure FTD” cases) are currently underway. Professor Roger Chung Proteomic studies to identify the defects in protein degradation pathways caused by mutations in a newly identified familial ALS gene A/Prof Ian Blair, A/Prof Julie Atkin, A/Prof Mark Molloy, Dr Albert Lee (Macquarie University), Dr Justin Yerbury (University of Wollongong) Our team has recently identified new mutations in a gene responsible for ALS in an Australian family, and through international collaborations has identified mutations in this gene in Canadian, US and European ALS patients. This ALS gene encodes an E3 ligase (hereafter referred to as FBX), a protein that is directly involved in protein degradation and recycling in motor neurons. Our studies indicate that ALS mutations in this gene lead to abnormal accumulation of proteins within the cell, leading to neurodegeneration. Using mass spectrometry screening, we have identified putative substrates of FBX, and revealed specific biological networks that are impaired in motor neurons expressing mutations in this new ALS gene. Because abnormal protein degradation and the inappropriate accumulation of proteins inside motor neurons is observed in all forms of ALS, the outcomes of this project will lead to greater understanding of the molecular origins of the disease. Dr Bradley Turner Androgen receptor abnormalities in MND Victoria M. McLeod, Rebecca K. Sheean, Nirma D. Perera, Richard H. Weston, Bradley J. Turner Florey Institute of Neuroscience and Mental Health, University of Melbourne Androgens such as testosterone are essential factors for proper development, growth and survival of motor neurons. Androgens act on the androgen receptor (AR), leading to signalling of gene transcription underlying their trophic effects. There is increasing evidence that androgens may play a role in ALS, including the higher incidence of ALS in males and genetic defects in AR cause selective motor neuron loss in spinal bulbar muscular atrophy (SBMA), or Kennedy's disease. We therefore assessed a potential role for AR dysregulation in ALS models. AR transcription and protein expression were measured using qPCR and Western blot analyses in NSC-34 cells transfected with mutant SOD1 and spinal cords from SOD1G93A mice at presymptomatic and symptomatic ages. Serum androgen levels were also measured in SOD1G93A using LCMS. Cytoplasmic and nuclear AR distribution was determined in motor neurons of SOD1G93A mice using immunohistochemistry. ORAL PRESENTATIONS We showed that AR expression protein levels progressively decline in spinal cords of SOD1G93A mice from disease onset, leading to significant depletion at endstage compared to wild-type controls. AR was highly abundant in the cytoplasm of spinal motor neurons, but not present in astrocytes or microglia in wild-type mice. In contrast, AR was severely depleted in motor neurons of SOD1G93A mice. These findings suggest a potential role for AR loss in motor neurons in ALS for the first time. If supported, then ALS and SBMA may share a common mechanism of neuronal vulnerability conferred by loss of AR neurotrophic function. Associate Professor Julie Atkin Optimising the therapeutic properties of PDI Emma Perri1, Sonam Parkah2, Colleen Thomas1, Damian Spencer1, Begona Heras1, Julie Atkin1,2 1 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University 2 Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Macquarie University There is currently an urgent need to identify new therapeutic targets in ALS/MND. Protein misfolding is a characteristic pathological hallmark of ALS/MND, and chaperones prevent protein misfolding, suggesting that they could be protective. Consistent with this notion, we have identified that the chaperone protein disulphide isomerase (PDI) is protective in cellular/animal models of ALS/MND. However it remains unclear which features of PDI mediate this protection. However, previous studies have demonstrated that only a few residues dictate PDI function. PDI is the prototype of a protein family that performs two major functions; (i) chaperone activity, and (ii) formation/isomerization of disulfide bonds via disulfide-interchange activity, involving the active site –CXXC-. The aim of this study is to define the regions of PDI necessary for protection in ALS/MND, with the future goal of designing therapeutic compounds based on this activity. Using sitedirected mutagenesis, we generated a series of PDI mutants and expressed these in neuronal cells expressing mutant TDP-43, SOD1 or FUS, to determine if they are still protective compared to wildtype PDI. Some of these mutants express individual domains of PDI only (containing active site residues, chaperone or isomerase activities), whereas other mutants display altered redox properties or alterations in the cis-Proline loop (an important structural feature of PDI). In addition, other PDI family proteins are being examined for their protective activity to further probe these requirements. These experiments are allowing us to define the most favourable properties of PDI necessary for protection, which will be used in future drug design strategies. Dr Justin Yerbury Monitoring accumulation of ubiquitin chains in ALS – Developing a potential imaging tool for monitoring preclinical disease progression Justin Yerbury, Ian Blair, Darren Saunders Currently there are no effective treatments for MND. Although many drugs have showed promise in the laboratory none have translated to become symptom-slowing drugs in human trials. It has been proposed that motor neuron dysfunction may begin much earlier than previously thought, and potentially years before physical symptoms appear. This means that we may need to better define the effective treatment window in MND. The ultimate aim of this project is to develop an imaging molecule that would allow the detection of cellular dysfunction well before symptom onset. Given the association of ubiquitin positive inclusions with all forms of ALS we have characterised the role of ubiquitin in inclusion formation in ALS models. Further, we have designed and created an in-cell assay to use in small molecule screens to allow us to identify compounds that will identify ubiquitin positive inclusions in vivo. We believe that our screening assay will lead to the discovery of a molecule capable of binding inclusions and may in the future allow us to detect MND prior to symptom onset. Associate Professor Peter Noakes Progress into the contributions made by motor neurons and muscle in the progression of MND: human neuromuscular and muscle analyses Dr Rachel Tan and Peter G. Noakes, School of Biomedical Sciences, and Queensland Brain Institute, The University of Queensland There is growing evidence that abnormalities in the connections between motor nerves and muscle are an early disease event in MND. My talk will focus on how our human muscle-skin biopsy project has advanced the contribution of these abnormalities in MND. Our molecular and ORAL PRESENTATIONS structural analyses of neuromuscular junctions from early stage MND patients show defects in the depths of their post-synaptic folds, synaptic basal lamina structure, and a greater gap between the motor nerve terminal and its underlying muscle membrane. We have also observed invasion of terminal Schwann cells into the synaptic cleft, which may in part explain the lifting of the motor nerve terminal from the muscle membrane. All these defects are consistent with a loss of synaptic adhesion molecules – such as the synaptic laminins, which we are currently investigating. The growth dynamics of muscle precursor cells from MND patients show faster proliferation rates, but a delayed in cellular fusion into muscle cells. We have also noted that these muscles do not effectively form post-synaptic specializations induced by neural-agrin. We have made progress in the formation of our human neuromuscular circuit, in that we have been able to generate and functionally characterize upper and lower motor neurons from hiPSCs, and when cultured with muscle can from rudimentary neuromuscular connections. We have also been able to take the pre-synaptic mRNA transcripts that bind to TDP-43, fluorescently tag them, and inject them into our hiPSC derived motor neurons and show that these transcripts can concentrate at the distal terminal endings. Dr Jacqueline Leung The role of TDP43 in oligodendrocytes development Jacqueline YK Leung, Samuel T Dwyer, James C Vickers, Anna E King Wicking Dementia Research and Education Centre There G93A mSOD1 mouse model has provided compelling evidence that oligodendrocyte dysfunction may be a primary event in disease pathogenesis in this model, but it is still unclear if oligodendrocyte dysfunction is involved in human disease. Human ALS tissue is characterized by extensive axonal degeneration and myelin loss and TDP-43 aggregates are present in oligodendrocytes as well as neurons. Thus we have hypothesized that either gain or loss of TDP-43 function in oligodendrocytes results in myelin loss and axonal dysfunction in ALS. In neurons, TDP-43 pathology results in defective neurite outgrowth. In ths current study we are focused on identifying if TDP-43 plays a role in oligodendrocytes differentiation and maturation.. Oligodendrocytes differentiate from precursor cells (OPCs) through a series of stages that involes process outgrowth and branching as well as the transport of mRNA required for myelin formation. In order to investigate the role of TDP-43 in oligodendrocyte differetetiation, OPCs have been grown in culture and the expression and localization of endogenopus TDP-43 has been examined as the cells differentiate. Additionally OPCs have been transduced with TDP-43 lentiviral constructs to alter the expression of TDP-34 and the effect on process outgrowth and branching examined.The results suggest that the TDP43 expression is altered through development of OPCs and that it may play a role in OPC development. Determining the role of TDP-43 pathology in oligodendrocyte health could present novel therapeutic targets for ALS. Dr Rachel Tan Cerebellar neuronal loss in ALS cases with ATXN2 intermediate repeat expansions Rachel H Tan, PhD1,2, Jillian J Kril, PhD3, Ciara McGinley, BSc3, Mohammad Hassani, BSc(Hons)1, Masami Masuda-Suzukake, PhD4, Masato Hasegawa, PhD4, Remika Mito, BSc(Hons)3, Matthew C Kiernan, DSc FRACP5, Glenda M Halliday, PhD1,2 1 Neuroscience Research Australia, Sydney 2 UNSW Medicine, University of New South Wales, Sydney 3 Discipline of Pathology, Sydney Medical School, The University of Sydney 4 Department of Neuropathology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan 5 Brain and Mind Centre, Sydney Medical School, The University of Sydney, Australia Objective: Despite evidence suggesting the cerebellum may be targeted in amyotrophic lateral sclerosis (ALS), particularly in cases with repeat expansions in the ATXN2 and C9ORF72 genes, the integrity of cerebellar neurons has yet to be examined. The present study undertakes a histopathological analysis to assess the impact of these repeat expansions on cerebellar neurons and determine if similar cerebellar pathology occurs in sporadic disease. Methods: Purkinje and granule cells were quantified in the vermis and lateral cerebellar hemispheres of ALS cases with repeat expansions in the ATXN2 and C9ORF72 genes, sporadic disease, and sporadic progressive muscular atrophy (PMA) with only lower motor neuron degeneration. Results: ALS cases with intermediate repeat expansions in the ATXN2 gene demonstrate a significant loss in Purkinje cells in the cerebellar vermis only. Despite ALS cases with expansions in the C9ORF72 gene having ORAL PRESENTATIONS the highest burden of inclusion pathology, no neuronal loss was observed in this group. Neuronal numbers were also unchanged in sporadic ALS and sporadic PMA cases. Interpretation: The present study has established a selective loss of Purkinje cells in the cerebellar vermis of ALS cases with intermediate repeat expansions in the ATXN2 gene, suggesting a divergent pathogenic mechanism independent of upper and lower motor neuron degeneration in ALS. We discuss these findings in context of large repeat expansions in ATXN2 and spinocerebellar ataxia type 2, providing evidence that intermediate repeats in ATXN2 cause significant albeit less substantial spinocerebellar damage compared with longer repeats in ATXN2. Dr Parvathi Menon Insights into ALS pathophysiology from patterns of disease progression Dr Parvathi Menon, Westmead Hospital, University of Sydney Prof Steve Vucic, Westmead Hospital, University of Sydney Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder of motor neurons in the spinal cord, brainstem, and motor cortex, for which there is no cure . Currently, there is debate as to whether ALS begins centrally, peripherally or whether it is multifocal in onset. Further, the pattern of disease spread remains uncertain with proposed mechanisms including focal onset with contiguous spread while others have suggested multifocal onset with local spread. The present study prospectively assesses cortical and lower motor neuron (LMN) dysfunction detected by clinical and sensitive neurophysiological techniques in multiple body regions to determine patterns of disease onset and spread in ALS. In addition, the contribution of cortical hyperexcitability to the pattern of disease spread was assessed, specifically whether cortical hyperexcitability precedes LMN dysfunction and underlies the spread of disease. At the current stage of the project clinical and neurophysiological measures of LMN involvement in three body region have been combined with cortical excitability studies over representative muscles in the regions in elen ALS patients. More importantly five age matched normal subjects have undergone cortical excitability measurements from the target muscles to understand the changes unique to ALS pathophysiology. Early findings of the study will be presented and insights into ALS pathophysiology will be discussed. Dr James Howells Dysfunctional properties of single motor units in ALS University of Sydney Abnormalities in the excitability of lower motor neurons in ALS have been extensively examined using threshold tracking techniques. Several studies have found evidence for a down-regulation of K+, and an up-regulation of Na+ conductances. The results as a whole however have been somewhat mixed. Threshold-tracking studies test the excitability of a fixed fraction of the maximal compound action potential, and for most disease processes this accurately reflects the excitability of all axons of the same modality within a nerve. However in ALS, degeneration of individual motor units is likely to change their excitability, with hyperexcitability and reinnervation believed to impact on their viability. In ALS, the disease process itself presents the opportunity to study single motor units, and in particular the large units which result from reinnervation of denervated muscle fibres. Measurements of the excitability of single motor units in the hand were undertaken in 8 subjects, and mathematical modelling was performed to examine the underlying biophysical changes. ALS single motor unit recordings were more varied in nature when compared to normal single motor units from a previous study. Significant differences were observed in the excitability of ALS single motor units compared to normal controls. The excitability of individual motor units in ALS is heterogeneous, with abnormalities correlated with disease progression. The underlying mechanisms were best modelled by an increase in the Na+/K+-ATPase pump current, and a near halving of slow K+ conductances. The relative proportions of these changes are likely to vary between individual motor units, and with disease progression. ORAL PRESENTATIONS Dr Catherine Blizzard Synaptic Dysfunction: An Early Mechanism Of TDP-43 Pathogenesis In ALS? Catherine A Blizzard1, EE Handley1, Dawkins E1, Clark R1, Turner BJ2, and Dickson TC1 1 Menzies Institute for Medical Research, University of Tasmania 2 The Florey Institute of Neuroscience and Mental Health, University of Melbourne ALS can be frequently characterised by the presence of cytoplasmic aggregates of the RNA binding protein TDP-43. Abnormal localisation and function of TDP-43 is likely to be a critical component of this disease, as mutation of the TARDBP gene (encoding TDP-43), including the A315T mutation, is sufficient to cause familial ALS. The associated neurodegeneration was proposed to result from a toxic gain of TDP-43 function within the cytoplasmic inclusions, or a detrimental loss of normal TDP-43 function from the nucleus. However, resent research indicates that TDP-43 may also play an underappreciated role at the synapse. To probe TDP-43 misprocessing at the synapse we turned to the TDP-43A315T: Thy1-YFP transgenic mouse. Our data indicates that there is a significant (P<0.05) reduction in total spine density in the motor cortex of TDP-43A315T: Thy1-YFP transgenic mice relative to Thy1-YFP controls that develops between postnatal day 30 and 60. This spine loss significantly precedes excitatory neuronal cell loss, which is only present by day 90. Our current in vitro studies using primary cortical neurons with the TDP-43A315T mutation has demonstrated that whilst there was no significant difference in mean dendrite outgrowth or dendrite complexity there was a significant reduction in spine formation. Drawing upon this data we propose that TDP-43 misprocessing may play a pathogenic role in neuronal communication and potentially synaptic plasticity, occurring early in disease progression. Understanding the role that TDP-43 plays in synaptic dysfunction may reveal new therapeutic windows for intervention in TDP-43 proteinopathies. Associate Professor Tracey Dickson Inhibitory Regulation of Motor Neurons: a new target mechanism for ALS? Rosie Clark, Mariana Brizuela, Catherine Blizzard, Tracey Dickson Menzies Institute for Medical Research, University of Tasmania There is considerable evidence from many areas of clinical and basic medical research that in MND motor neurons may be dying due excitotoxicity. We and others have new evidence that this toxic cascade may initially be triggered by the death or dysfunction of another type of neuron in the brain – the interneuron. We are developing a novel primary culture method of specifically growing interneurons and/or motor neurons, derived from transgenic mice developed to model MND, in primary culture. This chimeric culture approach will allow us to determine if the presence of abnormal or pathogenic interneurons can lead to abnormal motor neuron function and pathology. Initial investigations have utilised FACS to purify interneurons from SOD1/GADGFP E15 embryos, and combine with wildtype principal neurons. Similarly we will purify principal neurons from SOD1/Thy1YFP embryos and combine them with wildtype interneurons. Using this approach we consistently obtain cultures of >80% purity, that contain viable neurons that polarise appropriately and grow well in culture to 14 days in vitro, at which point they show evidence of cytoskeletal, synaptic and functional maturation. Purified neurons are combined to recapitulate the interneuron:projection neuron ratio observed in unsorted cultures and will be utilised to investigate synaptic composition and characterisation of electrophysiological properties. We would expect chimeric cultures, where mutant SOD expressed only in GABAergic interneurons, to show evidence of abnormal connectivity and electrophysiology. Not only will these studies provide important insight into the mechanisms responsible for ALS, but they would also provide a high throughput model for later assessing potential therapeutic interventions. Dr Shuyan Ngo Investigating the mechanisms underlying defective energy metabolism in MND Ngo S.T1,2,3,4, Steyn F.J1,4, Lee K1, Mantovani S1, Woodruff T.M.1, Rene F5,6, Loeffler, JP5,6, Sullivan J.D7. Henderson RD2,3,7, and McCombe PA3,4,7 1 School of Biomedical Sciences, The University of Queensland, St Lucia, Queensland 2 Queensland Brain Institute, The University of Queensland, St Lucia, Queensland 3 Department of Neurology, Royal Brisbane & Women’s Hospital, Herston, Queensland 4 University of Queensland Centre for Clinical Research, The University of Queensland, Herston, Queensland, 5 INSERM, U1118, Mécanismes Centraux et Périphériques de la Neurodégénérescence, Strasbourg, France. 6 Université de Strasbourg, UMRS1118, Strasbourg, France 7 School of Medicine, The University of Queensland, Herston, Queensland While the primary pathology in MND is the death of motor neurons, there is evidence that defective energy metabolism affects disease pathogenesis. We hypothesise that in MND, the state of metabolic flux modifies disease course, and ORAL PRESENTATIONS modulation of metabolic homeostasis will aid in sustaining survival. We have studied mouse models and human subjects to investigate how altered metabolic homeostasis impacts disease outcome. We have found that defects in glucose metabolism occur in the brains and muscle of mouse models of MND. In muscle, this drives a switch towards the use of fat as an energy substrate. Because metabolic flux underpins the generation of energy to maintain cell survival, we have developed targeted strategies to ameliorate defective glucose metabolism, with the aim to prevent MND pathology. By modulating glucose metabolism in two mouse models of MND, we have prevented metabolic defects and have improved motor deficit. To translate observations in mice to humans, we have studied subjects with MND. Analysis of muscle biopsies suggest defects in muscle glucose metabolism in MND patients. Moreover, MND patients have altered expression of circulating proteins that demonstrate metabolic responses that match altered demand on fat as an energy substrate. Our studies establish the disease-promoting role of defective energy metabolism in MND and pave the way for future studies aimed at improving metabolic capacity to prevent the death of neurons and muscle in neurodegenerative disease. Associate Professor Trent Woodruff The innate immune system as a therapeutic target in motor neuron disease Woodruff TM1, Lee JD1, Deora V1, Vollert A1, McAlary L4, Yerbury JJ4, Mantovani S1, Henderson RD5, McCombe PA3,5, Noakes PG1,2 1 School of Biomedical Sciences, 2 Queensland Brain Institute, 3 University of Queensland Centre for Clinical Research, The University of Queensland 4 llawarra Health and Medical Research Institute, Wollongong 5 Department of Neurology, Royal Brisbane and Women's Hospital, Herston, Queensland Inflammation is a key immune process in the body’s natural defence against infection. However, when chronically activated in the absence of infection, it can also lead to progressive tissue damage. One emerging consensus coming from MND research in recent years, is the concept that immune over-activation, leading to excessive inflammation, can accelerate the progression of the disease. Several studies have also documented that peripheral blood monocytes can infiltrate the spinal cord of MND sufferers, where it is thought they mediate inflammatory damage leading to accelerated motor neuron death. Our group is investigating innate immune pathways that may mediate the onset of neuroinflammation in MND. We have identified two key pathways, inflammasome and complement, which are chronically activated in mouse models of MND, with some evidence for activation in MND patient blood. With our collaborators, we have also developed novel, orally active drugs which can block these inflammatory pathways. One of these compounds shows therapeutic benefit in the SOD1G93A mouse model, and is now poised for human clinical trials. This presentation will provide an overview of the evidence for innate immune activation in MND, and highlight potential therapeutic targets for future clinical translation. Professor Steve Vucic & Dr Fiona McKay Safety and biological efficacy of narrow-band UVB phototherapy in ALS McKay, F.1, Menon, P.2, Fernandez-Peñas, P.3, Byrne, S.4, Turner, B.5, Booth, D.1, Stewart, G.J.1, Vucic, S.2 1 Westmead Millennium Institute, University of Sydney; Departments of 2Neurology and 3 Dermatology, Westmead Hospital; 4Infectious Diseases & Immunology, University of Sydney; 5MND Laboratory, Florey Institute of Neuroscience and Mental Health Recent evidence suggests a role for regulatory T cells (Tregs) in neuroprotection, neurogenesis and slowing of disease progression in animal models of amyotrophic lateral sclerosis (ALS; Beers et al, 2011). In human studies, higher numbers of circulating Tregs levels correlate with slower disease progression (Henkel et al, 2013). Exposure to the UV wavelengths of light increases the numbers of circulating FoxP3+ Tregs and serum vitamin D, also associated in a small study with slower progression (Karam et al, 2013). The aim of this Phase IIA trial is to investigate whether narrow-band ultraviolet B (NB-UVB) therapy in ALS patients 1. is safe and feasible; and 2. can increase Treg numbers and serum vitamin D. Methods: ALS patients with ALSFRS-R ≥38 will be randomised to receive NB-UVB phototherapy (3 times per week for 12 weeks) in addition to standard-of-care (n = 10), or standard-of-care only (n = 10). Blood will be collected at baseline, months 1, 3, 6, 9, and 12. Safety will be determined by adverse event assessment and clinical assessment (baseline, months 3, 6,12). Results: Nine participants were enrolled Aug-Sep 2015, and randomised to phototherapy (n = 4), or standard-of-care only (n = 5) and the trial is ongoing. Phototherapy has been safe and well-tolerated thus far, with high rates of compliance. One minor adverse event (skin rash) did not require cessation of therapy. Immunological analyses will be performed at study completion. If successful, this preliminary study would support a larger trial to investigate whether phototherapy slows progression of disease. ORAL PRESENTATIONS Dr Robert Henderson Biomarkers in MND Henderson RD, McCombe PA; University of Queensland Centre for Clinical Research and Royal Brisbane & Women’s Hospital There is a need for effective and practical biomarkers to assess disease progression in MND and for use in clinical trials. This study focusses on blood biomarkers. Phosphorylated neurofilament heavy has been identified as a promising biomarker of axonal injury and is elevated in the CSF of human and animal models. In this study we have examined serial studies in 98 MND patients from the Royal Brisbane & Women’s Hospital multidisciplinary MND clinic and 61 controls.65 MND patients were deceased at the end of the study and a total of 223 samples were obtained. Neurofilament heavy was elevated in comparison to controls with the elevation occurring early in the disease course and later falling. A correlation of higher levels with increasing age was found. A clear correlation with ALSFRS-R was not apparent. Higher levels occurred in those with shorter survival. The role of other potential blood biomarkers identified by preliminary work using mass spectroscopy will be discussed. Dr Anne Hodgen Decision support tools for motor neurone disease multidisciplinary care Anne Hogden1, David Greenfield1, Xiongcai Cai2, Jashelle Caga3 1. Australian Institute of Health Innovation, Macquarie University 2. School of Computer Science and Engineering, University of New South Wales 3 . Brain and Mind Centre, University of Sydney Objective: MND patients and families make numerous decisions for symptom management and quality of life as their condition deteriorates. Decisions may include: use of medication; assisted ventilation; artificial nutrition and hydration; and end-of-life care. MND challenges well-timed decision-making. Patients are often overwhelmed by their diagnosis, and take time to come to terms with their prognosis. Information needed to support their decisions is frequently confronting. Currently, there are no decision support tools available to guide MND patients through treatment and quality of life decisions. We will develop tools to help patients and families engage with health professionals to make well-timed and wellinformed decisions over the course of the disease. Methods: The project will use an iterative and consultative process to develop and validate decision support tools for MND multidisciplinary care. An expert stakeholder panel of MND patients, family members, health professionals and researchers will be formed to determine content, usefulness and feasibility of the tools. The tools will comply with International Patient Decision Aid Standards criteria, drawing on MND research, best practice and clinical guidelines. Expected outcomes: Completed decision support tools will inform patients of the benefits and risks of each option, the consequences of ‘doing nothing’, and clarify patients’ personal values. Tools that tailor MND information to decision-making will assist patients to make difficult decisions in a timely and informed manner, to improve patient outcomes and service delivery. The tools, and their process of development, have potentially wider clinical application for a range of degenerative neurological conditions. Dr Sharpley Hsieh My memories are important to me: autobiographical memory retrieval in MND MND not only results in disabling motor impairments but up to half of all patients show non-motor changes, which we know now to affect how patients think, behave and feel. In the realm of cognition, studies of memory show that patients have trouble remembering lists of words and passages of information. One aspect of memory that has not been tapped into at all is autobiographical memory, our personal memories of our own individual past. Autobiographical memories are contextually rich in detail, contain specific information of what we did and what had happened, is often emotionally laden and aids us in conceptualizing our own identity and in how we might see ourselves in the future. In this study, we found that recall of autobiographical memories was preserved in mild to moderate ALS. Interestingly, the phenomenology of these memories differs from age-matched adults. Memories in the last year, presumably those have occurred since ALS symptom onset, are far richer in detail with ALS disease progression and are weighted by the patient to be subjectively more important and more frequently rehearsed by the ALS patient than same aged adults. The phenomenological change in autobiographical memories in ALS can potentially be capitalized by multidisciplinary clinics who might consider adopting therapies that specifically encourage forms of life review. This technique is often used in other terminal illnesses and shown to support an individual’s unique identity through a terminal illness, improve patient psychological well-being, and is helpful as patients accepts and perhaps redefines purpose in their lives. POSTER PRESENTATIONS Monique Bax The impairment of the ubiquitin proteasome system in amyotrophic lateral sclerosis patient iPSC– derived neurons Monique Bax, Dzung Do-Ha, Rachelle Balez, Dr. Justin Yerbury, Dr. Lezanne Ooi Illawarra Health and Medical Research Institute, University of Wollongong Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative condition on the motor neurone disease- frontotemporal dementia spectrum. Ubiquitin-proteasome dysfunction is a common feature of ALS pathology. As it is not possible to culture primary ALS patient neurons, it is difficult to understand the dynamics of the ubiquitin proteasome system in affected neurons. Patient-derived induced pluripotent stem cells provide a dynamic system to study neurons with an ALS phenotype. We aimed to assess ubiquitin proteasome function using ALS and control patient iPSC-derived neurons. Here, we examined ubiquitin proteasome dysfunction in mRNA reprogrammed induced pluripotent stem celldifferentiated neurons from two patients with an E3 ubiquitin ligase mutation, a SOD1E101G patient and three control lines. Using a degron assay to measure the efficacy of the cells to degrade a proteasome degradation signal-tagged GFP we found an increase in protein retention in ALS affected neurons. Following treatment with a proteasome inhibitor MG132 (10 uM 16h), this protein retention was significantly increased in ALS neurons compared to controls. These results strongly implicate ubiquitin proteasome dysfunction in ALS pathology thus representing a viable target for future therapeutics. [email protected] Jashelle Caga Survival differences among ALS patients with different levels of apathy Jashelle Caga1,2; Martin R. Turner3; Sharpley Hsieh1; Rebekah M. Ahmed4; Emma Devenney4; Eleanor Ramsey1; Margaret C. Zoing1; Eneida Mioshi5; Matthew C. Kiernan1,2 1 Brain & Mind Centre, University of Sydney 2 Sydney Medical School, University of Sydney 3 Nuffield Department of Clinical Neurosciences, Oxford University, UK 4 Neuroscience Research Australia 5 Department of Psychiatry, University of Cambridge, Cambridge, UK Background: Apathy of varying severity is highly prevalent in amyotrophic lateral sclerosis (ALS). However, the degree to which it affects prognosis and overlaps with depression is unknown. The present study examined the relationship between level of apathy, mortality and survival time and whether apathy was linked to specific symptom clusters of depression. Methods: A cohort of 76 consecutive ALS patients attending specialised multidisciplinary clinics were classified according to level of apathy. ALS patients with comorbid frontotemporal dementia were excluded. The effect of clinical factors and apathy on mortality and survival time were analysed using univariate and multivariate methods. Results: 33% patients had no apathy, 48% had mild apathy and 19% had moderate-severe apathy. The majority of patients with moderate-severe apathy died during the study (p = 0.003) and had a median survival time of 21.7 months, significantly shorter than patients with mild apathy (46.9 months) and no apathy (51.9 months) (p = 0.0001). Apathy remained a significant predictor of survival even after controlling for clinical factors and symptom duration at study entry (hazard ratio 3.5, 95% confidence interval 1.9-6.5, p = 0.0001). Depression with demoralisation was not associated with level of apathy (p = 0.172) whereas depression with anhedonia was more common in patients with apathy than in those without apathy (p = 0.006). Conclusions: The presence of severe apathy is an independent, negative prognostic factor in ALS. [email protected] Jayden Clark Targeting Microtubules to Improve Outcomes in ALS Clark J1, Yeaman E1, Blizzard C1, Chuckowree J1, Dickson T1 1 Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia Alterations to key intracellular structures, such as microtubules, may impact on both the initiation and maintenance of ALS. In the mSOD1G93A mouse model key microtubule proteins, microtubule associated proteins (MAPs) and microtubule dynamics are altered. Utilising microtubule stabilising drugs to reduce microtubule dynamics may be a potential therapy to ameliorate motor neuron degeneration and associated motor phenotypes. We have used the microtubule stabiliser EpothiloneD (EpoD), which has shown promise in ameliorating the pathophysiology of disease models emulating Parkinson’s, Tauopathies and Schizophrenia. mSOD1G93A mice were treated with a high dose (2mg/kg) or a low dose (1mg/kg) of POSTER PRESENTATIONS EpoD. High dose treatment showed significant improvement to aspects of motor function (p<0.05). However, EpoD presented a toxic effect in motor function in late disease and decreased survival (p<0.05). Low dose treatment significantly improved all aspects of motor function (p<0.05), with not effect on survival (p>0.05). We posit that EpoD is a dose dependent disease-modifying agent that has both positive and negative effects on the ALS phenotype. This highlights the involvement of microtubules in ALS, a system that is still not well understood, but may offer additional targets for therapeutic intervention. Future studies will investigate the effect of varying EpoD concentrations on microtubule dependent functions, such as intracellular transport, MAPs alterations, tubulin modifications and isotype changes. Similarly, identifying the degree in which EpoD can alter microtubule dynamics in both cortical and spinal motor neurons will be undertaken. [email protected] Rosie Clark Intrinsic interneuronal vulnerabilities in the GAD67.SOD1 mouse cortex Rosie Clark, Mariana Brizuela, Catherine Blizzard, Tracey Dickson Menzies Institute for Medical Research Differential interneuron vulnerability has been previously identified in the cortex of amyotrophic lateral sclerosis (ALS) patients and familial animal models. Although the role of these key inhibitory neurons remains to be determined in ALS, identification of intrinsic interneuron vulnerabilities could enhance our understanding of their function in extrinsic regulation of excitability. Very little is known about the intrinsic vulnerabilities of interneuron sub-types, particularly in the context of altered excitability and excitotoxicity. The aim of this study was to provide a comprehensive overview of differential interneuronal vulnerability in vitro and in the context of known ALS mutations. For these analyses the GAD67.hSOD1 GFP mouse was used to characterise interneuron electrophysiological profiles in vitro, utilising patch-clamp recordings, immunohistochemistry and morphometric approaches in 15day cortical cultures. Vulnerability to known excitotoxins was subsequently assessed using western blotting techniques. Initial characterisation of interneuron subtypes in vitro suggests bipolar and multipolar calretinin-positive interneurons have differential electrophysiological profiles, with lower capacitance and higher membrane resistance demonstrated in bipolar cells (P<0.05). Furthermore, preliminary results from excitotoxic treatments, suggest calretinin-interneurons respond differentially to treatments compared to other calciumbinding populations. This study is the first to characterise differential interneuron electrophysiological profiles in the GAD67.hSOD1 mouse. Results suggest that a differential intrinsic vulnerability of calretinin-positive interneurons may contribute to disease processes in ALS, agreeing with a cortical subtype-specific interneuron phenotype previously identified in response to the SOD1 mutation. It remains to be determined how the dysregulation of this population contributes to disease, with future directions focused on exploring system effects. [email protected] Nicholas Cole Fishing to Better Understand the Biology of Motor Neurone Disease/Frontotemporal Dementia Nicholas Cole, Emily Don, Marco Morsch, Jack Stoddart, Yagiz Alp Aksoy, Rowan Radford, Alison Hogan, Jennifer Fifita, Isabel Formella, Andrew Badrock, Dasha Sayal, Gilles Guillemin, Julie Atkin, Ian Blair, Roger Chung, Garth Nicholson. Macquarie University Motor Neuron Disease Research Centre, Macquarie University, Sydney. Our approach is to use zebrafish, an established research model organism, to generate animal models that develop features of ALS/MND/FTD in order to understand the biology of the disease. Zebrafish offer some advantages for human disease research that compliment mammalian models. Transgenic technologies allow us to express known ALS/MND causing human genes in the fish in vivo whilst at the same time observing biology in the living organism in real time. In addition the zebrafish provides many elegant tools in order for us to investigate the biology of MND/FTD. An overview of our zebrafish projects and research strategy is presented. [email protected] POSTER PRESENTATIONS Vandana Deora Activation of the complement cascade and the NLRP3 inflammasome by hSOD1G93A protein Vandana Deora1, John D. Lee1, Luke McAlary2,3, Justin Yerbury2,3, Richard Gordon1, Trent M. Woodruff1* 1 School of Biomedical Sciences, University of Queensland, Brisbane 2 School of Biological Sciences, Faculty of Science, University of Wollongong, Wollongong, New South Wales 3 Illawarra Health and Medical Institute, University of Wollongong, Wollongong, New South Wales Motor neuron disease (MND) is an incurable neurodegenerative disorder characterised by a progressive loss of motor neurons in the motor cortex, brain stem and spinal cord. It leads to atrophy and weakness of bulbar, limb, and respiratory muscles. There is increasing evidence that activation of the innate immune system leading to chronic neuroinflammation can drive the progression of MND. Two key components of the innate immune system that have recently been proposed to play an important role in MND pathology and progression are the NLRP3 inflammasome and the complement system. The NLRP3 inflammasome is an intracellular protein complex that mediates the generation of mature IL-1 via caspase-1 activation, and has been recently implicated in Alzheimer’s disease pathology by responding to beta-amyloid fibrils. The complement system is a cascade of secreted molecules that reacts to pathogens and protein aggregates, which also has documented roles in MND. The current study aimed to determine if the NLRP3 inflammasome and the complement system are activated in response to a MND-relevant protein, SOD1G93A. Microglial cultures were treated with mutant SOD1G93A protein, and inflammasome activation determined by western blotting and ELISA. We found that soluble and aggregated mutant SOD1 protein triggers IL-1β secretion from LPS-primed microglia, as well as generating the cleaved form of caspase-1. These results indicate that activation of the inflammasome pathway, leading to increased expression of caspase-1, IL-1β and NLRP3 can occur in response to a MND mutant protein. In addition, we found that SOD1G93A protein aggregates also activated the complement cascade, generating the terminal complement component C5a from lepirudinplasma. Taken together, these findings suggest that NLRP3 inflammasome activation and C5a-signalling could be potential downstream pathological mechanisms triggered by neurotoxic proteins seen in MND. Inhibiting these innate immune pathways may thus be a means to slow propagative neuroinflammatory cell death in MND, and ameliorate disease progression. [email protected] Emma Devenney Psychosis in the Frontotemporal Dementia - Motor Neurone Disease continuum – clinical features and neuroimaging correlates Devenney E1,2,3, Irish M1,4, Hornberger M1,5, Mioshi E1,5, Halliday G1, Kiernan MC1,2,3, Hodges JR1,4, 1 Neuroscience Research Australia, Sydney, 2 Prince of Wales Clinical School, University of New South Wales, Sydney, 3Brain and Mind Research Institute, Sydney 4 School of Medical Sciences, University of New South Wales, Sydney 5University of Cambridge, United Kingdom Delusions and hallucinations were previously considered rare in frontotemporal dementia (FTD) and motor neurone disease (MND). More recently however they have been described in patients with these conditions, particularly those with TDP-43 pathology and the C9orf72 genetic expansion[1, 2]. This study aimed to determine the rate and severity of psychotic symptoms in a cohort of patients with FTD, with and without concomitant MND, and determine the neural correlates with respect to genetic status. Together 56 patients with FTD (20 with concomitant MND) were included. All carers underwent a carer-based interview, the neuropsychiatric inventory (NPI). All patients underwent clinical interview, neuropsychological assessments and MRI brain. An index of psychosis was calculated, taking into account the degree and severity of psychosis in each case. Voxel-based morphometry analysis was conducted to establish patterns of brain atrophy and determine correlations between the psychosis index and grey matter density decrease. The rate of psychosis was high across the FTD-MND continuum (35%). C9orf72 carriers were more likely to exhibit psychotic features than non-carriers (p<0.05). Symptoms were more marked in carriers as reflected in the significantly higher psychosis index (p<0.005). Voxel-based morphometry correlational analysis linked psychosis with key structures of the thalamo-cortico-cerebellar networks. The neural correlates of psychosis were similar in both carriers and non-carriers. This novel study established that psychosis is common in the FTD-MND continuum, and in particular in C9orf72 carriers. The role of neural networks, and specifically key structures within thalamo-cortico-cerebellar pathways, in addition to subcortical structures, appear critical to the generation of psychosis. [email protected] POSTER PRESENTATIONS Emily Don A Zebrafish Model of the MND Linked C9orf72 Hexanucleotide Repeat Expansion Emily K Don, Jack Stoddart, Marco Morsch, Serene Gwee, Alison Hogan, Sharron Chow, Isabel Formella, Rowan Radford, Ian Blair, Julie Atkin, Garth Nicholson, Nicholas J Cole Australian School of Advanced Medicine, Health Sciences, Macquarie University Recently, the most common genetic cause of ALS was identified as a hexanucleotide (GGGGCC) repeat expansion in a non-coding region of the gene C9ORF72. While healthy individuals have 2-23 repeats, ALS patients can have up to 700-1600 repeats. This repeat expansion accounts for 20-80% of familiar and 5-15% of sporadic ALS in North American and European populations. However, it is currently unknown how this repeat expansion leads to the loss of motor neurons and the development of MND. As the repetitive nature of the C9ORF72 hexanucleotide repeat expansion causes inherent difficulties when generating animal models, the high-throughput capabilities and quick turnaround time of the zebrafish makes the zebrafish an ideal system in which to test several different approaches to model the MND linked C9ORF72 hexanucleotide repeat expansion. We therefore plan to make several different stable transgenic zebrafish lines of the MND linked C9ORF72 hexanucleotide repeat expansion in order to examine the mechanisms by which the repeat expansion causes MND. We will generate zebrafish expressing short or long repeat expansions either ubiquitously or specifically in the motor neurons, in addition to generating lines which allow for inducible expression of the repeat associated proteins. It is hoped that the flexibility of this approach will generate a clinically relevant model of the MND linked C9ORF72 hexanucleotide repeat expansion to allow for observation and characterisation of disease progression at a molecular and behavioural level. [email protected] Kimberley Duncan Characterising the functional domains of an MND candidate protein Kimberley A. Duncan1, Shu Yang1, Kelly L. Williams1, Katharine Y. Zhang1, Emily P. McCann1, Alison L. Hogan1 and Ian P.Blair1 1. Faculty of Medicine and Health Sciences, Macquarie University, Sydney The only known causes of MND are gene mutations, several of which are intrinsic to protein degradation pathways. Dysfunctional protein degradation could lead to abnormal protein aggregation in neurons, a hallmark of ALS pathology. Our laboratory recently identified MND-linked mutations in a gene encoding an E3 ubiquitin ligase (E3UL) protein. E3UL mediates the destruction of aberrant proteins by the ubiquitin proteasome system (UPS). E3UL has three functional domains including a cyclin box for substrate binding and a PEST domain (proline (P), glutamic acid (E), serine (S) and threonine (T) rich sequence) responsible for destabilising the protein to ensure proper protein turnover. MND-linked mutations have been found in several functional domains of this E3UL, including the PEST domain. While several E3UL binding substrates have been discovered, the E3UL protein has still not been fully characterised in neuronal cells. We transfected a series of E3UL deletion constructs into neuronal cell lines. We then investigated features of MND pathogenesis including cytotoxicity and sub-cellular localisation and protein aggregation of transfected proteins. The ΔPEST construct led to increased nuclear expression of the protein and aggregate formation in both cell lines. Both ΔPEST and Δcyclin box led to a significantly higher levels of cellular toxicity. Deletion of the PEST region of E3UL recapitulates some of the MND cellular features associated with MNDlinked E3UL mutations, including mis-localisation and protein aggregation. In addition, cells with aggregates are significantly more toxic than the wild type, supporting the pathogenic role of protein aggregation in MND. [email protected] Michelle Farrar Dysfunction of the motor cortex heralds onset of ataxia in Machado-Joseph Disease Michelle A. Farrar1,2, Steve Vucic2,3, Garth Nicholson4, Matthew C. Kiernan2,5 1 Discipline of Paediatrics, School of Women’s and Children’s Health, UNSW Medicine, The University of New South Wales, 2Neuroscience Research Australia, 3Westmead Hospital and Western Clinical School, University of Sydney, 3ANZAC Research Institute, University of Sydney, 4Brain & Mind Research Institute, University of Sydney, Sydney, Australia Background and aims: Machado Joseph Disease(MJD) is an inherited neurodegenerative disorder characterized by cerebellar ataxia and variable expression of clinical features beyond the cerebellum, including amyotrophy and motor neurone disease. To gain further insights into disease pathophysiology, , the present study explored motor cortex function in MJD to determine whether cortical dysfunction was present and if this contributed to the development of clinical manifestations. POSTER PRESENTATIONS Methods: Clinical phenotyping and longitudinal assessments were combined with central (threshold-tracking transcranial magnetic stimulation) and peripheral(nerve excitability) techniques in 11 genetically characterized MJD patients. Results: Clinical severity varied, ranging from presymptomatic in 2 patients to severe (ICARS score range 059, median 27). Short-interval intracortical inhibition(SICI) was significantly reduced in presymptomatic (5.3±0.5%) and symptomatic MJD patients (-1.3±1.4%) compared to healthy controls (10.3±0.7%, P<0.0001), evident prior to clinical onset of ataxia and related to worsening severity (R=-0.67, P<0.05). SICI reduction was accompanied by decreases in resting motor threshold and cortical silent period in pre-symptomatic patients and inversely related to severity of clinical ataxia. CMCT was also significantly prolonged in all MJD patients (symptomatic MJD 7.5±0.4 ms; pre-symptomatic MJD 6.2±0.5 ms controls 5.3±0.2 ms, P<0.0005) and a feature of neurodegeneration related to clinical severity (R=0.72, P=0.01). Markers of peripheral motor neurodegeneration and excitability were not associated with cortical hyperexcitability or ataxia. Conclusion: Simultaneous investigation of clinical status and central and peripheral nerve behaviour has established that cortical dysfunction is a critical driver of neurodegeneration in patients with MJD, that precedes the development of cerebellar symptoms, ataxia and peripheral neurodegeneration. [email protected] Natalie Farrawell Ubiquitin homeostasis and SOD1 aggregation in ALS Natalie Farrawell1,2, Justin Yerbury1,2, Darren Saunders3 1. Illawarra Health and Medical Research Institute, Wollongong 2. Faculty of Science, Medicine and Health, University of Wollongong, Wollongong 3. Faculty of Medicine, University of New South Wales Amyotrophic lateral sclerosis (ALS) pathology is marked by the accumulation of protein inclusions within motor neurons. The composition of these inclusions varies considerably depending on whether the disease is sporadic or familial and the genetics of the familial forms. Despite this variation, a common feature across all forms of ALS is the presence of ubiquitin-positive inclusions. However, the mechanisms of inclusion formation and ubiquitination have not yet been described. Here we describe a series of experiments to probe the role of ubiquitin in SOD1 aggregation. Co-transfection of NSC-34 cells revealed that SOD1-GFP always co-aggregates with ubiquitin. When immunofluorescence was performed on these SOD1-GFP aggregates, both K48 and K63 poly-ubiquitin chains were detected. SOD1 aggregates detected in G93A SOD1 mouse spinal cord were also found to contain K48 polyubiquitin chains. To test if mutant SOD1 affects the rate of UPS activity we co-expressed SOD1 with the proteasome reporter GFPu. Levels of GFPu fluorescence were found to be significantly higher in cells expressing SOD1A4V compared to SOD1WT suggesting UPS dysfunction. To further probe ubiquitin homeostasis we used fluorescence recovery after photobleaching analysis which revealed that when mRFP-Ub was incorporated into SOD1A4V-GFP inclusions, the diffusion pattern of remaining soluble Ub was similar to that observed when expressed with soluble SOD1WT. Interestingly, the diffusion pattern of mRFP-Ub was altered in cells with soluble SOD1A4V containing no visible aggregates. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction prior to aggregate formation and that ubiquitin positive inclusion formation is an important target for monitoring pathological changes in ALS. [email protected] Jennifer Fifita Disease gene discovery in familial amyotrophic lateral sclerosis Jennifer A Fifita1, Kelly L Williams1, Katharine Zhang1, Garth A Nicholson1,2, Dominic Rowe1, Ian P Blair1 1 Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2 Molecular Medicine Laboratory, Concord Hospital, Sydney, NSW The majority of ALS cases occur sporadically, however 10% of cases show familial inheritance. Genetic studies of families has identified over 20 ALS genes, accounting for about 60% of familial cases. We aim to identify novel gene mutations causing ALS in the remaining 40% of Australian families with unknown gene mutations. We apply both unbiased and hypothesis driven gene discovery strategies to analyse exome sequence data from Australian familial ALS cases. We have completed whole exome capture and sequencing of probands from 74 ALS families, including ten individuals from three multigenerational ALS families. Custom bioinformatics analysis of these families has produced small lists (5, 10, and 18) of novel candidate gene POSTER PRESENTATIONS mutations. All variants were prioritised for functional studies based on factors such as protein predictions, conservation across species and gene function. The top-ranking candidate from Family One has undergone functional studies to examine potential pathogenicity. Our ALS gene discovery strategies also include mutation analysis of identified functional candidate genes and recently published ALS genes. This is illustrated by our recent analysis of two recently reported ALS genes MATR3 and TBK1. The penetrance of mutation-linked disease in familial ALS varies substantially, ranging from classic Mendelian inheritance to apparently sporadic disease. As such, familial ALS genes were also examined in apparently sporadic ALS cases. Rapid sequencing of 624 sporadic ALS cases has been completed to identify variation in known ALS genes, including a novel gene identified by our laboratory. The identification of novel ALS causing mutations is critical to increase our knowledge of ALS pathology, and provide insight into the development and testing of novel therapies. [email protected] Nimeshan Geevasinga Revisiting early diagnosis in ALS Geevasinga, Nimeshan 1, Menon, Parvathi 1, Loy, Clement1, Yiannikas, Con2, Simon, Neil G3, Kiernan, Matthew 4, Vucic, Steve 1 1 Western Clinical School, University of Sydney, Sydney, Australia, 2Royal North Shore Hospital, St Leonards, University of Sydney, 3Prince of Wales Clinical School, University of New South Wales 4Brain and Mind Research Institute, University of Sydney Objectives: The currently utilized diagnostic Awaji criteria in Amyotrophic Lateral Sclerosis (ALS) can result in a diagnostic delay of up to ten months. Subsequently we utilized a three pronged approach, an individual patient data (IPD) meta-analysis was undertaken, we then undertook the first prospective multicenter study looking at the current diagnostic criteria. Finally we looked at the addition of a novel Transcranial magnetic stimulation (TMS) technique as an objective biomarker of upper motor neuron dysfunction, to further improve the current criteria. Methods: An IPD meta-analysis was performed after communicating with key researchers and authors who published diagnostic studies looking at the Awaji criteria. In total IPD was available for 1086 individuals across 8 published studies. Statistical modelling was undertaken using SAS (9.3). The first prospective multicenter study looking at the Awaji criteria was undertaken in Sydney, Australia, according to the STARD criteria in 369 patients. Finally, a prospective multicenter study was undertaken looking at the diagnostic utility of Threshold tracking TMS in 333 patients. Results: An IPD meta analysis of 1086 individuals revealed that the current Awaji criteria were more. In the multicenter prospective study, the Awaji criteria were compared in a cohort of 369 patients. Specificity was 100% across both criteria. By objectively measuring evidence of UMN dysfunction, an extra 34% of patients could be diagnosed at the first visit. Conclusion: Whilst an individual patient data meta-analysis revealed a better sensitivity for the Awaji criteria when compared to the older rEEC criteria, there were still a number of patients that could not be classified early. The first prospective multicenter study looking at the Awaji criteria, revealed that the sensitivity of the Awaji criteria was lower than expected. Finally by utilizing an objective neurophysiological marker of UMN dysfunction, the diagnosis of ALS could be made earlier, when will then hopefully translate into earlier recruitment into clinical trials, ideally within the critical time frame for therapeutic response. [email protected] Nimeshan Geevasinga Riluzole exerts short-term effects on cortical hyperexcitability in sporadic ALS Geevasinga, Nimeshan 1, Menon, Parvathi 1, Karl Ng2, Kiernan, Matthew C 3, Vucic, Steve 1 1 Western Clinical School, University of Sydney, Sydney, Australia, 2Royal North Shore Hospital, St Leonards, University of Sydney, 3Brain and Mind Research Institute, University of Sydney, Sydney, Australia Objectives: Riluzole is the only neuroprotective agent to date shown to be effective in amyotrophic lateral sclerosis (ALS). The mechanism by which riluzole exerts neuroprotective effects is via antagonism of glutamate, recent studies in ALS patients have established partial normalization of cortical hyperexcitability. Given the modest therapeutic benefits, the duration of riluzoles effect in ALS is probably short and remains to be fully elucidated. Consequently, the present study assessed longitudinal effects of riluzole on cortical excitability in a cohort of sporadic ALS patients. Methods: Studies were longitudinally undertaken on 18 sporadic ALS patients, with cortical excitability assessed at baseline (prior to initiation of riluzole), at 4, 8, and 12 weeks post riluzole initiation. Motor evoked POSTER PRESENTATIONS potentials were recorded over the right abductor pollicis brevis muscle. Statistical analysis was undertaken with a Wilcoxson paired analysis. Results: At baseline, cortical hyperexcitability was evident in the sporadic ALS cohort, as indicated by a marked reduction in SICI (3.5%). Riluzole therapy resulted in a marked increase in SICI at 4 weeks (8.4SEM%, P < 0.01), which was maintained at 8 weeks (9.4% SEM). Interestingly, at 12 weeks, SICI again reduced to baseline values (SICI12 week 4.6%) despite ongoing riluzole therapy. There were no significant changes in other TMS parameters such as resting motor threshold, motor evoked potential amplitude or cortical silent period duration. Conclusion: The findings in the present study indicated that riluzole normalizes cortical excitability in ALS, although this cortical effect is limited. Given that cortical hyperexcitability is an important pathophysiological mechanism in ALS, the modest therapeutic benefits of riluzole could in part be explained by the short-term effects of riluzole on cortical hyperexcitability. Importantly, TMS may be utilised in future clinical trials to assess the effects of potential therapeutic agents on cortical function at early stages of drug development. [email protected] Emily Handley Spine Loss is an Early Pathogenic Event in the TDP-43A315T Mouse Model of ALS Emily E Handley1, Dawkins E1, Clark R1, Turner BJ2, Dickson TC1 and Blizzard CA1 1 Menzies Institute for Medical Research, University of Tasmania 2 The Florey Institute of Neuroscience and Mental Health, University of Melbourne TDP-43 is the major component of inclusions pathologically characterising ALS, with protein mutations identified in familial forms of disease. Whilst previous research has focused on the nuclear role of TDP-43 and toxicity of cytoplasmic aggregates, recent evidence indicates TDP-43 mis-processing may have a pathological synaptic role. The current study aimed to characterise synaptic pathology in TDP43A315T:YFP-H mice at postnatal days 30, 60 and 90. Cortical protein expression was investigated using western blots and immunohistochemistry at P90 with pre- and post-synaptic markers. Dendritic spines were investigated in 20μm coronal sections using confocal microscopy and Neurolucida software. At P90 there was a reduction in the mean number of YFP positive pyramidal neurons (p < 0.05) and in spine density within the TDP-43A315T:YFP-H motor cortex (p < 0.05). Quantification of spine morphology identified specific loss of mushroom spines within the motor cortex at this time point. Additionally, at P90 there was a reduction in spine density within the TDP-43A315T:YFP-H somatosensory cortex (p < 0.05), with no significant loss of YFP positive pyramidal neurons. Alterations at P90 were not associated with changes in global pre- or postsynaptic protein expression. Interestingly, dendritic spine changes were identified between P30 and P60, with reduced spine density in the motor cortex of TDP-43A315T YFP-H mice at P60 prior to cell loss. These findings suggest TDP-43A315T mediated alterations to spine density may play an early role in the progression of ALS. Identifying early occurring disease events is crucial to developing viable therapeutic strategies needed to combat this devastating disease. [email protected] Alison Hogan Generation of novel zebrafish models of MND using traditional and emerging transgenic techniques Alison L. Hogan1, Emily K. Don1, Kelly L. Williams1, Garth A. Nicholson1,2, Nicholas J.Cole1, Ian P. Blair 1 1 Faculty of Medicine & Health Sciences, Macquarie University, Sydney. 2 Concord Clinical School, ANZAC Research Institute, Sydney The only known cause of motor neuron disease (MND) are gene mutations. Ongoing identification of MNDlinked genes allows development of transgenic in vitro and in vivo models that reflect some of the pathological features of the disease. Such models are essential to unravel the pathophysiology of MND, however, no current model adequately mimics the disease. We are developing novel disease models in the zebrafish (Danio rerio), based on recently identified MNDlinked mutations in a E3UL gene. Zebrafish have the advantage of high fertility and fecundity with rapid maturation rates, resulting in efficient model development. We will generate multiple in vivo models using different transgenic techniques: Tol2 system: currently the most commonly used transgenic technique in zebrafish. These models will overexpress the E3UL transgene under a ubiquitous or motor neuron-specific promoter. Preliminary data however, suggests that overexpression of both wildtype and mutant E3UL is toxic in the developing zebrafish. - POSTER PRESENTATIONS - Tet-on system: an inducible system for delayed overexpression of the E3UL transgene until maturity. This strategy is likely to better reflect an adult-onset condition like MND. CRISPR-Cas9 system: a rapidly emerging technique, which allows targeted modification of the endogenous E3UL gene. Mutant E3UL will be expressed at physiologically accurate levels, potentially producing a more phenotypically accurate model. Utilising multiple techniques will provide us with an optimal model for use in functional studies aimed at furthering our understanding of the pathophysiology of MND and provide information that can be used to inform the design of future mammalian models of disease. [email protected] Janine James What’s in a stress granule? Janine James, Jeffery Liddell, Diane Moujalled, Anthony White TDP-43, FUS, hnRNP (etc) are all RNA binding proteins, are all involved in MND and are all involved in stress granules. Stress granule mechanisms are likely to be involved in MND disease pathogenesis. It is not currently known what roles these proteins have in stress granules in conditions of cell stress, and if they all have the same role within the stress granules. By studying stress granules induced by different types of cell stress (energy depletion, heavy metal stress, proteasomal inhibition) we have shown that there are different types of stress granules. This study has also shown that using super-resolution microscopy, there are differing localisations of TDP-43 and FUS within stress granules. The distinct different localisations of TDP-43 and FUS within stress granules suggests that stress granules may have sub-structure, and that FUS and TDP-43 may have different roles within stress granules. [email protected] Tongcui Jiang Neurite outgrowth and spine formation in primary cortical neurons overexpressing human TDP-43A315T T.C Jiang, Handley E.A, Young K.A, Dickson T.C and C.A Blizzard Menzies Institute for Medical Research, University of Tasmania TDP-43, a DNA-/RNA binding protein, is the most frequently aggregated protein in the pathological cytoplasmic inclusions that characterise ALS and mutations to it’s gene cause familial forms of disease. Recent studies have determined that TDP-43 and mutant TDP-43 function may play an important role at the neuronal synapse. We utilised the YFP:TDP-43A315T mouse model to investigate how overexpression of mutant human TDP-43 effects the formation of synapses in vitro. Primary cortical neurons, derived from individual E15.5 embryos were grown to relative maturity, 15 days in vitro (DIV). To investigate axonal outgrowth, WT and TDP-43A315T cortical neurons were grown in microfluidic chambers and live microscopy at 5 DIV was performed. There was no difference (p>0.05) in axonal outgrowth and growth cone areas, indicating that axonal dynamics were not affected by overexpression of the mutant TDP-43. Quantification of dendrites (MAP2 immunolabelling) demonstrated no significant (p<0.05) difference between wild type (WT) and TDP-43A315T primary neurons in total dendrite length, mean length, dendritic branching number and branching order at 3,5,10 and 15 DIV. However, tracing of YFP positive WT and TDP43A315T cortical neurons at 15 DIV demonstrated a significant (p<0.05) decrease in dendritic spines in the TDP-43A315T cortical neurons in comparison to WT controls. These results indicate that the TDP-43A315T mutation may play a role in the formation of postsynaptic structures in cortical neurons. Unravelling the mechanisms that render neurons in the cortico-motor system vulnerable to TDP-43 misprocessing and pathology will be imperative in the pursuit of identifying novel therapeutic interventions. [email protected] Mark Laidlaw A Temporal Association between Accumulated Petrol (Gasoline) Lead Emissions and Motor Neuron Disease in Australia Mark A.S. Laidlaw1*, Dominic B. Rowe2, Howard W. Mielke3, Andrew S. Ball1 1 Centre for Environmental Sustainability and Remediation (EnSuRe), School of Applied Sciences, RMIT University 2 Department of Neurology, Faculty of Medicine and Health Sciences, Macquarie University 3 Tulane University School of Medicine, New Orleans, Louisiana, USA, 70112. POSTER PRESENTATIONS Background: The proportion of total deaths in Australia from motor neuron disease (MND) has increased 337% between 1979 and 2012. It is clear that genetics could not have played a causal role in the increased rate of MND deaths over such a short epoch. We postulate that environmental factors must be responsible for this increase rate. Methods: The association between historical petrol lead emissions and MND death trends in Australia between 1979 and 2011 was examined using linear regression. Results: Regression results indicate positive correlations between a 20 year lag of petrol lead emissions and age-standardised female death rate (R2 = 0.55, p < 0.0001), male age standardised death rate (R2 = 0.48, p < 0.0001) and percent all cause death attributed to MND (R2 = 0.96, p = 1.47 E-23). Conclusion: Historical petrol lead emissions are associated with increased MND death trends in Australia. Further examination of a lag between exposure to petrol lead emissions and MND is warranted. [email protected] Isabella Lambert-Smith A collection of GFP fusion proteins reveals responses in the proteostasis network to TDP-43, FUS and SOD1 in a yeast model of ALS Isabella Ann Lambert-Smith1, 2, 3, 4, Lu Cao3, 4, Daniel Bean3, 4, Darren Saunders5, Steve Oliver3, 4, Justin J. Yerbury1, 2, Giorgio Favrin3, 4 1. Illawarra Health and Medical Research Institute, Wollongong 2. Faculty of Science, Medicine and Health, University of Wollongong 3. Cambridge Systems Biology Centre, University of Cambridge, Cambridge, CB2 1GA, UK 4. Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK 5. Kinghorn Cancer Centre and Cancer Research Program, Garvan Institute of Medical Research, Sydney A striking feature of ALS is its molecular heterogeneity, both in terms of the genes harbouring mutations in different people with ALS, as well as the distinct types of inclusion bodies containing different aggregated proteins. Within this heterogeneity, however, evidence points to a dysfunctional protein homeostasis (proteostasis) environment. There are hundreds of proteins involved in maintaining proteostasis; a crucial step in understanding pathogenesis in ALS is narrowing down on the responses of key molecules and pathways to the ALS gene mutants. This study aimed to screen for genes that are differentially expressed in response to heterologous expression of human TDP-43, FUS and SOD1 in Saccharomyces cerevisiae. DsRed-tagged human wild-type TDP-43, FUS, SOD1 as well as A4V mutant SOD1 were expressed in a selection of 120 strains from the GFP-tagged yeast library; strains were selected based on involvement of the fusion proteins in conserved proteostasis pathways, and pathways suspected to be involved in ALS pathogenesis. Expression of the GFP-tagged genes was monitored using a fluorescence plate reader throughout logarithmic growth and into stationary phase. Using this model we have identified distinct changes in the eukaryotic cell in response to wild-type TDP-43, FUS, SOD1 and A4V SOD1. Pathways implicated are widespread across the proteostasis environment and include transcription, stress granule function, the UPS, ER stress, autophagy, aggresome formation, and the oxidative stress response. These findings point us to commonality in the dysfunction caused in cells by TDP43, FUS and SOD1, as well as to the distinct perturbations caused by each disease protein. [email protected] Linda Lau Establishing plutipotent stem cell-derived spinal motor neurons for screening CSF toxicity in ALS Lau CL1, Niclis J1, Weston RH2, Talman P2 & Turner BJ1 1 Florey Institute of Neuroscience and Mental Health, University of Melbourne 2 Department of Neurosciences, Barwon Health Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer a powerful model system to generate and differentiate motor neurons for screening toxicity and potential therapeutic agents in ALS. The overall aim of this project was to adapt a reproducible protocol to generate human spinal MNs for testing of sensitivity to toxicity of cerebrospinal fluid (CSF) from MND patients and controls.Aims: 1. To establish and generate highly pure mature spinal MNs from hESCs. 2. To evaluate spinal MNs by their morphology (cell body and neurites), neurochemical and transcriptional markers (HB9, Islet1, ChAT, TUJ1) and electrophysiology. 3. To examine the sensitivity of MNs to patient CSF (MND, neurological and non-neurological controls). POSTER PRESENTATIONS Spinal motor neurons were generated and differentiated from H9 hESC cells using a published protocol (Du et al (2014)) which produces MNs in the shortest time frame (over 30 days), includes four differentiation stages. Initial differentiations were carried out at different cell densities to determine the optimal density for differentiation. Immunocytochemistry for SOX1 and HOXA3 revealed higher densities were optimal for differentiation. Preliminary data shows neuroepithelial cells (NEPs) were successfully induced from H9 hESCs, which stain positive for SOX1 and these NEPs were caudal NEPs, staining positive for HOXA3. These cells were then successfully differentiated into MN progenitors (MNPs), which stained positive for OLIG2. These MNPs can be passaged and expanded at least twice and stained positive for OLIG2 and showing similar morphology with initial differentiation. Differentiations were then continued for another 17 days and staining showed CHAT and TUJ1 positive cells. Cell vulnerability to CSF samples from MND, neurological and non-neurological control patients was examined using PI/Annexin V staining. [email protected] John Lee Opposing roles for complement C3aR and C5aR1 in the disease progression of hSOD1G93A mice John D. Lee1, Peter G. Noakes1, 2 and Trent M. Woodruff1 1 School of Biomedical Sciences, the University of Queensland 2 Queensland Brain Institute, the University of Queensland The complement system, which is an integral component of innate immunity, has been implicated in the pathogenesis of motor neuron disease (MND). Complement activation generates the bioactive fragments C3a and C5a, which modulate the inflammatory response by binding to their receptors C3aR and C5aR1. Our previous studies in hSOD1G93A rats and mice demonstrated that C5aR1 has a pro-inflammatory function. However, the contribution of C3aR to disease progression in MND is still unknown. The current study aimed to extend the findings of C5aR1 and determine the function C3aR in the disease progression of MND using hSOD1G93A mice lacking C5aR1 (hSOD1G93AxC5aR1-/-) and C3aR (hSOD1G93AxC3aR-/-). Behavioral tests were conducted to observe any differences in motor symptoms with gliosis and cytokine expressions also measured in these animals. This was done using quantitative PCR at defined disease stages. There was an increase in survival of hSOD1G93AxC5aR1-/- mice, whereas hSOD1G93AxC3aR-/- mice showed significantly reduced survival relative to hSOD1G93A mice. There was an improvement in behavioral deficits in hSOD1G93AxC5aR1-/- mice. By contrast, hSOD1G93AxC3aR-/- mice showed worsened behavioral deficits compared to hSOD1G93A mice. Levels of gliosis and cytokine expressions were reduced in hSOD1G93AxC5aR1-/- mice, whereas there was increased gliosis in hSOD1G93AxC3aR-/- mice relative to hSOD1G93A mice. These results indicate that C3a and C5a generated during complement activation have opposing anti- and pro-inflammatory activities during disease progression in hSOD1G93A mouse. Hence modulating complement activation in MND should ideally be targeted towards downstream C5a inhibition, in order to avoid blocking any endogenous protective effects of upstream factors such as C3a. [email protected] Jeffrey Liddell Activation of Nrf2 is implicated in the disease-attenuating activity of CuII(atsm) Liddell JR1, Kanninen KM2, Duncan C1, Vähätalo S2, Kärkkäinen V2, Koistinaho J2, Crouch PJ1, White AR1. 1 Department of Pathology, University of Melbourne 2 Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland We have demonstrated that diacetyl-bis(4-methylthiosemicarbazonato)CuII (CuII(atsm)) significantly attenuates disease symptoms and improves survival in multiple animal models of amyotrophic lateral sclerosis (ALS). The concomitant decrease in oxidative damage and astrogliosis in these models is consistent with activation of the neuroprotective transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), responsible for regulating a multitude of antioxidant genes. This study seeks to elucidate the involvement of Nrf2 in the neuroprotective effects of CuII(atsm). We find that CuII(atsm) induces nuclear accumulation of Nrf2 in cultured astrocytes. This is accompanied by induction of Nrf2 targets including heme oxygenase 1 and glutamate cysteine ligase, the latter increasing the cellular content and export of the critical antioxidant glutathione from astrocytes, but not neurons, indicating glial-specific activation of Nrf2. These effects are conserved in cells of human origin, but blocked in astrocytes cultured from Nrf2-deficient mice. Coadministration of the cell-permeant metal chelator TPEN blocks the induction of Nrf2 and its downstream targets, whereas the copper-free H2(atsm) ligand has no effect, indicating delivery of bioavailable copper is POSTER PRESENTATIONS required for induction of Nrf2. These results demonstrate that CuII(atsm) activates the transcription factor Nrf2 and upregulates antioxidants in cultured astrocytes. Finally, analysis of spinal cord and brain tissue from transgenic ALS-model mice shows CuII(atsm) administration increases Nrf2 mRNA and decreases its negative regulator Keap1. These actions may contribute to the neuroprotective and disease attenuating activity of CuII(atsm) observed in vivo, and indicates that Nrf2 may be a valuable therapeutic target for the treatment of ALS. [email protected] Luke McAlary Unfolding the Mystery: Common Misfolded Intermediates Captured with Mass Spectrometry Luke McAlary1,2, Andrew Aquilina, Justin Yerbury1,2 1. Biological Sciences, University of Wollongong 2. Illawarra Health and Medical Research Institute Mutations in the cytosolic, superoxide scavenging enzyme Superoxide dismutase 1 (SOD1) are implicated in amyotrophic lateral sclerosis. Mutations in SOD1 are thought to lead to misfolding and aggregation. Due to the number of mutations that are all associated with ALS, it is believed that there is a common misfolded intermediate responsible for cellular dysfunction and death. The aim of our work is to investigate the unfolded intermediate states that SOD1 can potentially occupy when destabilised through mutation, metal loss, and disulphide reduction. To achieve this we purified recombinant SOD1 variants associated with ALS and unfolded their native forms using EDTA and DTT. After unfolding, the variants were examined using analytical gel-filtration chromatography to assess oligomeric distribution. Native mass spectrometry coupled with ion-mobility was then utilized to deconvolute the mixed structural species present for each variant. Analytical gel-filtration chromatography showed that variants treated with DTT and EDTA had an increased population of monomeric SOD1. When fractions across the peak were analysed using native mass spectrometry, it was revealed that unfolded monomeric species were present at the typical dimeric elution volume for nearly all variants, although the relative abundance of these unfolded monomers was different for each variant. We attribute the different abundance of the unfolded monomeric species in each SOD1 variant to the differential susceptibility of said variants to disulfide reduction and metal chelation. We propose that this differential susceptibility to reduction and chelation may explain the differences in aggregation propensity, and as a consequence SOD1 variant dependent patient survival time. [email protected] Emily McCann Genetic analysis workflow in the Macquarie University MND Research Centre Emily P. McCann1, Kelly L. Williams1, Jennifer A. Fifita1, Lorel Adams1, Dominic B. Rowe1 and Ian P. Blair1 1 Faculty of Medicine and Health Sciences, Macquarie University Approximately 10% of all MND cases present with a family history of the disease, with the remainder occurring sporadically. To date, gene mutations are the only known cause of MND. One third of familial cases, and 5% of sporadic cases, are attributable to hexanucleotide repeat expansions in C9ORF72. A further 10-20% of MND families harbour pathogenic mutations in SOD1. At the Macquarie University MND Research Centre, we have implemented a genetic analysis workflow that begins with blood collection in the clinic and automated DNA extraction. DNA samples collected from all MND patients are screened for pathogenic expansions in C9ORF72. Familial MND samples are also screened for mutations in SOD1. We have developed optimised screening procedures for both genes, combining repeat primed PCR and custom TaqMan SNP genotyping for C9ORF72 and modified PCR protocols followed by direct sequencing for SOD1. Mutation screening of new Australian MND families (n=24) and sporadic MND cases (n=110), has resulted in the identification of 3 MND families (11.1%) and 2 sporadic cases (1.8%) with a pathogenic expansion in C9ORF72. Subsequent genealogy investigation of one of these sporadic cases identified a first-degree relative with dementia. Five MND families (18.5%) were determined to have a causative SOD1 mutation. Surprisingly, we have observed significantly lower proportions of C9ORF72 repeat expansions than the values reported in the literature for Australian MND cases. Identifying causative gene mutations in both familial and sporadic MND patients is crucial as it allows patients to be classified into appropriate downstream research cohorts. [email protected] POSTER PRESENTATIONS Diane Moujalled Phosphorylation of hnRNP K by cyclin-dependent kinase 2 controls cytosolic accumulation of TDP-43 Diane Moujalled1, Janine James1, Jeffrey Liddell1, Shu Yang2, Katherine Zhang2, Tobias M. Boeckers3, Christian Proepper3, Ian Blair2 and Anthony R. White1 Affiliation(s): 1. Department of Pathology, The University of Melbourne, Victoria 2. Australian School of Advanced Medicine, Macquarie University 3. Institute of Anatomy and Cell Biology, Ulm University, Germany Tar DNA binding protein 43 kDa (TDP-43) belongs to the family of heterogenous nuclear ribonucleoproteins (hnRNPs) and has been identified as the major pathological protein of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U). Phosphorylation of hnRNPs by kinases may be a central factor in driving neurodegeneration in ALS by altering the physiological components of the hnRNPs and affecting protein–protein interactions, which may have deleterious effects pathologically. In the present study, we used cell models and human ALS spinal cord motor neurons to investigate how Cyclin dependent kinase-2 (CDK2) modulates the accumulation of TDP-43. We found that selective inhibitors of CDK2 abrogated the cytosolic accumulation of TDP-43. Inhibition of CDK2 phosphorylation blocked phosphorylation of hnRNP K, preventing its incorporation into Stress granules (SGs). Confirmation of the importance of hnRNP K in TDP-43-positive SG formation was obtained through mutation of key phosphorylation sites (Ser216 and Ser284) on hnRNP K, this inhibited hnRNP K and TDP-43 colocalisation to SGs. siRNA-mediated inhibition of hnRNP K expression attenuated TDP-43 positive SGs. Analysis of patient motor neurons from sporadic and familial ALS cases demonstrated a substantial decrease in CDK2 and hnRNP K expression. Our findings are the first to ascribe a prominent change to hnRNP K and CDK2 in human ALS spinal cord tissue. This demonstrates a potential key role for these proteins in ALS and TDP-43 accumulation. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention. [email protected] Jane Parkin Kullmann ALS Quest: Initial Results from an Internationally Accessible Web-Based Questionnaire to Discover Risk Factors for Motor Neurone Disease Roger Pamphlett, Discipline of Pathology, Sydney Medical School Jane Parkin Kullmann, Brain and Mind Centre, The University of Sydney Epidemiological studies of motor neurone disease (MND) using paper-based questionnaires have failed to find widely accepted risk factors for the disease. Online questionnaires have considerable advantages over paper-based ones, but few online epidemiological studies into neurodegenerative diseases have been undertaken. We have designed a Web-based questionnaire to identify environmental risk factors for MND that can be used to compare risk factors in multiple countries. New and modified questions have been added from the Australian MND DNA Bank paper-based questionnaire, from literature searches, and from validated MND questionnaires supplied by other investigators. The questionnaire addresses many risk factors that have already been proposed for MND, as well as a number that have not yet been rigorously examined, such as stress, personality type, and dental history. The survey is being translated into a number of languages which will allow most people around the world to answer in their own language. After the questionnaire had been online for 4 months it had 379 respondents, compared to only 46 respondents for the same initial period using a paper-based questionnaire. Preliminary results from an analysis of the data are in line with previously-identified risk factors for MND such as exposure to certain chemicals and prenatal exposure to testosterone (as indicated by index to ring finger ratios). This questionnaire has the potential to identify robust MND risk factors especially when they are found in multiple countries. [email protected] Nirma Perera Rilmenidine increases mTOR-independent autophagy enhancing disease progression in a mouse model of MND Perera ND1, Sheean RK1, Beart PM1, Horne MK1, Turner BJ1 1 Florey Institute of Neuroscience and Mental Health, University of Melbourne Autophagy eliminates misfolded proteins and damaged organelles linked to neurodegeneration. Rilmenidine, a clinically approved imadazoline receptor agonist, induces autophagy allowing clearance of mutant huntingtin (1). Misfolded superoxide dismutase 1 (SOD1) accumulates in motor neurons in MND. Induction of autophagy with rilmenidine is a potential therapeutic approach to enhance POSTER PRESENTATIONS degradation of mutant SOD1 as imadazoline receptors are expressed by spinal cord motor neurons. We aimed to profile autophagy induced by rilmenidine and its impact upon disease progression in SOD1 G93A mice. SOD1G93A mice received rilmenidine (10 mg/ml, ip, 4x week) from 60 days of age. Mice were examined for weight loss, motor function and survival. Spinal cords and brains were analysed for expression of autophagy markers, motor neuron survival, glial cell activation, and mutant SOD1 level and aggregation. Rilmenidine upregulated microtubule associated light chain-3 II and reduced voltage-dependent anion channel 1 levels in spinal cord, indicative of autophagy activation. Soluble mutant SOD1 levels were diminished in spinal cords of rilmenidine treated SOD1G93A mice consistent with autophagy. Despite this, disease onset was unaltered by rilmendine treatment, survival was significantly reduced (P<0.05) and disease progression accelerated in male SOD1G93A mice. Rilmenidine induces mTOR-independent autophagy and enhances disease progression in mutant SOD1 mice, suggesting that autophagy activation contributes to pathology in this MND model. (1) Rose C et al (2010) Hum Mol Genet 19: [email protected] Mona Radwan Determining the pathomechanism of C9ORF72 Repeat-Associated Non-ATG (RAN) translation products Mona Radwan1, Bradley J Turner2, Gavin Reid1 and Danny M Hatters1. 1 Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne 2 Florey Institute of Neuroscience and Mental Health, University of Melbourne; Centre for Neuroscience, University of Melbourne GGGGCC repeat-expansion mutations in the first intron of the C9ORF72 gene are the leading genetic causes of motor neuron disease (MND) and frontotemporal dementia (FTD). The mutations lead to bidirectional transcription and non-canonical translation in 6 reading frames, which include 5 different dipeptide repeat polymers (DPRs). These DPRs accumulate in human brain and have recently been suggested to be the major cause of dysfunction in animal models of disease. Our goal was to determine the mechanism of toxicity caused by each of the individual DPRs (polyGA, GR, AP, PR and GP) using cell biology, RNA transcriptome sequencing and proteomics approaches. Using artificial cDNA constructs of short (10 repeats) and long (101 repeats) DPR sequences, we have characterized their impact to neuroblastoma cell culture models of disease. PolyGA remained diffusely throughout the cytoplasm in the short length but formed cytoplasmic aggregates in the expanded form. PolyGR localized predominately in the nucleus as puncta in short length but became restricted to the cytoplasm in expanded form. PolyPR formed puncta in the nucleus for both short and expanded forms. PolyAP and GP proteins remained diffusely distributed throughout the cytoplasm in both lengths. All of the expanded forms led to a reduction in survival rates, most notably the arg-rich DPRs. The interactome of the arg-rich DPRs included proteins involved in ribosome biogenesis, RNA-processing and spliceosome suggesting these DPRs interfere with RNA processing machinery. We anticipate our efforts to yield a better understanding of the fundamental modes of toxicity and hence enable new therapeutic targets to be identified. [email protected] Sarah Rea SQSTM1 mutations associated with ALS, Frontotemporal dementia and Paget’s disease of bone lead to dysfunctional autophagy and altered cell signalling Melanie Sultana*1,2, Nathan Pavlos3, Alice Goode4, Robert Layfield4, Sarah Rea*1,2 1 Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital 2 Harry Perkins Institute of Medical Research, University of Western Australia 3 School of Surgery, University of Western Australia 4 School of Biomedical Science, University of Nottingham *indicates that these authors contributed equally to the work presented Background: Mutations in the SQSTM1/p62 (p62) gene have been reported as associated with Frontotemporal Dementia (FTD) and the motor neurone disease amyotrophic lateral sclerosis (ALS). p62 is a “ubiquitin” or “autophagy” receptor and is one of several such receptors implicated in FTD/ALS, Paget’s disease of bone (PDB) or the rare syndrome of “Inclusion body myopathy with FTD and PDB”. p62 has regulatory roles in various cell signalling events including; transcription factor activation and apoptosis, protein degradation by both autophagy and the ubiquitin proteasome system. Methods: We used luciferase assays to determine the affect of mutant p62 expression on NF-stimulated cells. We investigated POSTER PRESENTATIONS p62/LC3 interaction by co-immunoprecipitation and confocal microscopy. Additionally, we investigated affects on autophagosome maturation into protein degrading autophagolysosomes using mCherry-GFP-LC3 with p62 constructs and confocal microscopy to determine the ratio of immature autophagosomes (red) and mature autophagolysosomes (yellow) that formed in cells expressing either mutant or wild type p62. Results and Conclusions: We observed that p62 UBA mutants significantly increase NFphysical interaction between p62 and the autophagy marker LC3. We also observe that mutant proteins attenuate autophagosome maturation. By contrast, expression of wild type SQSTM1/p62 promotes autophagosome maturation. Together, our data suggests p62 UBA mutant proteins are defective mediators of the final stages of autophagy and this may be important for NFspecific protein substrates. Dysfunctional autophagy and NFneurodegeneration. Our study provides insights into pathologies associated with mutations in p62. [email protected] Mary-Louise Rogers Target gene delivery to neonatal p75NTR expressing motor neurons Kevin S Smith1, Mary-Louise Rogers1 1 Department of Human Physiology, Centre for Neuroscience, Flinders University Targeted gene therapy for MND and the childhood spinal muscular atrophy (SMA) have huge potential. Our group has developed immunogenes comprising an internalizing antibody to a cell surface receptors conjugated to a carrier which can bind and condense DNA/RNA. The neurotrophin receptor-p75 (p75NTR) is highly expressed in neonatal motor neurons highlighting the potential entry point for SMA treatments, as well as damaged adult motor neurons, including MND. Our current immunogene consists of an antibody to p75NTR (MLR2) conjugated to a PEGylated polyethylenimine (PEIPEG12), which upon binding plasmids e.g pVIVO2-GFP forms immunogene-MLR2-PEI-PEG12-pVIVO2GFP. We aimed to characterise the ability of this immunogene to transfect neonatal motor neurons. Methods: Embryonic motor neurons were isolated from C57BL/6J (B6) mice and GFP expression checked after application of MLR2-PEI-PEG12-pVIVO2. p75NTR expressing motor neurons were quantified from PND1-7 and 14 in B6 mice. Finally, neonatal mice were injected intraperitoneally (i.p) with MLR2-PEI-PEG12pVIVO2 or PEI-PEG12-pVIVO2 and ChAT positive motor neurons checked for GFP. Results: MLR2-PEI-PEG12-pVIVO2-GFP transfected primary motor neurons with GFF. p75NTR expression was highest at PND1 (92.6%,n=3). Transfection of 25% of spinal motor neurons (n=5 mice) identified by ChAT also expressed GFP 48h after i.p injections at PND1 of MLR2-PEI-PEG12-pVIVO1-GFP. PEI-PEG12pVIVO2-GFP was unable to transfect lumbar motor neurons, indicating p75NTR is requisite Conclusions: We have shown that MLR2-PEI-PEG12-pVIVO2 has the ability to specifically transfect p75NTR expressing motor neurons, in neonatal mice. Further work is needed to apply this technique to SMA and MND mice for delivery of therapeutic genes. [email protected] Darren Saunders Defining the Neuronal Ubiquitome in ALS Jessie McKenna1, Phil Poronnik4, Kristen Mitchell3, Monique Bax3, Sharad Kumar5, Natasha Boase5, Natasha Harvey5, Lezanne Ooi3, Justin Yerbury3, Darren Saunders1,2. 1 Kinghorn Cancer Centre, Garvan Institute of Medical Research; 2School of Medical Sciences, UNSW; 3Illawarra Health and Medical Research Institute, University of Wollongong; 4School of Medical Sciences, University of Sydney; 5Centre for Cancer Biology, Uni SA Genetic and biological evidence implicates dysfunction of the Ubiquitin Proteasome System (UPS) in the etiology of ALS. A hallmark of ALS pathology is the accumulation of ubiquitin-tagged protein aggregates within motor neurons. We hypothesise that mutations in ALS genes disrupt Ub homoestasis, either directly or through sequestration of Ub into protein aggregates. This compromises vital Ub-dependent cellular functions and ultimately results in cell death. We are addressing this hypothesis using a multi-faceted approach, integrating unique model systems with powerful proteomics and functional imaging to define the role of ALS genes in modulating neuronal Ub homeostasis. This approach is providing unprecedented, detailed understanding of Ub homeostasis in cells expressing mutant ALS genes. We have mapped the Ub-modified proteome (ubiquitome) in cultured motor neurons derived from induced pluripotent stem cells (iPSCs), identifying ~1500 ubiquitlyated proteins across various functional pathways including the UPS. By investigating dynamic changes in the ubiquitome of fibroblasts, iPSCs and neurons we have also identified key pathways involved in iPSC reprogramming and neuronal differentiation. Furthermore, ubiquitomics analysis of embryonic mouse brain detected ~1000 ubiquitylated proteins, showing significant POSTER PRESENTATIONS overlap with iPSC-derived neurons. Importantly, we have observed significant changes in the ubiquitome of NSC-34 cells expressing either WT or mutant (A4V) SOD1. Collectively, our data indicate a significant role for Ub in the regulation of a wide variety of cellular functions vital for neuronal function and demonstrate that fALS-associated mutations disrupt Ub homeostasis. Targeting Ub homeostatic pathways might be a promising therapeutic avenue for ALS. [email protected] Rebecca Sheean Expansion of Tregs by interleukin-2/antibody complexes slows disease progression in mutant SOD1 mice Rebecca Sheean1 Fiona McKay2 Erika Cretney3 Stephen Nutt3 Karlene Scheller1,4 Nirma Perera1 Justin Yerbury5 Steve Vucic6 Bradley Turner1 1. Florey Institute of Neuroscience and Mental Health 2. Westmead Millennium Institute 3. Walter and Eliza Hall Institute of Medical Research 4. LaTrobe University, Bundoora, VIC, Australia 5. Illawarra Health and Medical Research Institute, University of Wollongong 6. Westmead Hospital Background: The peripheral immune system is implicated in modulating glial cell activation, motor neuron survival and disease progression in ALS. Activation of T-regulatory cells (Tregs) may be a disease modifier of ALS and transplantation of Tregs is neuroprotective in mutant SOD1 mice. Specific expansion of Tregs over harmful T-effectors was optimised using interleukin-2 (IL-2)/IL-2 antibody (IL-2 mAb) complex with rapamycin (rapa). Here, we investigate the therapeutic potential of IL-2/IL-2 mAb complexes in mutant SOD1 mice. Methods: SOD1G93A mice (P60, n=10 male/female per group) were administered IL-2/IL- mAb complex (1 µg/5 µg) + rapa (1 mg/kg), rapa alone or vehicle 2-3 times weekly. Weight loss, motor function and survival were examined. T-cell populations in peripheral blood and spleen were determined via FACS. Spinal cords were analysed for motor neuron counts and glial activation using immunohistochemistry. Results: Treatment with IL-2/IL-2 mAb + rapa significantly prolonged survival of male SOD1G93A mice (p<0.01) compared to rapa and vehicle groups. IL-2/IL-2 mAb significantly elevated CD4+ FoxP3+ Tregs in blood and spleens of SOD1G93A mice, but not CD8+ T-cells, confirming selective Treg expansion. Furthermore, these Tregs showed increased CTLA4 and GITR expression, consistent with a suppressor phenotype. Lastly, there was reduced glial cell activation in spinal cords of SOD1G93A mice treated with IL2/IL-2 mAb + rapa. Conclusion: We demonstrate IL-2/IL-2 mAb complexes efficiently stimulate selective expansion of suppressor Tregs leading to attenuated glial cell activation, inflammation and increased survival in mutant SOD1 mice. These data suggest that this novel approach may be an effective and feasible therapeutic strategy for ALS. [email protected] Kazumoto Shibuya Underling pathomechanisms of motor neuronal hyperexcitability in sporadic and familial ALS Kazumoto Shibuya,a Susanna B. Park, a, b, c Nimeshan Geevasinga, d William Huynh,a, b Neil G. Simon, c Parvathi Menon, d James Howells, a Nidhi Garg,a Yu-ichi Noto, a Yuta Iwai,e Garth A. Nicholson,f Steve Vucic, d Satoshi Kuwabara,e Matthew C. Kiernana, b, c a Brain and Mind Research Institute, University of Sydney b Neuroscience Research Australia c Prince of Wales Clinical School, University of New South Wales d Westmead Clinical School, University of Sydney e Department of Neurology, Graduate School of Medicine, Chiba University, Japan f Department of Neurology, Concord Hospital, Central Clinical School, University of Sydney Motor neuronal hyperexcitability potentially contributes to motor neuron death in amyotrophic lateral sclerosis (ALS). To reveal pathomechanisms of motor neuron hyperexcitability in ALS, threshold tracking transcranial magnetic stimulation and multiple excitability measurements were performed in 130 healthy controls (HC), 83 sporadic ALS (sALS) patients, 12 patients with superoxide dismutase-1 (SOD-1) mutations, 16 C9orf72 hexanucleotide repeat expansions and 2 FUS mutations. Compared to HC, in peripheral nerve excitability sALS had greater strength-duration time constant (p < 0.05), depolarizing threshold electrotonus (p < 0.0001) and superexcitability (p < 0.05) and smaller late subexcitability (p < 0.05). In cortical excitability, sALS had reduced short interval intracortical inhibition (p < 0.0001). These indices were not significantly different in SOD-1 and C9orf72, compared to sALS. These findings suggest both sALS and familial ALS have increased POSTER PRESENTATIONS sodium currents, decreased potassium currents, rendering to peripheral nerve hyperexcitability. In addition, all ALS patients demonstrated decreased intracortical inhibition. It is already reported that TDP-43 protein increases firing frequency of action potentials in a motor neuron like cell line and mutant SOD motor neurons also demonstrated increased excitability. Motor neuronal hyperexcitability may be a common feature in both sporadic ALS and familial ALS, associated with different pathological basesand, and may be common therapeutic effector site for treatment of ALS, such as riluzole. [email protected] Frederik Steyn Metabolic dynamics in MND: Assessment of metabolic flux in patients reveal breakdown in energy supply and demand Steyn FJ1,2, Ioannides Z1,3, Henderson RD3, Ngo ST1,2,3,5 and McCombe PA1,3,4 1 University of Queensland Centre for Clinical Research, The University of Queensland 2 School of Biomedical Sciences, The University of Queensland 3 Department of Neurology, Royal Brisbane & Women’s Hospital 4 School of Medicine, The University of Queensland 5 Queensland Brain Institute, The University of Queensland Background: Motor Neurone Disease (MND) is a fatal neurological disease for which there is currently no cure. It is thus critical to develop strategies that alleviate or slow disease progression. In MND, metabolic effects have been shown to modify the course of disease. We assessed metabolic changes that may contribute to the progression of disease. Methods: Patients and control subjects were recruited from the MND clinic at the RBWH. After an overnight fast, patients attended the MND research clinic at UQCCR for metabolic assessment. Body composition was assessed using air displacement plethysmography (BodPod; COSMED). Resting energy expenditure (REE) and the thermogenic effect of food [TEF – response to a standardised meal (Sustagen, Nestle)] were assessed by indirect calorimetry using gas exchange analysis (Quark RMR, COSMED). Disease severity was assessed using the Amyotrophic Lateral Sclerosis Functional Rating scale (ALSFRS-R). Results: Hypermetabolism was present in patients with MND. When controlling for fat free mass (FFM), MND patients tend to use more energy relative to a control population. Resting energy use in MND patients increased relative to symptom severity and the TEF of MND patients decreased relative to symptom severity. Conclusions: Results from this study suggest that energy needs in patients escalate with disease severity. In addition, the capacity of patients to digest, absorb, process or store energy may worsen with disease severity. Collective impairments in the capacity of patients to meet energy needs may exacerbate energy deficit, potentially accelerating disease progression leading to early death. [email protected] Jack Stoddart An In Vivo And Behavioural Study Of Als-Associated Protein, Tdp-43 In Transgenic Zebrafish Jack J. Stoddart, Isabel Formella, Andrew P. Badrock, Emily K. Don, Marco Morsch, Garth Nicholson, Roger S. Chung, Nicholas J. Cole. Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, Macquarie University Amyotrophic lateral sclerosis (ALS) is a progressive muscle wasting disorder, characterized by the degeneration of upper and lower motor neurons. There is currently no cure and the disease is fatal 2-5 years post onset. An improved understanding of ALS at a cellular level is essential in order to identify disease pathways, leading to motor dysfunction, that can be targeted pharmacologically. Zebrafish are a unique model organism, enabling in vivo study at such a cellular level. Through ease of genetic manipulation and single-cell resolution, we hope to shed some light on the underlying mechanisms of ALS-associated proteins and possibly even elucidate their role in ALS pathology. The DNA/RNA binding protein, Transactive Response DNA-binding protein 43kDa (TDP-43) is a component of ubiquitinated cytoplasmic inclusions, present in around 97% of ALS cases. A number of causative mutations in the TARDBP gene have also been identified in both familial and sporadic ALS patients. We have developed a stable transgenic zebrafish specifically expressing the zebrafish ortholog of TDP-43 (zTDP-43) in motor neurons. In this study, a mutation associated with sporadic ALS, Q331K, was compared to wild-type TDP-43. We conducted cellular analysis of these transgenic zebrafish, assessing the localization of zTDP-43 in motor neurons, as well as examining possible motor phenotypes. Elucidating these key aspects may better our understanding of ALS and take a step toward unveiling the complex neurobiology underlying the disease. [email protected] POSTER PRESENTATIONS Xin Tan Traumatic brain injury, Motor Neuron Disease, and TDP43 Xin L Tan1, Bradley Turner2, Terence J O’Brien1, Sandy R Shultz1 1 Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, 2 The Florey Institute of Neuroscience and Mental Health Motor neuron disease (MND) is a neurodegenerative condition that is pathologically characterized by the progressive death of motor neurons and the presence of protein inclusions consisting of TAR DNA-binding protein 43kd (TDP-43). Sporadic MND, where the aetiology of the disease remains largely unknown, accounts for the large majority of MND cases. Traumatic brain injury (TBI) has been identified as a risk factor in the development of MND, and motor neuron loss, corticospinal tract degeneration, and TDP-43 pathologies, which are consistent with features of MND, have been observed in individuals with a history of TBI. However, the potential pathological mechanisms linking TBI and MND are poorly understood. Here we administered either a fluid percussion injury or sham injury to transgenic mice that overexpress TDP-43 or wild-type mice. After a one-week recovery, mice underwent behavioural testing to assess cognitive, motor, and emotional impairments before brains were collected for post-mortem analysis. TDP-43 mice given a TBI had worse cognitive and motor deficits compared to their wild-type counterparts. While all mice given a TBI had significant neuronal death, it was worse in TDP-43 mice given a TBI. Notably, all mice given a TBI also had increased expression of phosphorylated TDP-43 relative to their sham-controls, with TDP-43 mice given a TBI having more than all other groups. We are currently conducting analyses to further probe other TDP-43 pathologies after TBI, however these initial finding suggest that TDP-43 phosphorylation may be detrimental in the aftermath of TBI. [email protected] Hannah Timmins Identifying links between neurophysiological markers and patient reported outcomes in ALS Hannah C. Timmins1, Benjamin C. Cheah 2, Cindy S.Y Lin 3, Steve Vucic 4, Matthew C. Kiernan 1, Susanna B. Park 1 1 Brain and Mind Centre, University of Sydney 2 Neuroscience Research Australia and Prince of Wales Clinical School, University of New South Wales 3 School of Medical Sciences, University of New South Wales, 4 Westmead Clinical School, University of Sydney Objective: To identify links between neurophysiological and functional outcome measures in ALS. Methods: Clinical function was assessed in 53 ALS patients (age 54±10.3 years) via ALSFRS-r scale, hand grip strength and Forced Vital capacity (FVC). Compound muscle action potential (CMAP) amplitude, F-wave persistence, neurophysiological index (NI) were measured from the ulnar nerve, recording at the Abductor Digiti Minimi (ADM) muscle. Results: The total ALSFRS-r scale demonstrated moderate positive correlations with CMAP amplitude (r =.354, p =.016), F-wave persistence (r =.362, p =.014) and NI (r =.431, p =.003). Measures of neurophysiological function were strongly positively correlated with the fine-motor subscale of the ALSFRS-r scale (CMAP r =.579, p <.001, NI r =.579, p <.001). Average grip strength showed a strong positive correlation with CMAP amplitude (r =.553, p < .001), and NI (r =.570, p < .001), and a moderate positive correlation with F-wave persistence (r =.300, p =.040). FVC demonstrated a moderate positive correlation with CMAP amplitude (r =.302, p =.041), and NI (r =.386, p =.008). Conclusions: Neurophysiological measures correlate with functional outcomes in ALS, specifically those relating to fine-motor function. Significance: Identifying links between objective neurophysiological markers and patient reported outcomes are important to assess the impact of clinical interventions. [email protected] POSTER PRESENTATIONS Mehdi Van den Bos Cortical hyperexcitability in ALS: A heterogeneous process Mehdi A J van den Bos MBBS1, Parvathi Menon PhD1, 2, Nimeshan Geevasinga MBBS1, 2, Matthew C Kiernan DSc2, 3, Steve Vucic PhD1, 2 1 Department of Neurology, Westmead Hospital; 2 Westmead Clinical School, University of Sydney; 3 Brain and Mind Centre, University of Sydney Objective: Cortical hyperexcitability, as heralded by reduced short interval intracortical inhibition (SICI) has been identified as an intrinsic feature of amyotrophic lateral sclerosis (ALS). However, in approximately 20% of patients cortical excitability may be normal, suggesting the presence of pathophysiological heterogeneity. Consequently, the present study assessed for the presence of clinical, functional and neurophysiological differences in ALS phenotypes, as defined by cortical hyperexcitability. Methods: Cortical excitability was assessed in 62 ALS patients. Clinical, functional and neurophysiological parameters were compared between ALS patients with cortical hyperexcitability (SICI < 5.5%) versus those with a “normal” level of cortical excitability (SICI > 5.5%). Results: In ALS patients with features of cortical hyperexcitability, there appeared to be an absence of SICI in the early phase (interstimulus interval [ISI] 1-3 ms) with a transition to facilitation at later ISIs (3.5-7 ms). In contrast, ALS patients with normal SICI demonstrated similar levels of inhibition across early and late SICI phases. Of further relevance, ALS patients with abnormal SICI exhibited increased mortality in follow-up. Conclusion: Dysfunction in cortical inter-neuronal circuits probed by short latency paired-pulse TMS appears to capture some of the pathophysiological heterogeneity in ALS and may have direct implications for survival. [email protected] Adam Walker Clearance of pathological cytoplasmic TDP-43 allows functional recovery in new mouse models of sporadic ALS/F Adam K. Walker1, Krista J. Spiller1, Guanghui Ge1, Allen Zheng1, Yan Xu1, Melissa Zhou1, Kalyan Tripathy1, Linda K. Kwong1, John Q. Trojanowski1,2, Virginia M.-Y. Lee1,2 Macquarie University Accumulation of phosphorylated cytoplasmic TDP-43 inclusions accompanied by loss of normal nuclear TDP-43 in neurons and glia of the brain and spinal cord are the molecular hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the role of cytoplasmic TDP-43 in disease pathogenesis remains unclear, due in suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (∆NLS) under the control of the neurofilament heavy chain (NEFH) promoter. Expression of hTDP-43∆NLS in these ‘regulatable NLS’ (rNLS) mice resulted in the accumulation of insoluble, phosphorylated cytoplasmic TDP-43 in brain and spinal cord, loss of endogenous nuclear mouse TDP-43 (mTDP-43), brain atrophy, muscle denervation, dramatic motor neuron loss, and progressive motor impairments leading to death. Notably, suppression of hTDP-43∆NLS expression by return of Dox to rNLS mice after disease onset allowed clearance of TDP-43 pathology, increased in nuclear mTDP-43 levels, and prevented further motor neuron loss. rNLS mice back on Dox also showed a significant increase in muscle innervation, rescue of motor impairments, and dramatic extension of lifespan. Importantly, even after neurodegeneration and onset of motor dysfunction, removal of cytoplasmic TDP-43 and the concomitant return of nuclear TDP-43 led to neuron preservation, muscle reinnervation and functional recovery. Clearance of pathological TDP-43 is therefore beneficial even at late disease stages, indicating that targeting cytoplasmic TDP-43 is a worthy avenue for therapeutic development in ALS and FTLD-TDP. [email protected] Robyn Wallace Direct Conversion of Adult stem cells to induced neuronal and Muscle cells Padraig Strappe, Gayle Petersen, Nicole Dawson, Robyn Wallace School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW Development of a patient-derived in vitro disease model for MND will allow screening of novel therapeutics, assessment of potential MND-causing genes and to further our understanding of disease mechanisms. A rapid direct reprogramming technique has been developed using lentiviral vector delivery of key transcription factors to produce neuronal like cells and separately skeletal muscle cells through trans-differentiation of adult bone marrow and adipose derived stem cells. Delivery of Brn2, Ascl1, and Mytl1 (BAM) transcription factors to adult stem cells resulted in the formation of induced neuronal cells with typical bipolar morphology and formation of extensive networks together with POSTER PRESENTATIONS specific expression of the beta-III-Tubulin protein and increased gene expression of Map2 and synapsin. The conversion efficiency was typically 15% of the total cell population, which was further enhanced through a combination of a third transcription factor (NeuroD1) and the use of an extracellular matrix substrate and culture in a novel 3D scaffold. Myoblast like cells were produced through MyoD1 over-expression in adult stem cells, resulting in the generation of elongated and fused multinucleated cells co-expressing the muscle specific filament protein Desmin. The next step will be to include motor neuron specific factors to produce functional motor neurons with the potential to form neuromuscular junctions and ultimately to use MND patient derived cells. Compared to current methods that require conversion to induced pluripotent stem cells (iPSCs), this transdifferentiation technique has the advantage of being faster, more efficient and involves less genetic modification of cells. [email protected] Daniel Whiten Clusterin ameliorates TDP-43 pathology Daniel Whiten, Mark Wilson Illawarra Health and Medical Research Institute, University of Wollongong Clusterin is a normally secreted glycoprotein that functions as a molecular chaperone in extracellular fluids. It has also been suggested that clusterin influences several aspects of intracellular proteostasis, such as autophagy by stabilising the LC3-Atg3 heterocomplex. Here, we show that clusterin is able to protect against TDP-43 pathology in cultured neurons (Neuro-2a). TDP-43 is heavily implicated in the pathology of motor neuron disease, a fatal and untreatable neurodegenerative disease characterised by the presence of cytoplasmic inclusions. In an ER stress dependent manner, the overexpression of clusterin was found to reduce the number of inclusions formed by M377V TDP-43-GFP. This effect was still observed when either autophagy or the ubiquitin-proteasome system was inhibited. Clusterin was also found to co-localise with TDP-43-GFP and LC3 in autophagosomes, as well as with both M377V and C-terminal fragment (Δ216414) TDP-43-GFP in inclusions. Additionally, clusterin was shown to inhibit the aggregation of full-length TDP-43 in vitro. These results suggest that clusterin is able to protect against the misfolding of both intracellular and extracellular proteins. Therefore, it appears likely that clusterin plays a role in the pathogenesis of a wider range of diseases than previously believed. [email protected] Rafaa Zeineddine SOD1 protein aggregates activate micropinocytosis in neurons to facilitate their entry Rafaa Zeineddine1, Dr Justin Yerbury1 1 Illawarra Health and Medical Research Institute, University of Wollongong Faculty of Science, Medicine and Health, School of Biological Sciences, University of Wollongong Amyotrophic lateral sclerosis is an enigmatic adult-onset neurodegenerative disease that is characterized by focal onset of symptoms and spread of molecular pathology that has been likened to the transmission of the infectious prion protein. Cell-to cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated. Using motor neuron-like cell lines, we demonstrate that aggregates of SOD1 interact with the cell surface triggering the activation of the Rho GTPase Rac1, leading to membrane ruffling and fluid phase uptake in neurons that defines macropinocytosis and that this facilitates aggregate entry into cells. In addition, we show that these aggregates can escape macropinosomes to be deposited in the cytosol. We also show that SOD1 aggregates are capable of transferring from cell-to-cell allowing propagation of aggregation to proceed. Thus, we conclude that in addition to basic proteostasis mechanisms, pathways involved in the activation of macropinocytosis are key determinants in the spread of pathology in these misfolding diseases, similar to the prion protein. [email protected] POSTER PRESENTATIONS Katharine Zhang Functional pipeline for determining pathogenicity of candidate gene mutations causing motor neuron disease Katharine Y. Zhang1, Jennifer A. Fifita1, Shu Yang1, Kelly L. Williams1, Garth A. Nicholson1,2, Dominic B. Rowe1 and Ian P. Blair1 1 Faculty of Medicine & Health Sciences, Macquarie University 2 Molecular Medicine Laboratory, Concord Hospital Gene mutations are currently the only known cause of MND. Around 10% of MND cases demonstrate inheritance of disease with known gene mutations accounting for two-thirds of these familial MND cases. The identification of new MND-linked genes provides further insight into the cellular processes contributing to pathology, such as the formation of TDP-43-positive aggregates in affected neurons. Our disease gene discovery pipeline includes analysis of whole exome sequence data from Australian familial MND cases using custom bioinformatics strategies and scripts to produce lists of novel candidate gene mutations, followed by in silico prioritisation to identify high priority variants. The pipeline moves from genetics to cell biology through functional-based studies of candidate genes to identify specific mutations resulting in MND pathology. In vitro strategies involve mutant gene expression in neuronal cell lines to investigate the effect of candidate mutations on cell viability and candidate protein mislocalisation. Specific to MND pathology, co-expression of mutant genes with TDP-43 allows for examination of characteristic TDP-43 co- and mis-localisation. Patient fibroblasts are used to examine whether candidate proteins undergo changes such as altered expression level and mislocalisation. Furthermore, the presence of aggregates positive for candidate proteins is also examined in patient spinal cord sections. In one Australian MND family, the genetics workflow has identified five potential causative gene mutations. The in vitro functional pipeline is currently underway to pinpoint the disease-causative gene. This combined pipeline facilitates the identification of new disease genes, providing further diagnostic tools and insight into disease mechanisms. [email protected]