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Transcript
MND Australia Research Meeting 21 October 2016 The Queensland Brain Institute The University of Queensland QLD 4072 Generously supported by: FRIDAY 21 OCTOBER ORAL PRESENTATIONS 9.00 am Registration 9.30 am Welcome David Ali, President, MND Australia 9.40 am Opening address Professor Pankaj Sah, Director, Queensland Brain Institute, The University of Queensland 9.50 am Introduction Professor Matthew Kiernan, Chair, MNDRIA Research Committee 10.00 am – 11.15 am Session 1 Chair: Professor Simon Foote Dr Nicholas Cole, NSW Elucidating the pathogenesis of sporadic motor neurone disease using zebrafish models Dr Adam Walker, NSW Investigating disease processes in a new TDP-43 mouse model of MND A/Prof Greg Neely, NSW A functional genomic approach to identify new motor neuron disease genes and drug targets Novel stress responsive subnuclear bodies, paraspeckles, play a role in neuronal cell health and may be part of a common pathway perturbed in MND DNA damage and neurodegeneration in ALS Dr Archa Fox, WA A/Prof Julie Atkin, NSW 11.15 am – 11.40 am Morning Tea 11.40 am – 1.00 pm Session 2 A/Prof Danny Hatters, VIC Chair: Associate Professor Peter Noakes Dr Marco Morsch, NSW A new method to measure proteostasis capacity: a potential biosensor for pre-onset neurodegenerative disease Identifying mechanisms responsible for ubiquitylation of TAR DNA-binding protein 43 (TDP-43) in ALS pathogenesis Transfer of ALS protein aggregates in the zebrafish spinal cord Dr Darren Saunders, NSW Disrupted ubiquitin homeostasis in ALS Dr Albert Lee, NSW Dr Shu Yang, NSW 1.00 pm – 2.00pm Lunch Aberrant protein degradation as a common pathogenic mechanism in MND 2.00pm – 3.30 pm Chair: Associate Professor Tracey Dickson Session 3 Dr Peter Crouch, VIC Copper malfunction is common to sporadic MND and animal models of familial MND A/Prof Peter Noakes, QLD Dysregulation of complement in a new mouse model of MND, and the therapeutic effect of selective C5aR1 antagonism in hSOD1G93A MND model mice The relationship between traumatic brain injury and motor neurone disease, and their potential treatment with a PERK-inhibitor Development of novel immunogenes to improve growth factor support for motor neurons Development of an immunogene delivery approach for SMN restoration in MND Dr Sandy Schultz, VIC Dr Mary-Louise Rogers, SA Dr Bradley Turner, VIC Dr Justin Yerbury, NSW 3.30pm – 4.00 pm Afternoon Tea 4.00 pm – 5.30.pm Session 4 A/Prof Tracey Dickson, TAS Dr Catherine Blizzard, TAS Dr Nimeshan Geevasinga, NSW Dr Michael Lee, NSW Dr Derik Steyn, QLD 5.30 pm Development of a biocompatible functionalised liposome drug delivery system to increase efficiency of delivery to motor neurons Chair: Professor Dominic Rowe Subtype-specific alteration of inhibitory circuits in the primary motor cortex in motor neuron disease: a cellular basis for cortical pathophysiology TDP-43 mediated synaptic alterations in the pathogenesis of ALS Cortical excitability in familial C9orf72 ALS patients Is resistance exercise therapeutic in ALS? A pilot study of exercise-induced fatigue on cortical excitability Metabolic and gut dynamics in MND: Identifying novel strategies to meet energy needs in MND Close 6.00 pm – 8.00 pm POSTER SESSION AND DRINKS RECEPTION POSTER PRESENTATIONS Name and Poster No. Institution Title 27. Dr Beben Benyamin Queensland Brain Institute, University of Queensland School of Biomedical Sciences, University of Queensland Trans-ethnic methylome-wide association analysis of ALS and its clinical phenotypes Cortical neuronal glucose metabolism is impaired at onset of G93A disease in the superoxide dismutase 1 (hSOD1 ) mouse model of amyotrophic lateral sclerosis (ALS) The nexus between SOD1 structure and cell dysfunction in amyotrophic lateral sclerosis Prior elevated inflammatory status is neuroprotective for adult rat motoneurones Hexanucleotide expansions activate the NLRP3 inflammasome and the complement cascade Tracking the pattern of cortical dysfunction in MND 30. Dr Karin Borges 13. Autumn Bricker University of Melbourne 21. Shane Deery Anatomy and Pathology, University of Adelaide School of Biomedical Sciences, University of Queensland Brain and Mind Centre, University of Sydney 19. Vandana Deora 34. Dr Thanuja Dharmdhasa 5. Dr Emily Don A Tol2 Gateway compatible toolbox for the study of the nervous system and neurodegenerative disease The utility of next generation sequencing in children and young people with inherited motor neurone disorders 10. Natalie Farrawell Medicine & Health Sciences, Macquarie University Paediatrics, School of Women’s & Children’s Health, University of NSW Paediatrics, School of Women’s & Children’s Health, University of NSW IHMRI, University of Wollongong 33. Dr Alessandra Ferri Victoria University 9. Dr Isabel Formella Medicine & Health Sciences, Macquarie University Medicine & Health Sciences, Macquarie University Queensland Brain Institute, University of Queensland Medicine & Health Sciences, Macquarie University Effect of a tailored training program on individuals with amyotrophic lateral sclerosis Oxidative stress as a tool to understand motor neuron death 28. Dr Michelle Farrar 35. Dr Michelle Farrar 14. Jasmin Galper 29. Dr Fleur Garton 2. Alison Hogan 32. Dr Anne Hogden 6. Dr Elinor Hortle 15. Dr Linda Lau 23. Andi Lee 22. Dr John Lee 18. Dr Jacqueline Leung 31. Dr Rui Li 8. John Manion Australian Institute of Health Innovation, Macquarie University Medicine & Health Sciences, Macquarie University Florey Institute of Neuroscience & Mental Health, University of Melbourne School of Biomedical Sciences, University of Queensland School of Biomedical Sciences, University of QLD Wicking Dementia Research and Education Centre, University of Tasmania School of Biomedical Sciences, University of Queensland University of Sydney Counting the uncounted costs of motor neurone diseases in children Ubiquitin homeostasis in a SOD1 model of ALS Ubiquitin proteasome system dysfunction as a biomarker for the diagnosis and prognosis of MND Integrated analyses of exome and methylation genomic data sets for insight into the etiology of sALS Expression of MND-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype User-centric design of decision support tools for MND Creating an Inducible zebrafish model of TDP-43 mislocalisation Establishing human pluripotent stem cell-derived spinal motor neurons to model motor neuron disease Mutation of TDP43 leads to disrupted transmission and morphology at neuromuscular junctions A protective role for complement C3aR activation in SOD1G93A mice Role of TDP43 in oligodendrocytes development and its implication in the disease progression in ALS. Assessing skeletal muscle bioenergetics in situ: characterising metabolic perturbations in a mouse model of motor neuron disease A functional genomic approach to identify new Motor Neuron Disease (MND) genes and drug targets POSTER PRESENTATIONS Name and Poster No. Institution Title 7. Victoria McLeod Characterising androgen and androgen receptor dysregulation in the SOD1G93A mouse 17. A/Prof Ken Rodgers Florey Institute of Neuroscience & Mental Health, University of Melbourne Florey Institute of Neuroscience & Mental Health, University of Melbourne Bio21 Molecular Science and Biotechnology Institute, University of Melbourne Department of Biomedical Sciences, Macquarie University University of Technology Sydney 12. Dr Hamideh Shahheydari Medicine & Health Sciences, Macquarie University 3. Dr Rebecca Sheean Florey Institute of Neuroscience & Mental Health, University of Melbourne Medicine & Health Sciences, Macquarie University Medicine & Health Sciences, Macquarie University Anatomy and Pathology, University of Adelaide Medicine & Health Sciences, Macquarie University 4. Nirma Perera 11. Mona Radwan 24. Stephanie Rayner 16. Vanessa Tan 25. Ingrid Tarr 20. Anu Tennakoon 1. Dr Amanda Wright 26. Katharine Zhang Medicine & Health Sciences, Macquarie University Targeting autophagy protein degradation pathway to improve motor neuron health in MND The role of aggregation-prone C9orf72 poly(GA) RANtranslated protein in motor neuron disease pathogenesis Investigating the role of cyclin F in ALS disease pathology Proteomic analysis identifies multiple links between cyanobacterial toxins and sporadic MND (sMND) Rab1 reverses both autophagy dysfunction and ER–Golgi trafficking defects in ALS Characterisation of the SMNΔ7 mouse model of spinal muscular atrophy (SMA). Kynurenine pathway metabolites as progression markers for MND Genome-wide analysis of DNA methylation in a disease discordant MND twin cohort Changes in brainstem cytokines in normal ageing and Motor Neurone Disease. Mice with reduced GluA2 Q/R site RNA editing show synaptic changes but no modification to AMPA receptor trafficking or assembly Mutation analysis and immunopathological studies of HNRNPA1, HNRNPA2B1 and HNRNPA3 in Australian motor neuron disease cohorts Follow us on Twitter #MNDmeeting16 @MND_RIA @mndaustralia ABSTRACTS Oral Presentations Session 1 Fishing for an understanding of the biology of MND with zebrafish Dr Nicholas Cole, Macquarie University Our approach is to use zebrafish, an established research model organism, to generate animal models that develop features of ALS/MND in order to understand the biology of the disease. Zebrafish offer several advantages for human disease research that compliment mammalian models. For example zebrafish are transparent and transgenic technologies allow us to express known ALS/MND causing human genes in the fish in vivo whilst at the same time observing motor neurons in real time. We have made some progress with the generation of zebrafish expressing normal, mutant and mislocalised forms of FUS, TDP, and CCNF. We are characterising these animal models and will report on our progress to date. Investigating disease processes in a new TDP-43 mouse model of MND Dr Adam Walker, PhD, Macquarie University The brain and spinal cord pathology of almost all MND patients is characterised by the presence the RNA/DNAbinding protein TDP-43. However, the causes and consequences of TDP-43 pathology development remain unclear, largely due to a lack of valid mammalian in vivo models of disease. We recently created transgenic TDP-43 mice expressing cytoplasmically-targeted TDP-43 (called ‘regulatable NLS’ or rNLS mice) which develop both ALS-like TDP-43 pathology and disease phenotype. In order to define the temporal and regional biochemical changes occurring in these mice, we have performed high-coverage advanced quantitative sequential windowed dataindependent acquisition of total high-resolution mass spectra (SWATH-MS) mass spectrometry. Using this technique, we profiled proteomic changes in brain and spinal cord of transgenic rNLS TDP-43 mice compared with litter-matched controls over the disease course. Quantitative data were obtained from multiple peptides for >1500 proteins derived from soluble and insoluble protein fractions, and of these >60 proteins were detected with >1.5x statistically significant difference between transgenic rNLS TDP-43 mice and controls, covering novel proteins and those previously associated with MND. Pathway analysis also showed biochemical processes that are altered even very early in disease. These studies have revealed valuable insights into the drivers of and responders to TDP-43 pathology and MND pathogenesis, revealing a set of protein changes associated with disease onset and progression. These findings provide insights for continuing biochemical, cell biology and in vivo studies into potential new therapeutic avenues and biomarkers for MND. A functional genomic approach to identify new motor neuron disease genes and drug targets A/Prof Greg Neely, University of Sydney Defective synaptic transmission at the neuromuscular junction (NMJ) contributes to motor neuron disease. However, our understanding of the regulation of synaptic transmission remains incomplete. Through bioinformatics analysis, we identified 420 candidate synaptic regulators that may play an essential role in development or function of the NMJ. Using RNAi we specifically knocked down each gene in the fruit fly motor neuron and identified animals with defects in NMJ development or motor function over their lifespan. In parallel, we are investigating genes that when targeted can suppress a MND phenotype (expression of TDP-43 in motor neurons) in the fly. So far we have identified 100 new synaptic genes that have an essential role in motor neuron function. Gene ontology analysis of these hits suggests associations with establishment of protein localisation and potassium ion transport in the presynaptic motor neuron. We are continuing our gene identification efforts and will further examine candidate genes for synaptic development, synaptic function, and an ability to suppress MND pathology in vivo. Novel stress responsive subnuclear bodies, paraspeckles, play a role in neuronal cell health and may be part of a common pathway perturbed in MND Dr Archa Fox, University of Western Australia Studies on MND genetics and cells have revealed that MND is associated with protein aggregation. But why do some of our proteins have this aggregation-behaviour? We and others have shown that proteins require this behaviour to form useful healthy granules that help cells cope with stress. One type of granule is found in the cell nucleus and is called a ‘paraspeckle’. One possibility is that in MND, nerve cells are killed when healthy granules, such as paraspeckles, instead switch to disease-causing aggregates that are toxic to the cell. This could be a double blow to the cell – losing a useful granule that helps with stress and gaining a toxic byproduct at the same time. Here I will present progress on this project that aims to test the idea that the paraspeckle granule is helping nerves cope with stress using a variety of different approaches. DNA damage and neurodegeneration in ALS A/Prof Julie Atkin, La Trobe University Hexanucleotide (G4C2) repeat expansions in a noncoding region of C9ORF72 are the major cause of familial ALS (~40%) and FTD (~20%). The C9ORF72 repeat expansion undergoes RAN translation, resulting in expression of five dipeptide repeat proteins (DRPs). Whilst it remains unclear how mutations in C9ORF72 lead to neurodegeneration in ALS/FTD, recently, nucleolar stress and R loop formation were implicated. These events can occur during normally, but they damage DNA and hence seriously threaten genomic integrity. DNA damage occurs in many forms, but double-stranded breaks (DSBs) are the most cytotoxic lesions, and the cell activates the DNA damage response (DDR) with the aim of repairing this damage. In lumbar spinal cord motor neurons from C9ORF72 ALS patients we demonstrated significant up-regulation of markers of DDR compared to control subjects: phosphorylated histone 2AX (γ-H2AX), p-ATM, cleaved PARP-1 and 53BP1. Similarly, significant up-regulation of γH2AX was detected in neuronal cells expressing poly(GR) 100 and poly(PR)100 compared to controls, confirming that DNA damage is triggered by the C9ORF72 DRPs. FUS was recently demonstrated to play an important function in DNA repair processes, and we have obtained exciting new evidence that TDP-43 plays a similar role to FUS. Wildtype TDP-43 protected cells from DNA damage induced by hydrogen peroxide or etoposide, whereas mutant TPD-43 was not protective, suggesting that wildtype TDP-43 plays a functional role in DNA repair mechanisms. However, this function is lost by mutant TDP-43 in ALS. This study therefore demonstrates that DNA damage is an important and novel pathogenic mechanism in ALS. Session 2 A new method to measure proteostasis capacity: a potential biosensor for pre-onset neurodegenerative disease A/Prof Danny Hatters, University of Melbourne Protein-folding homeostasis (proteostasis) in humans is controlled by a network of about 800 proteins. While proteostasis can dynamically respond to stresses, it can also become depleted in protein misfolding diseases. A challenge has been defining the depth of proteostasis buffering capacity and how it changes when stimulated or challenged. Here we devised a methodological framework for achieving this goal. A biosensor was made from a tunably-foldable barnase kernel that quantitatively sampled quality control engagement through binding to the unfolded barnase state. The impact of upregulating and suppressing proteostasis on the balance of folded, unfolded and aggregated barnase states was tracked in individual cells using a fluorescence resonance energy transfer scheme coupled to flow cytometry. Application of the method to two mutants of superoxide dismutase 1 (SOD1) revealed they dysregulated proteostasis, but in an unexpected manner. Wild-type and both mutants triggered an increase in latent chaperone capacity to engage unfolded proteins in mammalian cells, which is consistent with SOD1’s activity in scavenging reactive oxygen species to lower baseline demand for proteostasis and in the case of the mutants an activation of a stress response to increase chaperone activity. However, the mutants (A4V and G85R) simultaneously conferred a deficiency in quality control capacity to suppress protein aggregation, indicating an imbalance in proteostasis compared to wild type. The extent of the imbalance correlated with the severities of these mutants in disease progression rates. This scheme opens new paths for biosensors of disease risk and mechanistic insight to proteostasis systems. Identifying mechanisms responsible for ubiquitylation of TAR DNA-binding protein 43 (TDP-43) in ALS pathogenesis Dr Albert Lee, Macquarie University The recently identified mutations in the CCNF gene are a novel cause of ALS/FTD. CCNF encodes Cyclin F, an E3 ubiquitin ligase that forms a part of a SCF complex that binds to proteins for ubiquitylation and degradation by the UPS. We focused on the p.Ser621Gly (S621G) missense mutation that segregated across multiple generations in S621G an Australian family with ALS/FTD. Experimental expression of the CCNF mutation led to defective protein S621G degradation and signature features of ALS pathogenesis in vitro. We investigated the effect of the CCNF mutation in neuronal cells and determined that the Lys48-specific ubiquitylation of protein substrates was elevated WT compared to the CCNF control. This was reflected in the expression of RRM2 (a known Cyclin F substrate) that S621G WT was ubiquitylated mostly with Lys48-ubiquitin in CCNF -transfected cells while RRM2 in the CCNF control 6 lysates were ubiquitylated with both Lys48- and Lys63-ubiquitin. Additionally, we also identified that Ser621 as a (Cyclin F) phosphorylation site that when occupied by phosphate, reduced the Lys48-ubiquitylation activity of the SCF S621G complex by ~0.74-fold (n=3, p<0.01) compared to the CCNF mutant indicating that the Ser621 phosphorylation site is a key regulator of Cyclin F E3 ligase activity. It is postulated that the formation of TDP-43 aggregates is a key step in ALS pathogenesis despite the mechanisms being poorly understood. We have evidence to indicate that Cyclin F and TDP-43 associate as binding partners to form inclusions within neuronal cells. These findings highlight new mechanisms of Cyclin F for sustaining neuronal cell homeostasis. Transfer of ALS protein aggregates in the zebrafish spinal cord Dr Marco Morsch, Macquarie University The clinical progression of ALS suggests a pathological spread of neurodegeneration through the nervous system. The mechanisms that are responsible for the spread of neurodegeneration are not known, but are of intense interest because they represent a feasible therapeutic target to halt or delay disease progression. This study investigated the hypothesis that ALS proteins have propagating characteristics, such that insoluble aggregates can transfer between cells and seed aggregation and degeneration in non-affected cells. Our aim was to explore in a living animal model (in vivo) the propagation and clearance of ALS aggregates. We have expressed ALS aggregates in single motor neurons by injecting zebrafish embryos with DNA constructs of fluorescent wild type and mutant TDP-43 (M337V mutation), or SOD1 (G93A). We applied a UV laser ablation assay to kill the expressing neurons and observe their fate in the spinal cord of zebrafish. This novel approach allowed us to visualise the enduring ALS aggregates after ablation and track their movement and progression throughout the spinal cord in real-time. Our data provides strong evidence that ALS aggregates can survive after the death of a motor neuron. We furthermore demonstrate the intercellular transfer of these aggregates within the spinal cord. Microglial cells get rapidly recruited to the dying neuron and clear the neuronal debris including ALS proteins. We visualized this clearance of neuronal remnants by microglia in real time and characterised the process of microglial engulfment, involving the formation of phagosomes by activated microglia. Disrupted ubiquitin homeostasis in ALS Dr Darren Saunders, University of NSW Mounting genetic and functional evidence supports a role for dysfunctional ubiquitin (Ub) homeostasis in the pathology of ALS. The most striking evidence to date is the widespread presence of Ub-positive inclusions in motor neurons. We hypothesise that mutations in ALS-associated genes disrupt Ub homeostasis, either directly or through sequestration of Ub into protein aggregates. These changes result in altered Ub distribution and subsequent depletion of free Ub, compromising vital Ub-dependent cellular functions and ultimately result in cell death. We are addressing this hypothesis using a multi-faceted approach, integrating unique model systems with powerful proteomics to define the role of ALS genes in modulating neuronal Ub homeostasis. We have mapped the Ubmodified proteome (ubiquitome) in a panel of patient-derived fibroblasts, induced pluripotent stem cells (iPSCs) derived from these cells, and iPSC-derived motor neurons. We identified changes in ~1500 ubiquitlyated proteins across various functional pathways. Ubiquitome profiles clearly delineate disease state in fibroblast samples and also clearly define differentiations state in the fibroblast > iPSC > lineage, revealing key pathways involved in iPSC reprogramming and neuronal differentiation. Importantly, we have also observed significant changes in the ubiquitome of NSC-34 cells expressing either WT or mutant (A4V) SOD1. Our data indicate a significant role for Ub in regulating a variety of cellular functions vital for neuronal function and demonstrate that fALS-associated mutations disrupt Ub homeostasis. This approach provides unprecedented, detailed understanding of dynamic Ub homeostasis in ALS and supports targeting Ub homeostatic pathways as a promising therapeutic avenue for ALS. Aberrant protein degradation as a common pathogenic mechanism in motor neuron disease Dr Shu Yang, Macquarie University The primary molecular pathways underlying ALS remain unclear. Recently, the discovery of mutations in ubiquilin 2 (encoded by UBQLN2) and cyclin F (encoded by CCNF) suggests that defective protein degradation plays a critical role in MND. Ubiquilin 2 and cyclin F play different roles in the ubiquitin-proteasome system (UPS): ubiquilin 2 mediates delivery of ubiquitinated protein substrates to the proteasome, whereas cyclin F, as an E3 ubiquitin ligase, recognises substrates and transfers activated ubiquitin to the substrate as part of the ubiquitination process. Using ALS-linked mutations in these two genes, we have strong evidence that UPS dysfunction is a common pathogenic mechanism in MND, This includes the identification of ubiquilin 2-positive inclusions in non-UBQLN2 MND patient 7 tissues. In this study, we aim to investigate the possibility of using an assay of UPS dysfunction, in readily available MND patient fibroblast cells, as a biomarker for diagnosis and prognosis. These cells are easily obtained from skin and grow in a short time frame, making them an ideal candidate for the development of short-term diagnostic or prognostic tools. We found that upon UPS inhibition, these cells developed cellular pathologies similar to those seen u in MND patient neurons, such as aggregate formation and accumulation of ubiquitinated proteins. Using a GFP UPS reporter assay, we found that UPS impairment can be found in a subset of MND patient fibroblasts. Together, our results suggest that abnormal protein degradation can be used as a common biomarker in many MND cases and skin fibroblasts have potential for short-term, rapid diagnostic and prognostic purposes. Session 3 Copper malfunction is common to sporadic MND and animal models of familial MND Dr Peter Crouch, University of Melbourne The metallo-complex copper-ATSM is protective in mutant SOD1 mice and selectively accumulates in the motor cortex of sporadic MND patients. In vitro and in vivo studies indicate these positive outcomes may be related to the compound’s ability to selectively modulate bioavailable copper. A focus of our current work is to better understand the mechanisms that may underpin the therapeutic and imaging outcomes for copper-ATSM and their significance in the pathogenesis of MND. Data presented herein demonstrate that copper malfunction in the central nervous system is common to familial MND model mice and human cases of sporadic MND. Pseudo-longitudinal data obtained from the mice show the magnitude of copper malfunction escalates with the progression of symptoms and analysis of human tissue revealed the copper-dependent processes are affected in sporadic MND. Improving bioavailable copper in mice via genetic and therapeutic approaches is protective. Moreover, a decrease in specific activity of the cupro-enzyme ceruloplasmin in the human motor cortex is associated with an increase in iron which is amenable to detection via MRI, and biomarkers for altered ceruloplasmin manifest in the CSF. Overall, we have established that copper malfunction unifies human cases of sporadic MND and transgenic animal models of familial MND. Aspects of the copper malfunction are consistent with a proposed therapeutic mechanism of action for copper-ATSM and may also explain why PET-detectable copper-ATSM selectively accumulates in affected regions of the human CNS. Perturbations in iron homeostasis secondary to copper malfunction suggest novel biomarkers for clinical trials in MND. Dysregulation of complement in a new mouse model of MND, and the therapeutic effect of G93A selective C5aR1 antagonism in hSOD1 MND model mice A/Prof Peter Noakes, University of Queensland The complement system is up regulated in MND, with recent studies indicating the activation product C5a may accelerate disease progression, via its receptor C5aR1. We therefore aimed to examine the therapeutic effect of G93A selective C5aR1 antagonism in hSOD1 mice, the most widely used preclinical model of MND. Our developed C5aR1 orally active antagonist PMX205, entered the CNS at pharmacologically active concentrations, with G93A increased entry observed in hSOD1 mice as disease progressed in line with augmented blood-brain-barrier G93A breakdown. hSOD1 mice treated with PMX205 prior to disease onset had significantly improved hind-limb grip strength, slower disease progression and extended survival, compared with vehicle treatment. PMX205 treatment beginning 3 weeks following disease onset also significantly extended survival and reduced disease progression. We have also extended our investigations of complement (C5a-C5aR1 and up stream members) to a non-SOD1 Q331K MND animal model – the TDP43 transgenic mouse. Our aim is to determine if this mouse would be useful as a second preclinical model for examining the therapeutic benefits of targeting complement in slowing MND-like symptoms. This mouse develops MND like symptoms later in life, similar to the late life onset in humans. In this mouse, we have observed that complement is dysregulated during the early stages of MND symptoms. In particular, we see an early up regulation of complement components including C5a and its receptor C5aR1. Together these findings provide us with the opportunity to extend and validate the blocking of C5aR1 in the therapeutic treatment of MND. The relationship between traumatic brain injury and motor neurone disease, and their potential treatment with a PERK-inhibitor Dr Sandy Schultz, University of Melbourne Traumatic brain injury (TBI) is a common neurodegenerative disease that has been linked to the later onset of motor neurone disease (MND), however a causal relationship between these conditions remains controversial. We recently 8 found that experimental TBI in rodents results in persisting motor deficits, progressive atrophy of the motor cortices, reduction in motor cortex neurons, fewer spinal cord motor neurons, and increased expression of muscle atrophy markers. These findings resemble some of the pathological and functional features common in MND and support the notion that TBI can result in a progressive disease process bearing similarities to MND. We also found a number of TAR DNA-binding protein 43 (TDP-43) pathologies that were associated with the degenerative aftermath of TBI. TDP-43 pathologies may induce neuronal toxicity via activating the unfolded protein response, and we have previously found that activation of the unfolded protein response occurs after TBI. A new drug, GSK2606414, can mitigate the neurotoxic effects of the unfolded protein response in other neurodegenerative models by inhibiting PERK. We are currently investigating GSK2606414 treatment in rodent models of TBI and/or MND. Development of novel immunogenes to improve growth factor support for motor neurons Dr Mary-Louise Rogers, Flinders University Background: Targeted gene therapy to motor neurons including growth factors genes has potential as a treatment for motor neuron disease. Our group has developed non-viral gene delivery system called immunogens and shown delivery of reporter genes to motor neurons from the circulation in neonatal mice using an antibody to p75NTR (MLR2) conjugated to PEGylated-polyethylenimine (PEI-PEG12). Objectives: We now wish to determine immunogene dosing and length of gene expression in vivo. Methods: MLR2-PEI-PEG12-pVIVO2-GFP was constructed as previously reported. Neonatal C57BL/6 mice were injected intraperitoneally with different doses of immunogene (n=6-9 mice) and after euthanising, the percent of spinal motor neurons expressing GFP and ChAT determined. Expression was also determined, 72, 96, 144, 168, and 240-hours after delivery (n=3-9 mice). Insulinlike Growth Factor (IGF-1) and Glial Derived Growth Factor (GDNF) was delivered to motor neurons. Results: 26.2±0.6% (n=9) of motor neurons in neonatal mice (n=7) identified by ChAT also expressed GFP 72h after intraperitoneal injection of MLR2-PEI-PEG12 (150µg) carrying pVIVO2 (116µg). Double the dose increased this to 48.7±1.1% (n=8) and persisted for 7-days. GDNF and IGF-1 delivery to motor neurons was shown. Conclusion: Proof of concept for immunogenes has been demonstrated in neonatal mice, such that near 50% of motor neurons can be transfected from the circulation and transfection persists 7 days. Further work is needed determine if immunogenes can be translated to effective growth factor therapy for mice with MND. Development of an immunogene delivery approach for SMN restoration in MND Dr Bradley Turner, University of Melbourne Deficiency of the widely expressed survival motor neuron (SMN) protein causes selective loss of spinal motor neurons and infant death in spinal muscular atrophy (SMA). Given that low levels of SMN confer vulnerability of spinal motor neurons, we determined whether SMN expression is also abnormal in ALS. Our evidence from spinal cords of ALS patients and genetic mouse models demonstrates SMN depletion occurs in motor neurons and may contribute to ALS pathogenesis. Neuronal SMN overexpression in mutant SOD1 and TDP-43 mice significantly delayed symptom onset, extended lifespan and improved spinal motor neuron survival. Our results therefore suggest that restoration of SMN levels may be beneficial for ALS. In an effort to build a non-viral gene therapy for ALS, we have employed a novel approach using immunogenes as vectors for targeted motor neuron delivery of SMN from the periphery. Bicistronic plasmids encoding human SMN and GFP were conjugated to antibodies targeting neurotrophic receptors expressed by motor neurons. Systemic injection of immunogenes into neonatal mice demonstrates: (1) broad targeting of cervical, thoracic and lumbar motor neuron populations, (2) efficient immunogene transduction of ~50% motor neurons and (3) sustained immunogene expression lasting 7 days in spinal cords of mice. The impact of SMN immunogenes on disease onset, progression, neurodegeneration, and SMN levels and function is currently under investigation in mouse models of MND. Our data demonstrating widespread, efficient and prolonged immunogene targeting of motor neurons in the spinal cord highlight the promise of immunogenes to correct inadequate SMN expression levels for ALS. Development of a biocompatible functionalised liposome drug delivery system to increase efficiency of delivery to motor neurons Dr Justin Yerbury, University of Wollongong Despite an increase in the number of novel drugs under preclinical and clinical investigation for MND, the lack of response to therapy is largely due to ineffective delivery of the therapeutic to the diseased site. The delivery of drugs, especially gene therapies, across the blood brain barrier (BBB) and on into motor neurons has proven particularly challenging. A novel approach to drug delivery is therefore clearly required in the context of MND. 9 Liposomes (small phospholipid vesicles) have been used successfully to deliver of a variety of drug payloads, including chemotherapy drugs, oligonucleotides, DNA and proteins, due to their high degree of biocompatibility and stability. Further, surface-functionalised liposomes have been shown to cross the BBB and deliver genes to the nervous system including neurons. Despite this, no such nanotechnology has been developed for the treatment of MND. In this study, transferrin (Tf) or anti-transferrin receptor (TfR) antibody functionalised liposomes were prepared containing pEGFP or SOD1 oligonucleotide using thin-film rehydration and extrusion and their biophysical properties characterised. Dynamic light scatting found the particles to be monodisperse with a hydrodynamic diameter of 120130 nm. The encapsulation efficiency of supercoiled pEGFP was 5.4-fold greater than relaxed pEGFP (22.2% vs 4.1%), while the encapsulation efficiency of SOD1 oligonucleotide was 31.9%. TfR-dependant uptake into NSC34 cells was confirmed by real-time fluorescent imaging of GFP or western blot of SOD1. Altogether these results demonstrate for the first time the feasibility of targeting TfR on the surface of motor neurons as a novel therapeutic strategy to improve drug delivery in MND. Session 4 Subtype-specific alteration of inhibitory circuits in the primary motor cortex in motor neuron disease: a cellular basis for cortical pathophysiology A/Prof Tracey Dickson, University of Tasmania In amyotrophic lateral sclerosis (ALS), a loss of motor neuron function defines the disease, however, increasingly, cortical hyperexcitability is recognised as a prominent event, often proceeding motor neuron degeneration. While many factors may be attributed to this altered pathophysiology, a possible candidate, the interneuron has largely been overlooked. In a systematic immunohistochemical study of interneuron subsets, we demonstrate regionG93A specific pathological alteration in end-stage ALS cases and in SOD1 transgenic mice. Investigations are performed in motor and control somatosensory cortex from presymptomatic to end-stage disease. We report two distinct interneuron populations are altered in the motor cortex and both exhibit presymptomatic, but contrasting G93A + pathology. In SOD1 mice, relative to controls, NPY interneuron number was decreased (~17%) prior to motor + symptom onset (8 weeks), but had increased by end-stage (~30%). In contrast, calretinin interneurons had altered branch complexity at 8 weeks, with progressive reductions in cell number (~31%) from symptom onset (16 weeks). + + Interneurons were unaltered in the somatosensory cortex, suggesting that NPY and calretinin populations drive a motor-specific inhibitory phenotype early in disease. In human ALS brain tissue calretinin-interneuron pathology was recapitulated in a proportion of cases, and positively correlated with the extent of cortical motor neuron pathology in + all cases. Calretinin GABAergic neurons play a crucial role in cortical disinhibition, by regulating other interneurons, + whereas NPY populations are coupled to circuit excitability. Therefore, their differential involvement is likely to alter the motor cortex inhibitory circuit at early stages of disease, contributing to pathophysiology and motor neuron vulnerability within this region. TDP-43 mediated synaptic alterations in the pathogenesis of ALS Dr Catherine Blizzard, University of Tasmania ALS can be pathologically identified by the development of large neuronal cytoplasmic protein aggregates that most frequently contain TDP-43 (transactive response DNA binding protein 43 kDa). Abnormal localisation and functioning of TDP-43 is likely to be a critical component of this disease, as mutation of the TDP-43 gene, is sufficient to cause familial ALS. We report that TDP-43 plays an important role in dendritic spine formation in the + cortex. The density of spines on YFP pyramidal neurons in both the motor and somatosensory cortex of Thy1-YFP mice, increased significantly from postnatal day 30 (P30), to peak at P60, before being pruned by P90. By A315T comparison, dendritic spine density was significantly reduced in the motor cortex of Thy1-YFP::TDP-43 transgenic mice prior to symptom onset (P60), and in the motor and somatosensory cortex at symptom onset (P90). Morphological spine-type analysis revealed that there was a significant impairment in the development of basal A315T mushroom spines in the motor cortex of Thy1-YFP::TDP-43 mice compared to Thy1-YFP control. Furthermore, reductions in spine density corresponded to mislocalsation of TDP-43 immunoreactivity and lowered efficacy of synaptic transmission as determined by electrophysiology at P60. Our current in vitro studies using primary cortical A315T neurons with the TDP-43 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. 10 Cortical excitability in familial C9ORF72 ALS patients Dr Nimeshan Geevasinga PhD, University of Sydney Background: The recently identified C9ORF72 gene expansion is recognized as the most common cause of familial ALS (FALS). The mechanisms by which hexanucleotide gene expansions in the C9ORF72 gene lead to neurodegeneration is complex. Cortical hyperexcitability, as reflected by the threshold tracking transcranial magnetic stimulation (TTTMS) technique, is an early feature in SOD1 mutation familial ALS patients. Consequently, the present study explored cortical dysfunction in affected carriers and non-affected carriers with the C9ORF72 repeat expansion. Methods: Cortical excitability studies were undertaken on two cohorts with the C9ORF72 repeat expansion. A symptomatic cohort, who manifested disease (6 males and 4 females, age range 41-78), and an asymptomatic mutation carriers (9 females and 1 male, age range 26-78). Patients were compared with agematched controls. Results: Short-interval intracortical inhibition was significantly reduced in c9orf72 FALS and sporadic ALS patients [SALS] (P<0.0001), as was the cortical silent period duration (FALS P<0.02; SALS; P<0.01). Central motor conduction time was prolonged (FALS P<0.05; SALS; P<0.0001) and motor evoked potential amplitude was increased in both ALS groups. Resting motor threshold (RMT) was significantly reduced amongst FALS patients (P<0.01) but not in SALS patients whilst a reduction in the RMT was also seen in asymptomatic carriers (P<0.01). There were no significant differences in cortical excitability in asymptomatic mutation carriers when compared to controls. Conclusions: Cortical hyperexcitability appears to be a feature of the pathophysiological process in patients with the C9ORF72 gene expansion, potentially contributing to c9orf72 FALS pathophysiology. Significance: FALS patients with the C9ORF72 gene expansion and SALS patients share a common pathophysiological process of cortical hyperexcitability. Is resistance exercise therapeutic in ALS? A pilot study of exercise-induced fatigue on cortical excitability Dr Michael Lee, University of Technology Sydney Fatigue is a common and debilitating symptom of ALS and can negatively impact on quality of life and the capacity to exercise. The underlying mechanisms remain elusive. A better understanding of the mechanisms mediating fatigue has important clinical implications for future exercise-based strategies for ALS. As such, the aim of our study was to investigate effects of exercise-induced fatigue on intracortical circuits in 15 healthy volunteers (7 females; mean age of 24±3.3 years-old) using established threshold-tracking transcranial magnetic stimulation. Subjects performed 3 sets of 4 different fatiguing exercises involving the thenar muscles on 4 separate occasions (order was randomised): 1) 2-min sustained maximal contractions, 2) 2-min sustained submaximal contractions, 3) 2-min intermittent maximal contractions and 4) 2-min intermittent submaximal contractions. Short-interval intracortical inhibition (SICI) was measured at baseline, immediately after fatiguing exercise, and at 10, 30 and 45 minutes after the last voluntary contraction. SICI declined progressively following sustained maximal and submaximal fatiguing contractions at both inter-stimulus intervals (ISI) of 1 ms and 3 ms (35-60%; p>0.05), and remained depressed for 45 minutes. In contrast, intermittent maximal and submaximal contractions increased SICI at ISI of 3ms (30-65%; p>0.05) (but not at ISI of 1ms), and remained elevated for 45 minutes. In conclusion, fatigue induced by different exercise modalities (sustained V.s., intermittent contractions) and intensity (maximal V.s., submaximal) produced different modulatory effects in SICI. Exercises involving submaximal intermittent contractions maybe therapeutically useful in ALS patients by restoring normal level of cortical excitability. Similar studies in ALS patients are currently underway. Metabolic and gut dynamics in MND: Identifying novel strategies to meet energy needs in MND Dr Derik Steyn, University of Queensland Background: Changes in energy balance that result in weight loss may contribute to worse prognosis in MND. Objective: Building on our existing multicenter study, we seek to develop a comprehensive understanding of factors that impair energy balance in MND. Methods: MND (n=35) and Control (n=35) participants were recruited at the RBWH, and attended a research clinic at UQCCR. Body composition was assessed by air displacement plethysmography (BodPod; COSMED). Energy expenditure (EE) was determined by indirect calorimetry (Quark RMR, COSMED). Blood glucose levels were measured prior to (fasting) and 15 minutes following consumption of a mixed meal (Sustagen, Nestle). Disease severity was gauged by ALSFRS-R and respiratory function tests. Disease duration was determined relative to symptom onset and time since diagnosis. Faecal samples were collected for amplicon based profiling (16S gDNA; ACE, UQ) and assessment of gut microbiota diversity. Results: EE varied greatly between MND and control participants, and hypermetabolism was observed in 43% of MND cases. Metabolic rate in MND did not increase relative to worsening functional scores or the duration of disease, but increased relative 11 to a worsening in blood glucose clearance. Measures for gut microbiota are ongoing. Conclusions: Energy needs in MND vary considerably, with increased energy needs occurring alongside impairments in blood glucose control. When considered alongside reports of impaired insulin function and muscle glucose use, data suggest that increased metabolic needs in MND patients occur due to deficiencies in processes that mediate glucose uptake and/or use. Follow us on Twitter #MNDmeeting16 @MND_RIA @mndaustralia 12 ABSTRACTS Poster Presentations 27. Trans-ethnic methylome-wide association analysis of ALS and its clinical phenotypes Dr Beben Benyamin, University of Queensland Genome-wide analyses of DNA methylation have identified epigenetic marks associated diseases, but few have been reported for ALS. To identify methylation probes associated with ALS and the clinical phenotypes of age and site of onset, we conducted a methylome-wide association analysis in whole blood using the Illumina HumanMethylation450 array in a discovery cohort from China (461 cases and 198 controls) and in validation cohort of European ancestry from Australia (147 sALS cases and 120 unrelated controls). We tested for associations between methylation probes and disease or clinical phenotypes in the discovery cohort by fitting age, sex, plate, plate position, cell counts and principal components as covariates. We then attempted to replicate our findings in the independent validation cohort. In the discovery cohort we did not identify any probes associated with case-control status. A predictor based on the top 100 associated probes in the discovery cohort was not associated with disease status in the validation cohort. We identified three probes associated with age of onset (correcting for chronological age), but none associated with onset site. No significant methylation probes may reflect an absence of probes with effect sizes that our sample size is powered to detect. Our preliminary analyses identified some laboratory protocols that impacted on methylation status; this required us to implement very stringent control of identified and nonidentified confounding factors, which may have removed signal due to true associations. Larger samples will be needed to draw strong conclusions about association of DNA methylation with ALS. 30. Cortical neuronal glucose metabolism is impaired at onset of disease in the superoxide G93A dismutase 1 (hSOD1 ) mouse model of amyotrophic lateral sclerosis (ALS) Dr Karin Borges, University of Queensland Although altered glucose uptake has been shown in various brain regions and spinal cord of patients with ALS, the specific biochemical changes in glucose metabolism in neuronal and astrocytic glycolytic and TCA cycle pathways 13 13 were unknown. We therefore investigated [1- C]glucose and [1,2- C]acetate metabolism in neurons and astrocytes G93A G93A in cortex extracts of hSOD1 mice at onset of disease (80 days). Wild-type (n=15) and hSOD1 mice (n=10) 13 13 were simultaneously injected with 543 mg/kg [1- C]glucose and 504 mg/kg [1,2- C]acetate (i.p.) followed by microwave fixation of the head 15 minutes later. Cerebral cortices were removed and extracted with methanol1 13 chloroform. HPLC, H and C NMR spectroscopy were used to quantify the total concentrations of metabolites, 13 amino acids and C labelling in cortex extract. We found reductions in the amounts of glutamate by 27% and 13 13 succinate by 23% (p=0.02). In addition, the labelling in glutamate ([4- C]glutamate), glutamine ([4- C]glutamine) 13 13 and GABA ([2- C]GABA) from [1- C]glucose was diminished by 30% (p<0.04). Also, levels of various glycolytic G93A 13 derived metabolites were reduced in hSOD1 mice cortex compared to control mice, namely [3- C]lactate by 13 53%, total lactate by 43%, [3- C]alanine by 45% and total alanine by 20% (p<0.03). This could be explained by impairments in glycolysis, however additional abnormalities in TCA cycle reactions or enzymes cannot be ruled out. 13 13 Astrocytic metabolism of [1,2- C]acetate was unchanged as evidenced by unaltered incorporation of C in [4,513 13 C]glutamate and [4,5- C]glutamine. In conclusion, cortical neuronal glucose metabolism is compromised, while G93A astrocytic TCA cycling appears to be normal in the hSOD1 mouse model of ALS at onset of disease. 13. The nexus between SOD1 structure and cell dysfunction in amyotrophic lateral sclerosis Autumn Bricker, University of Melbourne Amyotrophic Lateral Sclerosis (ALS) is an incurable and fatal disorder, which causes neuromuscular paralysis via motor neuron degeneration. This progressive syndrome greatly impacts the lifespan of afflicted individuals; the mean duration of survival being two to five years. The majority of cases are classified as sporadic (sALS) and the remainder 10% as familial (fALS). Within the fALS class, ~20% of the cases are due to autosomal dominant mutations in Cu,Zn-superoxide dismutase 1 (SOD1). A large number of point mutations (at many different sequence positions) cause disease, with large variation in disease progression rates. My project aims to investigate how the mutations in SOD1 propagate a gain of toxic function. My goal is to examine how mutations in SOD1 relate to dysfunctional changes in health of cell culture models of disease and through which proteins in the proteome this happens. While over 180 mutations in SOD1 exist, I will examine 10 mutants (mSOD1) that influence different structural aspects of SOD1. By using SOD1 mutants that have different structural features, I can further pinpoint 13 novel protein interactions to the defects in structure. This will indicate protein clusters that correlate with different SOD1 mutants and development of statistical analyses that will give clues on what different types of SOD1 mutants are interacting with the proteome. In this work I will use Neuro2a (Neuro2a), Neuroblastoma Spinal Cord 34 (NSC34) and SH-SY5Y neuroblastoma cell lines to evaluate an appropriate cell culture model. The cell culture model will measure the physiological dysfunction of SOD1 mutations on viability, stress, dysfunction and proteomic interactions. 21. Prior elevated inflammatory status is neuroprotective for adult rat motoneurones Shane Deery, University of Adelaide An increase in inflammatory cytokines has been implicated in the pathogenesis of MND. Whether these changes are neurotoxic or neuroprotective, however, is unclear. Here, we have artificially elevated levels of inflammatory cytokines in rats to determine whether this affects motoneuronal survival after nerve injury. Groups of 5-6 male Sprague-Dawley rats, aged 2 – 3 months, were injected intraperitoneally with lipopolysaccharide (LPS: 0.5mg/kg) or saline. After 24 hours, rats were anaesthetised and the facial nerve either crushed or avulsed at the stylomastoid foramen. After a further 28 days, rats were anaesthetised then perfused with 4% paraformaldehyde and numbers of facial motoneurones quantified using an optical disector method modified for use in the confocal laser scanning microscope. Results were compared with existing data on numbers of motoneurones surviving 28d after nerve crush (n=6) or avulsion (n=6) alone. Following either (i) nerve crush alone, (ii) saline then nerve crush, or (iii) LPS then nerve crush, approximately 1020% of motoneurones were lost by 28 days (p > 0.05). Following nerve avulsion alone, or saline then nerve avulsion, approximately 80% of motoneurones were lost by 28 days (p < 0.05). This loss was reduced to 40% when nerve avulsion was preceded by LPS (p < 0.05). Multiplex analyses of 12 cytokines revealed that this dose of LPS mimicked approximately 50% of the changes seen in ageing (24m) rats and was not associated with any significant sickness behaviour. Artificially inducing a low-grade systemic inflammatory response in adult rats protects 50% of avulsed facial motoneurones from death. 19. Poly glycine-alanine dipeptide repeats from C9orf72 hexanucleotide expansions activate the NLRP3 inflammasome and the complement cascade Vandana Deora, University of Queensland Expansion of a GGGGCC (G4C2) hexanucleotide repeat of the C9orf72 coding region is a common cause of motor neuron disease and familial frontotemporal lobar degeneration (ALS/ FTLD), however the pathomechanisms leading to neuronal degeneration are currently unclear. One consequence of C9orf72 expansions are the formation of dipeptide repeats, of which poly-GA dipeptides are the most common. Two key components of the innate immune system that have recently been proposed to play an important role in MND pathology are the NLRP3 inflammasome and the complement system. Our current study aimed to determine if the NLRP3 inflammasome and the complement system are activated in response to C9orf72 related poly-GA dipeptides. Primary microglial cultures were treated with poly-GA aggregates and inflammasome activation determined by IL-1 Elisa and western blots. We found that poly-GA aggregates trigger IL-1β secretion from LPS-primed microglia, as well as generating the cleaved form of IL-1 and caspase-p20. We also inhibited dipeptide-mediated inflammasome activation using specific NLRP3 inflammasome inhibitor, MCC950 or using NLRP3-deficient microglia. The complement system is a cascade of secreted molecules that reacts to pathogens and protein aggregates, which also has documented roles in MND. We found that poly-GA aggregates similarly activated the complement cascade, generating the terminal complement component C5a. Taken together, these findings suggest that NLRP3 inflammasome activation and C5a-signalling could be potential downstream pathological mechanisms triggered by neurotoxic proteins seen in C9orf72 cases of MND. Inhibiting these innate immune pathways may thus be a means to slow propagative neuroinflammatory cell death in MND. 34. Tracking the pattern of cortical dysfunction in MND Dr Thanuja Dharmadasa, University of Sydney Motor neurone disease (MND) represents a complex, progressive neurodegenerative process mainly affecting upper (corticomotor) and lower (spinal cord) motor neurones, but also involving extra-motor systems. The clinical, pathological and genetic heterogeneity has unmasked the multifactorial nature of MND, deviating classification from a single nosological entity to more a plethora of diseases. Such variability has made clinical trial designs less efficient and therapeutic modulation challenging, and may also imply differential responses to therapy. Phenotypic variability in MND is largely driven by differences in the vulnerability of upper (UMN) and lower (LMN) motor 14 neurones between individuals, resulting in distinct clinical subtypes and patterns of disease progression. Importantly, the clinical phenotype appears to provide significant prognostic insight, as well as impart critical information regarding the spread of disease. This project aims to assess the complex interplay of UMN and LMN dysfunction at onset and during the natural history of disease progression, to evaluate the differences seen across the clinical, pathological, cognitive and genetic spectrum of disease. However, cortical changes have not yet been comprehensively evaluated in the lower limbs, and longitudinal studies have been difficult to establish. We report differences in cortical function between dominant and non-dominant lower limbs in 30 healthy subjects using TT TMS and present preliminary results in patients with MND, classified according to their phenotype, with correlation to upper limb findings. This has significant impact to understanding the pattern of disease spread and synchronicity, and ultimately lends clues to the overall patterns of neuronal vulnerability in different phenotypes. Such understanding is critical for pathophysiological insights into MND and to guide targeted therapeutic intervention. 5. A Tol2 Gateway compatible toolbox for the study of the nervous system and neurodegenerative disease Dr Emily Don, Macquarie University Introduction: Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and to discover effective therapeutics. Zebrafish are emerging as a powerful model system for studying neurodegenerative disease due to their genetic similarities with humans and ease of transgenesis. Methods: Transgenesis in zebrafish was revolutionised by the advent of the Tol2 transposon system in zebrafish and the Multisite Gateway®-based Tol2 kit for the standard format modular assembly of vectors required for transgenesis. These tools have allowed for the generation of countless transgenic lines expressing fluorescent proteins and genes of interest in cell and tissue specific manners. Here, we build upon this work by presenting a Tol2kit compatible toolbox for the flexible generation of transgenic lines for the study of the nervous system and neurodegenerative disease. Results: We have generated a Tol2 Gateway compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores and compatibility for the introduction of genes of interest or disease linked genes. Conclusions: This toolbox will advance the rapid and flexible generation of zebrafish models to discover the biology of the nervous system and the disease processes that lead to neurodegeneration. 28. The utility of next generation sequencing in children and young people with inherited motor neurone disorders Dr Michelle Farrar, University of NSW Introduction: Next generation sequencing (NGS) enables testing of a selected panel of genes or the entire exome (WES) with demonstrated efficacy in several patient cohorts. The utility of NGS in children and young people with a genetic cause for motor neurone disorders (MNDs) has not been appraised. The present study assesses NGS in this group. Methods: NGS was performed in 31patients (29 families) aged 0-40 years with clinical, neurophysiological and pathological assessments diagnostic of MND from linked adult and paediatric neuromuscular clinics. Phenotypes included SMA, HSP and juvenile ALS. Toxic and inflammatory causes and 5qSMA and Kennedy’s disease were excluded. Genomic DNA was used for either WES or a custom-designed neuromuscular panel. Results: Conclusive genetic diagnoses were established in 4/7 57%) difficult to diagnose patient families by WES who had undergone extensive prior genetic testing; 3/4 had novel phenotypes and 4/4 diagnoses resulted in altered clinical management. Similarly, 8/17 (47%) consecutively recruited prospective MND patients received a molecular genetic diagnosis using a neuromuscular panel as a first tier genetic test. Two had atypical or novel phenotypes. Clinical management changed following diagnosis in 5/12 patients (42%). In addition 3 patient relatives received a genetic diagnosis following cascade testing with 2 having a high risk of recurrence in future pregnancies. Conclusion: This study supports the utility of NGS as a first tier test for children and young people with MND, enhancing rate and shortening time to diagnosis. NGS had a substantial impact on patient management and genetic counselling for the family and also expanded phenotypes. Clinical evaluation remains important in the implementation of NGS. 15 35. Counting the uncounted costs of motor neurone diseases in children Dr Michelle Farrar, University of NSW Background: In an era of emerging therapies, understanding the range and nature of costs incurred by families caring for a child with a motor neurone disease (MND) is important in improving care and providing a complete picture of the economic burden. Methods: A semi-structured interview exploring the effect caring for a child with MND had on family finances, employment, and career opportunities, as well as unmet supportive care needs for carers. Results: A total of 7 individual telephone interviews with primary carers of children with MND of varying ages were conducted (response rate: 78%). A diverse range of experiences were reported, including: significant financial and care-giving burdens, limited career opportunities for carers and a complex landscape of access to funding, equipment and resources. Additional financial resources were attained from family, donations, working overtime, accessing savings, and the opportunity costs in terms of foregone employment, purchases, holidays and leisure activities were substantial. Financial costs were combined with substantial emotional and social impacts. Participants also voiced determination and resilience, and called for continued efforts to improve supportive care services and information. Conclusions: The range and nature of financial costs encountered by families with MND is expansive and not typically quantified and recognised in the literature. These are combined with high levels of care-giving, limitations on employment and career progression, and psychosocial effects. Including the ‘uncounted costs’ of caring for a child with MND is essential in understanding disease impact, providing comprehensive supportive care and informing public policy to efficiently connect families with appropriate services, equipment and resources. 10. Ubiquitin homeostasis in a SOD1 model of ALS Natalie Farrawell, University of Wollongong A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitinated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS has not yet been described. Here we describe a series of experiments to examine ubiquitin homeostasis in a SOD1 model of ALS. Mass spectrometry analysis of the ubiquitinated proteome in transiently transfected NSC-34 cells A4V revealed that there were a greater number of ubiquitinated proteins in cells expressing SOD1 -GFP compared to WT A4V that of cells expressing SOD1 -GFP. The supersaturation scores of ubiquitinated proteins unique to SOD1 indicated that they are more susceptible to aggregation. To further probe ubiquitin homeostasis we used fluorescence recovery after photobleaching analysis which revealed that when mcherry-Ub was incorporated into A4V SOD1 -GFP inclusions, the rate of diffusion of remaining soluble Ub was slower than that of cells expressing A4V WT A4V soluble SOD1 or SOD1 . Levels of free Ub were also found to be lower in cells containing SOD1 -GFP inclusions when total levels of Ub were considered. Taken together, these results suggest misfolded SOD1 contributes to ubiquitin-proteasome system dysfunction and that ubiquitin homeostasis is an important target for monitoring pathological changes in ALS. 33. Effect of a tailored training program on individuals with amyotrophic lateral sclerosis Dr Alessandra Ferri, Victoria University The reduction or avoidance of physical activity by patients with amyotrophic Lateral Sclerosis (pALS) exacerbates the loss of physical function attributable to the disease itself, and sets up a deleterious cycle of progressive impairment. As one potential way to break this cycle is exercise training, the objective of this study was to evaluate the effects of a tailored moderate-intensity training program on functional capacities and strength in pALS. Seven pALS (50.8±6.0 y; mean±SD; M/F: 6/2) undertook 12 weeks of a combined aerobic-strength training, 3 times a week, at a moderate intensity. Before (T0) and at the end of the training program (T1), we evaluated the maximal exercise capacity, maximal strength for the leg extension exercise, functional capacity via the Timed Up and Go test (TUG) and the 6-min walking test (6MWT). From the 8 patients recruited, 7 completed the 12-week training program (1 dropout). The adherence was 85±6% and the satisfaction regarding the training program, evaluated by the Visual Analog Scale (VAS), was 8.8±0.8. Muscle aerobic capacity and strength increased respectively by 13% (p=0.12) and 70% (p=0.07). An improvement in the functional tests was noticed (9% in TUG and 2% in 6MWT), but without statistical difference. This study shows that 12 weeks of tailored, moderate-intensity, combined aerobic-strength training program maintains strength and functional motor skills and does not debilitate pALS. These results provide preliminary support for the role of exercise in the clinical management of pALS. Further studies with larger number of pALS are required to confirm our findings. 16 9. Oxidative stress as a tool to understand motor neuron death Dr Isabel Formella, Macquarie University Oxidative stress (OS) is known to be a prominent pathogenic mechanism involved in motor neuron (MN) degeneration. Evidence for increased oxidative damage was found in tissues, including CNS, from patients with motor neuron disease, and in the mutant superoxide dismutase 1 (SOD1) mouse model. SOD1 is a natural antioxidant against reactive oxygen species (ROS), which are a by-product of aerobic metabolism and play a crucial role in cell signaling processes. However, increased ROS levels can result in OS and cell damage. Despite the wellknown signaling properties of ROS, studying them has been limited by the inability to experimentally control local ROS levels. Here we present an animal model allowing for spatial and temporal control of ROS stimulation in vivo facilitating the investigation of ROS/OS during MN degeneration. While there is evidence from cell culture experiments that ROS can be selectively generated in MNs and furthermore can pass across the plasma membrane and disrupt neighboring cells, we currently lack the capability for non-invasive, single cell high-resolution real-time imaging of OS in existing animal models. We have designed transgenic zebrafish that selectively express the photosensitizer KillerRed, a genetically encoded, red fluorescent protein that produces ROS upon prolonged green light illumination, in spinal MNs. This new model allows us to induce KillerRed-mediated OS in a single MN, using confocal microscopy, and successively observe real-time generation of ROS followed by downstream effects including MN degeneration and apoptosis. 14. Ubiquitin proteasome system dysfunction as a biomarker for the diagnosis and prognosis of MND Jasmin Galper, Macquarie University In the diagnostic process, motor neuron disease (MND) is often confused with other neurological disorders and even non-neurological disorders. Diagnosis is only confirmed if symptoms are progressive, leading to an average diagnostic delay of one year, whereby irreversible neurological damage has occurred and the likelihood of receiving a benefit from clinical trials may substantially decrease. Through the sequencing of an MND family, our laboratory has identified MND causing mutations in two genes (UBQLN2 and CCNF) which are part of the ubiquitinproteasome system (UPS), a cellular pathway which tags and then degrades unwanted protein. This implicates UPS dysfunction as an underlying mechanism of MND. Preliminary data indicates that UPS dysfunction is evident in fibroblasts obtained via a superficial skin biopsy from familial MND patients. Fibroblasts can recapitulate features seen in MND patient neurons such as an accumulation of ubiquitinated proteins. Given that fibroblast attainment is relatively non-invasive and fibroblasts grow rapidly, we propose that the identification of UPS dysfunction in fibroblasts may be used as a diagnostic aid. Here we describe a flow cytometry assay of fibroblasts that measures the amount of an introduced reporter green fluorescent protein (GFP) that is degraded by the UPS. By comparing the amount of reporter GFP in both familial and sporadic MND patient and control fibroblasts, we will be able to ascertain UPS function, and whether UPS dysfunction is common to both sporadic and familial MND. If successful, this assay could quicken MND diagnosis and be utilised for assessing therapeutic efficacy. 29. Integrated analyses of exome and methylation genomic data sets for insight into the etiology of sALS Dr Fleur Garton, University of Queensland 1 Gene discovery has provided remarkable biological insights into ALS to date. Causative mutations in a number of genes have been identified in ALS which has the ability to enhance patient diagnosis, prognosis and treatment. Changes in DNA methylation may also accompany the mutation. This phenomeon has been reported in carriers of the C9orf72 expansion, but has not been explored for other ALS mutations. Establishing if differential methylation profiles exist with causal mutations could help identify new mutations whose contribution to causality is unclear. Here we use whole exome sequencing (WES) (Illumina HiSeq 2500) to identify ALS patients harbouring likely pathogenic mutations. In parallel, we assess the association of DNA methylation probes in the causative gene (Illumina HumanMethylation450 chip) to determine if methylation is also altered. We recruited patients and controls from the Royal Brisbane & Women’s Hospital (RBWH) and the Older Australian Twins Study. WES identified 8/111 patients carrying a genetic variant previously reported to cause ALS (in SOD1, TARDBP, SQSTM1, ANG). We found no evidence for differential methylation in carriers, either in the probe closest to the pathogenic variants, nor those closest to the gene’s transcriptional start sites. We demonstrate the clinical diagnostic capability of WES, which detected a causal variant in ~7% of Australian ALS cases; a capacity that is expected to improve with each new variant reported. Whole blood methylation profiles did not provide evidence of pathogenicity but may still be useful to detect regions of expansion repeats, which remains a limitation for short-read exome sequencing technology. 17 2. Expression of MND-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype Alison Hogan, Macquarie University Motor neuron disease (MND) is a rapidly progressing, fatal neurodegenerative disease. Approximately 10% of MND cases have a known family history of the disease and causal mutations in multiple genes have been identified, providing significant insight into MND pathogenesis. Our laboratory recently identified MND-linked mutations in CCNF, however the pathological mechanisms associated with these mutations are yet to be established. Proteomic analysis of a CCNF in vitro model identified several cellular pathways that were disrupted by expression of the mutant gene, including caspase-3 mediated cell death and axonal outgrowth. In this study we used zebrafish to examine whether these findings were reflected in vivo. Transient overexpression of MND-linked mutant CCNF in zebrafish led to a significantly increased number of caspase-3 positive cells and an increased level of cell death in the spinal cord. Additionally, a motor neuron axonopathy was demonstrated, characterised by shortened primary motor axons and an increased incidence of aberrant axonal branching. Consistent with these observations, a significantly impaired motor response to a light stimulus (photomotor response) was identified in the CCNF mutant zebrafish. This is the first assessment of an MND-linked CCNF mutation in vivo and our findings indicate that zebrafish models will be a useful tool to investigate the pathogenesis of CCNF-linked motor neuron degeneration. 32. User-centric design of decision support tools for MND Dr Anne Hogden, Macquarie University Background: This project concentrated on the development of decision support tools, for people with MND and their families to use in conjunction with health professionals. Currently, no tools are available to guide patients through the complex decisions that are needed during their care trajectory. The tools will prompt patients to consider the benefits and risks of symptom management options, and the consequences of declining treatment in light of their personal values. Aims: To improve the quality of life of Australian MND patients, we addressed the following research questions: 1. Which decision support tools are needed to support MND patients? 2. What is the optimal content, format and delivery of these tools in MND multidisciplinary care? Methods: Thirteen participants formed two expert panels: one comprising patients and carers; the other, MND clinicians and researchers, representatives of MNDA and MND NSW; and decision tool researchers. Results: Panel members identified 56 symptom management options that MND patients are commonly asked to consider. From these, six were selected as high priority for development into decision support tools. These were: gastrostomy/PEG insertion; assisted ventilation and withdrawal; genetic testing for patients and family members; end-of-life care location; communication equipment; and advance care planning. Optimal delivery mode is by mobile website, to allow access in specialised clinics, generalist services, home and facility-based care. Conclusion: Stakeholder consultation has been proved to ensure user-centric design. This lengthy, but comprehensive, process will result in tools that are both clinically feasible and useful to patients, families and health professionals. 6. Creating an Inducible zebrafish model of TDP-43 mislocalisation Dr Elinor Hortle, Macquarie University Almost all cases of ALS share a common neuropathology characterized by the mislocalistation of TAR-DNA binding protein (TDP-43) to the cytoplasm of cells, and the deposition of TDP-43-positive protein inclusions. Here we aim to gain further insight into the mechanisms behind ALS, by creating a zebrafish model of this pathology. We have created transgenic fish that ubiquitously express an inducible form of either WT human TDP-43, or human TDP-43 with a mutated nuclear localisation sequence, which causes the protein to be mislocalised to the cytoplasm. Further development of this model will allow us to study the ways in which TDP-43 mislocalisation is involved in neurodegeneration, and may provide an invaluable model for testing future ALS therapeutics. 15. Establishing human pluripotent stem cell-derived spinal motor neurons to model motor neuron disease Dr Linda Lau, University of Melbourne Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, offer a powerful model system to generate motor neurons (MNs) for screening toxicity and potential therapeutic agents in MND. The overall aim of this study was to adapt a reproducible protocol to generate highly pure human spinal MNs to model MND. Spinal MNs were generated and differentiated from H9 hESC cells using a published protocol (Du et al 18 (2014)) with slight modifications, and characterised by immunocytochemical analyses for neuroepithelial (NEPs), MN progenitor (MNPs) and mature MN markers. HOX and MN gene expression were also characterised by qPCR. + + Immunocytochemistry shows caudal NEPs (SOX1 , HOXA3 ) were successfully induced from hESCs. These cells + were then successfully differentiated into MNPs (OLIG2 ). Differentiations were then continued for another 17 days and staining showed HB9, ISL1, CHAT and TUJ1 positive cells, consistent with mature spinal motor neurons. qPCR data showed these cells expressed NEP gene (SOX1), caudal spinal MN genes (HOXA3, hindbrain; HOXC6, cervical; HOXC9, thoracic; HOXD11, lumbar), MNPs (OLIG2) and MN gene (HB9 and ISL1). This iPSC-derived spinal MN model will serve as an important platform to screen the toxicity of CSF from MND patients, identify factor(s) responsible for toxicity and agents that may mitigate toxicity. 23. Mutation of TDP43 leads to disrupted transmission and morphology at neuromuscular junctions Andi Lee, University of Queensland Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterised with the loss of motor neurons and degeneration of the neuromuscular junction (NMJ). Transactive response DNA binding protein 43 (TDP43) which is found in the nucleus has been shown to be involved in the pathological disease of ALS. TDP43 is involved in mRNA processing by splicing and transporting of mRNA transcripts. It has also been found at the presynaptic nerve terminal of the NMJ, suggesting a potential role of mRNA processing at the NMJ itself. The Q331K present study aimed to functionally and morphologically characterise the TDP43 mutation in the mouse model of ALS (Q331K), by comparing with non-transgenic wild-types (NTg) and wild-types expressing normal human TDP43 (Wild-type) at 3 months and 10 months of age. Results demonstrated early disruption of transmission Q331K properties at 3 month TDP43 NMJs with decreased frequency of spontaneous release, lower quantal content and higher intermittence, which suggest presynaptic defects. Perturbed transmission properties were more evident Q331K by 10 months at TDP43 NMJs with increased intermittence and decreased amplitudes of both spontaneous and evoked release. On the other hand, NTg and wild-type NMJs maintained normal transmission properties at both ages. Altered innervation patterns such as multiple innervations, partial denervation and altered axonal size were observed at these NMJs at 3 months which persisted at 10 months. Changes in primary axonal diameter size were Q331K also noted at TDP43 NMJs at both ages. These findings support that TDP43 mutation may have direct influence on NMJ function and morphology. 22. A protective role for complement C3aR activation in SOD1 Dr John Lee, University of Queensland G93A mice The complement system is an integral component of innate immunity and has been implicated in the pathogenesis of motor neuron disease (MND). Complement is activated in human MND and in mouse models, and its activation generates the bioactive fragment C3a, which can modulate the inflammatory response by binding to its receptor, C3aR. However, the contribution of C3aR to disease progression in MND is still unknown. The current study aimed G93A to determine the function of C3aR in the disease progression of MND using SOD1 mice lacking the C3aR G93A -/(SOD1 x C3aR ). Behavioural tests were conducted to observe any differences in motor symptoms, along with body weight and survival. Neuroinflammation was also measured in these animals by determining the degree of G93A -/gliosis and cytokine expression using quantitative PCR at defined disease stages. SOD1 x C3aR mice showed G93A G93A -/significantly reduced survival and body weight relative to SOD1 mice. SOD1 x C3aR mice also displayed G93A greater motor dysfunction, at an earlier age, compared to SOD1 mice. In line with this worsened disease progression, there was increased gliosis gene expression at later stages of disease, while the cytokine gene G93A -/G93A expression was increased in SOD1 x C3aR mice, relative to SOD1 mice at the earlier stages. These results indicate that the C3a generated during complement activation in MND, has anti-inflammatory activities in the G93A SOD1 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. 18. Role of TDP43 in oligodendrocytes development and its implication in the disease progression in ALS Dr Jacqueline Leung, University of Tasmania The TAR-DNA binding protein-43 (TDP-43) is one of the common pathogenic proteins involved in amyotrophic lateral sclerosis (ALS). The effect of altered TDP-43 expression or aggregation in neurons has been well studied. However, TDP-43 protein aggregates are also present in oligodendrocytes from patient’s post-mortem tissue. The effect of altered TDP-43 expression or its aggregation in oligodendrocytes is currently not known. The main aim of this study is to characterised the role of TDP-43 in oligodendrocyte 19 development from their precursor cells and their ability to myelinate. Primary oligodendrocyte precursor cell cultures were prepared from postnatal day 2 rats. Lentivirus transduction of GFP-tagged human TDP-43 was used to induce TDP-43 overexpression in cultured cells. The cells were grown in media to induce the formation of mature oligodendrocytes. Images were acquired from both live and immunocytochemically labelled cultures; To analyse cell morphology, tracing of the oligodendrocyte processes was performed using Image-J to quantitate the branching complexity, an indication of cellular maturity. At day 4 post transduction, there was a significantly (p<0.05) higher number of branches (indicating a more complex morphology) in the transduced oligodendrocytes compare to untransduced oligodendrocytes (n=10). These results suggest that TDP-43 overexpression accelerates the development of oligodendrocytes in vitro. This result supports our hypothesis that TDP-43 has an active role in oligodendrocytes development; however, further study is required to confirm its involvement in the myelination process. 31. Assessing skeletal muscle bioenergetics in situ: characterising metabolic perturbations in a mouse model of motor neuron disease Dr Rui Li, University of Queensland Motor neuron disease (MND) is a fatal neurodegenerative disease characterised by the progressive loss of motor neurons, muscle wasting and paralysis. While the fundamental cause for MND remains unknown, recent evidence suggests that impairments in skeletal muscle metabolism may contribute to disease progression. In order to define the pathophysiological nature of skeletal muscle bioenergetics in MND, we developed a method that allows for the G93A real-time assessment of metabolic flux in intact extensor digitorum longus (EDL) muscle fibres from the SOD1 mouse model of MND and age-matched wildtype (WT) mice at pre-defined stages of disease. In spite of the G93A remarkably decreased muscle size, we found that EDL muscle fibres from SOD1 mice exhibited basal respiration rates that were comparable to WT controls, indicating the basal energy demands do not decline with worsening muscle pathology. By assessing the flexibility and maximal capacity of EDL fibres to oxidise glucose or long-chain fatty acid as energy substrates, we demonstrated preferential use of fatty acid as an energy source, and G93A enhanced mitochondrial oxidation capacity in EDL fibres of SOD1 mice when compared to WT controls. Our data are in line with previous studies that report impaired glucose metabolism in MND, and suggest that metabolic adaptions towards fatty acid oxidation may occur to maintain the energy demands of skeletal muscle throughout disease progression. By assessing skeletal muscle bioenergetic profiles in situ, our study provides insight into metabolic pathways that may be modulated to potentially improve muscle symptoms in MND. 8. A functional genomic approach to identify new Motor Neuron Disease (MND) genes and drug targets John Manion, University of Sydney Defective synaptic transmission at the neuromuscular junction (NMJ) contributes to motor neuron disease. However, our understanding of the regulation of synaptic transmission remains incomplete. Through bioinformatics analysis, we identified 420 candidate synaptic regulators that may play an essential role in development or function of the NMJ. Using RNAi we specifically knocked down each gene in the fruit fly motor neuron and identified animals with defects in NMJ development or motor function over their lifespan. In parallel, we are investigating genes that when targeted can suppress a MND phenotype (expression of TDP-43 in motor neurons) in the fly. So far we have identified 100 new synaptic genes that have an essential role in motor neuron function. Gene ontology analysis of these hits suggests associations with establishment of protein localisation and potassium ion transport in the presynaptic motor neuron. We are continuing our gene identification efforts and will further examine candidate genes for synaptic development, synaptic function, and an ability to suppress MND pathology in vivo. 7. Characterising androgen and androgen receptor dysregulation in the SOD1 Victoria McLeod, University of Melbourne G93A mouse A gender bias towards higher male ALS incidence at younger age of onset, which is often reflected in mouse models of ALS, indicates potential underlying hormonal influences modulating disease progression. Androgens and signalling via the androgen receptor (AR) may influence ALS disease progression as evidenced by 1) high AR expression in motor neurons (MNs), 2) AR activation providing direct and indirect trophic support to MNs, and 3) mutations in the AR gene giving rise to selective lower MN vulnerability in spinal bulbar and muscular atrophy (SBMA). To provide insights into potential mechanisms driving sexual dimorphism in ALS, we have explored sex G93A hormone and receptor changes occurring in male and female SOD1 mouse tissues and the impact of blocking AR signalling on disease progression. Here, we provide a characterisation of AR and estrogen receptor (ER) 20 G93A expression levels and distribution in the lumbar spinal cord of SOD1 mice, compared to wild-types at ages P60 (presymptomatic), P90 (onset), P120 (late symptomatic) and P150 (endstage). Detectable serum androgen levels, including testosterone, were also characterised in male mice using liquid chromatography/mass spectrometry G93A analysis. We also administered the AR antagonist, flutamide (9.52 mg/d, SC), from P60 onwards to SOD1 mice via a sustained drug-release formulation. Systemic blockade of AR signalling from presymptomatic age was unable G93A to rescue motor deficits or modify disease progression in SOD1 mice, thereby highlighting the complexity of the steroid hormone systems which may be involved in driving sex-specific differences in ALS. 4. Targeting autophagy protein degradation pathway to improve motor neuron health in MND Nirma Perera, University of Melbourne Cytoplasmic accumulation and aggregation of misfolded proteins is a MND pathological hallmark, implicated in motor neuron death. Therefore, strategies that clear pathogenic proteins are likely to be beneficial. Autophagy pathway is a powerful modulator of intracellular protein degradation, targeting misfolded proteins, aggregates and damaged organelles. We aimed to screen clinically-approved drugs to stimulate autophagy in NSC-34 and stem cell models to find agents that can decrease mutant protein inclusion load for advancing to therapeutic testing in MND mouse models. NSC-34 cells were treated with candidate drugs and Western blotting or mCherry-EGFP-LC3B/p62 autophagy reporters were used to check autophagy marker expression to identify effective concentrations which were next used to treat cells expressing SOD1. Drugs were also investigated in human embryonic stem cell (hESC)derived motor neurons to investigate their ability to induce autophagy using Western blotting and fluorescence microscopy. We found that 10µM rilmenidine, 10µM nilotinib and 5µM tat-HA-ApoE beclin significantly increased LC3-II and decreased p62 in NSC-34 cells 24 hours after treatment indicating autophagy induction. Importantly, 10 uM rilmenidine and nilotinib significantly decreased mutant SOD1 protein and aggregates. Results in hESC motor neurons indicated that 10uM rilmenidine at 24 hours and 5µM nilotinib at 48 hours induces autophagy. Future studies will investigate drug effects on protein and aggregate levels of TDP-43, FUS and expanded C9ORF72, in hESC motor neurons as well as in MND patient derived induced pluripotent stem cells. Lead candidates will be advanced to established (SOD1) and novel (TDP-43 and expanded C9ORF72) MND mouse models to examine therapeutic potential. 11. The role of aggregation-prone C9orf72 poly(GA) RAN-translated protein in motor neuron disease pathogenesis Mona Radwan, University of Melbourne Neural inclusions of poly-(Gly-Ala), a protein generated form the non-canonical translation of the GGGGCC repeatexpansion mutations in the C9ORF72 gene, are abundant in the brains of patients with frontotemporal dementia and amyotrophic lateral sclerosis carrying this mutation (c9FTD/ALS). The contribution of the aggregation-prone polyGA protein to disease pathogenesis remains an open question. For this end, we designed artificial cDNA constructs to express EGFP-fused short (10× repeats) and long (101× repeats) polyGA chain. PolyGA remained diffusely throughout the cytoplasm in the short length but formed cytoplasmic inclusions in the expanded form with fast aggregation kinetics when expressed in neuroblastoma cell culture model of disease. The expression of poly(GA) 100 has led to a reduction in survival rates relative to cells expressing either the short polyGA chain or GFP only. We next used a proteomic approach for the identification of aggregate-interacting proteins to allow the process of aggregate formation to be investigated. The cytoplasmic aggregate particles were purified using a fluorescenceactivated cell sorter (FACS) by monitoring EGFP fluorescence. The resulting highly pure aggregates were subjected tryptic digestion followed by mass spectrometric sequence analysis. Poly(GA) inclusions are enriched in various proteins, including those involved in proteasomal protein degradation. It is anticipated that this knowledge will help to explain the role of aggregate interacting proteins in C9FTD/ALS pathogenesis. 24. Investigating the role of cyclin F in ALS disease pathology Stephanie Rayner, Macquarie University Our team has identified novel missense mutations in CCNF, some of which lead to ALS. At the protein level, cyclin F is a component of an E3-ubiquitin ligase which has four known substrates. A challenge when identifying new substrates is the transient binding between the ligase and its substrates. We aim to identify novel substrates of cyclin F using proximity-based biotinylation (BioID) and mass spectrometry (MS). We also aim to validate high confidence interactors using immunoprecipitations (IPs) and MS. BioID is a new methodology that involves fusing 21 mutant biotin ligase in frame with the protein of interest (cyclin F) in order to biotinylate proteins that come into close proximity. We generated a stably transfected, tetracycline-inducible HEK293 cell line that expresses the cyclin FBioID fusion protein. After tetracycline-induced protein expression, cells were treated with 50 µM biotin for 24 hours. After cell lysis, biotinylated proteins were enriched by streptavidin-bead pull-downs. Biotinylated proteins were digested with trypsin and analysed by LC-MS/MS. We identified 197 proximal proteins by LC-MS/MS. Bioinformatic analysis revealed potential interactors that clustered into several biological processes including protein refolding and synthesis, cell proliferation, and RNA-binding and processing. One of these potential binding partners has been validated using IPs and immunoblotting. Our preliminary results indicate that cyclin F interacts with proteins responsible for various cellular processes including protein refolding and RNA metabolism, which contributes to maintaining homeostasis. BioID-MS is the first step to expanding the roles Cyclin F plays, which aids in linking mutations in cyclin F to neurodegeneration. 17. Proteomic analysis identifies multiple links between cyanobacterial toxins and sporadic MND (sMND) A/Prof Ken Rodgers, University of Technology Sydney Background: Epidemiological studies from France, USA and Sweden support the view that exposure to cyanobacterial toxins increases the risk of developing sMND. Biomagnification of the cyanobacterial toxin βmethylamino-L-alanine (BMAA) through food chains has been reported, increasing the potential for human exposure. Aims: (a) To determine if BMAA and other cyanotoxins are present in the Australian environment (b) To perform proteomic analysis to identify mechanisms of toxicity that link human cyanotoxin exposure to sMND. Methods: Environmental samples were analysed by triple quadrupole mass spectrometry (MS). Multiplexed tandem mass tag (TMT) proteomics was performed on human neuroblastoma cells treated with BMAA, DAB and AEG to investigate their proteome response. Peptides were analysed by nano-flow LC coupled to tandem MS and quantified using TMT reporter ions. Results: BMAA, DAB and AEG, were present in a wide range of Australian environmental samples. On treatment of cells with BMAA and DAB, 200-300 proteins were differentially expressed. Mitochondria were targeted and proteins associated with ER and proteotoxic stress. Interactome pathway analysis revealed significant changes in proteins grouped as ‘neuromuscular disease’, ‘movement disorders’ and ‘disorders of basal ganglia’. Discussion: BMAA and DAB co-exist in nature and human exposure is likely. The combination of BMAA and DAB had additive effects and induced changes in a range of cellular pathways linked to the human neurological disease. As demonstrated recently in a vervet study, BMAA alone can cause neurofibrillary tangles and β-amyloid plaque-like deposits in the brain the combined neurotoxicity of BMAA and DAB however has not previously been investigated. 12. Rab1 reverses both autophagy dysfunction and ER–Golgi trafficking defects in ALS Dr Hamideh Shahheydari, Macquarie University Several diverse proteins are linked genetically or pathologically to neurodegeneration in ALS including SOD1, FUS and TDP-43. Mutant forms of these proteins inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells, and cause autophagy defects. Rab1 has a pivotal role in mediating intracellular membrane trafficking events, including ER–Golgi trafficking and autophagosome formation. However, the function of Rab1 in ALS remains unclear. The aim of this study is to investigate the effect of Rab1 overexpression on (i) ER–Golgi trafficking and (ii) autophagy, in neuronal cells expressing ALS-associated mutant proteins, (iii) the distribution of Rab1 in motor neurons of human spinal cord tissues from patients with sporadic ALS, (iv) and to identify novel Rab1-mimetic compound that could have therapeutic benefit in ALS. Rab1 overexpression in Neuro2a cells rescues inhibition of ER–Golgi transport and ER stress triggered by mSOD1, mTDP-43 and mFUS, and apoptosis and inclusion formation triggered by mSOD1. Rab1 also restored the inhibitory effects of mFUS on autophagosome and autolysosome formation. Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients and approximately 40% of the Rab1 inclusion-positive motor neurons co-localised with TDP43. We are currently investigating whether novel compounds that mimic Rab1 activity are protective in neuronal cells expressing mutant SOD1, TDP43 and FUS. We demonstrate that Rab1 rescued inhibition of ER–Golgi transport in cells expressing mutant SOD1, TDP-43 or FUS. This imply that restoring Rab1-mediated ER–Golgi transport and autophagy regulation is a novel therapeutic target in ALS. Finding therapeutic compound, which mimics Rab1 activity could be effective as a novel and broadly acting therapeutic agent in multiple forms of ALS. 22 3. Characterisation of the SMNΔ7 mouse model of spinal muscular atrophy (SMA) Dr Rebecca Sheean, University of Melbourne Background: Spinal muscular atrophy (SMA), the most common motor neuron disease in infants and children, is caused by the homozygous deletion or mutation in the survival motor neuron 1 (SMN1) gene and retention of the almost identical SMN2 gene. This results in the loss of SMN protein, leading to selective loss of lower motor neurons, progressive muscle weakness and death via respiratory failure. A number of mouse models of -/+/+ +/+ SMA have been generated, with the SMNΔ7 mouse (Smn ;SMN2 ;SMNΔ7 ) being the most widely utilised model of severe SMA (type I). We are the first group in Australia to import and establish a large breeding colony of SMNΔ7 mice. Here we characterise this model for future analysis of therapies for treatment of SMA. Methods: Disease progression of SMNΔ7 mice and age-matched het/wildtype mice (n≥18 mice/genotype) was analysed daily by measuring body weight, righting reflex and survival, determined by the onset of hind limb paralysis. Spinal cords and muscles were analysed for disease pathology at pre-symptomatic (P1, P3), symptomatic (P5-P9) and end stage (P12-P14) disease stages. Results: We report in accordance with previous findings, SMNΔ7 mice are indistinguishable from their littermates from birth but have reduced body weight, motor difficulties and NMJ abnormalities from P5. SMNΔ7 mice have significantly reduced survival (10 ± 2.6 days) compared to their wildtype and het littermates. No differences between wildtype and het mice were observed. Conclusion: The SMNΔ7 mouse model recapitulates many aspects of the human disease and therefore provides a good model for the pre-clinical trial of SMA therapies. 16. Kynurenine pathway metabolites as progression markers for MND Vanessa Tan, Macquarie University Background: MND diagnosis is hampered by a lack of suitable biomarkers, which also prevents biochemical monitoring of disease progression, limiting assessment of therapy efficacies during clinical trials. MND patients have been shown to have an altered KP as compared to normal controls, and this study further characterizes the KP in MND patients. Methods: Longitudinal serum samples from 59 MND patients were obtained from a clinical trial in France. KP metabolites were measured using ultra High Performance Liquid Chromatography and Gas Chromatography Mass Spectrometry, and regression analyses were performed to study association between KP and disease outcome adjusting for gender and age. Results: Preliminary data show that with ALSFRS significant correlation is observed with Kynurenic Acid (p=0.009, 95%CI: ± 0.6628), Tryptophan (p=0.036, 95%CI: ±0.968), Hydroxy Anthranilic Acid (p=0.03, 95%CI: ±1.171), Kynurenine (p=0.001, 95%CI: ±5.244). Kynurenic Acid strongly correlates with MMT scores (p=0.001, 95% CI: ±0.329). Further analysis is being done to better characterize the KP in patients longitudinally. This indicates that following changes in patient KP profile may provide a good measure of clinically associated disease progression. Conclusions: The KP shows potential for tracking disease development in ALS. This will allow for a biochemical technique that can monitor disease progression. This data has also allowed an in depth analysis of the KP changes through the course of disease. The role of immune modulating KP may be applied towards therapeutic considerations by using KP altering drugs that may lead to new therapeutic options for alleviating the clinical effects of MND. 25. Genome-wide analysis of DNA methylation in a disease discordant MND twin cohort Ingrid Tarr, Macquarie University Genetic discoveries have identified the causal genes for approximately 60% of hereditary MND cases in Australia, however there is unexplained variation in age of onset and progression of disease. This is observed even within patients harbouring identical mutations. Absence of genotype-phenotype correlation indicates that disease modifiers, including epigenetic modifications, could play a crucial role in disease manifestation. To investigate epigenetic modifiers in MND, we are examining DNA methylation profiles in a twin cohort comprising four twin sets that are discordant for MND, and include longitudinal sample collections during disease progression. One twin set harbours a C9orf72 repeat expansion, another set harbours a SOD1 p.I114T mutation, and the remaining two sets have no known mutation. gDNA samples have undergone genome-wide methylation analysis using the Illumina HumanMethylation 450K BeadChip. R packages wateRmelon, limma, DMRcate, and RnBeads have been used for QC and to perform biostatistics analyses. Initial analysis of predicted age due to methylation markers indicates that an affected twin and their unaffected cotwin may be biologically aging at different rates. Global methylation analysis shows no statistically significant differences between affected and unaffected twins, nor when compared to a control twin set. This was consistent across all classes of CpG regions. Differential methylation identified in this twin study will be supported using available methylation data from a validation cohort of 1000 MND patients and controls. Discovery of epigenetic 23 disease modifiers can provide insight into the causes of phenotypic variation in MND, will highlight disease-related pathways and thus identify new potential treatment targets. 20. Changes in brainstem cytokines in normal ageing and motor neurone disease Anu Tennakoon, University of Adelaide Age-related increases in inflammatory status, as measured by elevated levels of pro-inflammatory cytokines, have been implicated in the development of age-related neurodegenerative diseases including Motor Neurone Disease (MND). Analyses of cytokines in MND, however, tend to focus on individual cytokines and to compare MND tissue only with age-matched controls. This could lead to cytokine changes with normal ageing as well as cytokine interactions being overlooked. Here, 27 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, TNF-α, IFN-ϒ, FGF, G-CSF, GM-CSF, IFN-ϒ, MCP-1, MIP-1α, MIP-1β, Eotaxin, PDGF-1β, RANTES and VEGF) were analysed using multiplex technology in fresh frozen post-mortem brainstems of MND patients aged 60-68 years (n=6), and compared with those of ageing controls aged 48-86 years (n=6) and young adult controls aged 20-33 years (n=6). Levels of IL-6, IL-1β, IP-10 and MIP-1β were significantly increased in age-matched control brainstems compared to young adult control brainstems, but decreased in MND brainstems (p=0.0142, p=0.0150, p= 0.0718, and p=0.0022, respectively). Increased levels of this cytokine combination with normal ageing may be neuroprotective whereas their decrease in MND may be associated with motoneuronal degeneration. The source of these cytokines and nature of their interaction remains to be determined. 1. Mice with reduced GluA2 Q/R site RNA editing show synaptic changes but no modification to AMPA receptor trafficking or assembly Dr Amanda Wright, Macquarie University 2+ Background: Calcium (Ca )-permeable AMPA receptors can contribute to normal synaptic plasticity and 2+ neurodegeneration. AMPA receptors are Ca -permeable if they lack the GluA2 subunit or if GluA2 is unedited at the 2+ Q/R site. In addition to regulating AMPA receptor Ca -permeability, GluA2 RNA editing is thought to modulate AMPA receptor assembly and expression in vitro, however the role in vivo remains unclear. Methods: In this study, we examined mice engineered with a point mutation in the editing complementary sequence (ECS) of the gria2 gene, which is known to be required for efficient GluA2 RNA editing. Results: Mice heterozygous for the ECS mutation showed a 26% reduction in GluA2 RNA editing at the Q/R site and altered current-voltage relations, confirming expression of GluA2(Q)-containing AMPA receptors at the synapse. The reduced GluA2 RNA editing resulted in enhanced NMDA receptor-independent LTP, seizure vulnerability, altered dendritic morphology, reduction in spine density, loss of hippocampal CA1 neurons and behavioural deficits. However, in contrast to previous in vitro studies, we found no alterations to AMPA receptor composition and no changes to surface expression, glycosylation or endoplasmic reticulum retention of GluA2. This indicates that GluA2 is expressed normally in the heterozygous mice and the synaptic and phenotypic changes are not due to altered AMPA receptor trafficking or assembly. Conclusion: The mouse model developed adds to the understanding of how AMPA receptors modulate synaptic plasticity, and provides a useful tool to study altered RNA editing and neurodegeneration seen in numerous diseases such as stroke, Alzheimer’s disease, Huntington disease and ALS. 26. Mutation analysis and immunopathological studies of HNRNPA1, HNRNPA2B1 and HNRNPA3 in Australian motor neuron disease cohorts Katherine Zhang Gene mutations are currently the only known cause of MND, accounting for 60% of hereditary MND cases, and 5% of sporadic MND cases. Mutations in the genes that encode heterogeneous nuclear ribonucleoproteins hnRNPA1 and hnRNPA2/B1 (HNRNPA1, and HNRNPA2B1) have been reported in both familial and sporadic MND patients. These proteins, along with hnRNPA3, form the A/B subfamily of hnRNPs which are involved in numerous aspects of nucleic acid metabolism. Previous studies have reported that disease-linked mutations in these proteins enhance their prion-like behaviour, increase their nuclear clearance, and result in their accumulation in cytoplasmic aggregates and stress granules. In order to examine the involvement of these proteins in an Australian MND cohort, we analysed whole exome sequence data from 109 familial cases to identify variants in HNRNPA1, HNRNPA2B1, and HNRNPA3. We subsequently conducted targeted sequencing of the prion-like domains of these hnRNPs in 616 sporadic cases. No novel or known nonsynoymous variants were identified in our cohort. We carried out immunohistochemical analyses of hnRNPA1, hnRNPA2/B1, and hnRNPA3 in spinal cord tissues from MND patients and neurologically normal controls. No cytoplasmic inclusions positive for hnRNPA1 or hnRNPA2/B1 were observed in controls or patients. Patients with C9orf72 hexanucleotide repeat expansions showed a trend towards decreased 24 nuclear hnRNPA3 staining compared to controls and other MND cases. This study shows that although no hnRNP A/B gene mutations were present in Australian MND patients, immunohistology implicates hnRNPA3 in disease pathogenesis and warrants further study. Follow us on Twitter #MNDmeeting16 @MND_RIA @mndaustralia 25
