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Macquarie Neurodegeneration Meeting CENTRE FOR MOTOR NEURON DISEASE RESEARCH
19 July 2019
FACULTY OF MEDICINE AND HEALTH SCIENCES
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Our Sponsor
WE ARE INCREDIBLY THANKFUL TO OUR SPONSOR FOR THEIR SUPPORT OF THE MACQUARIE NEURODEGENERATION MEETING.
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In celebration of Olympus’s 100th anniversary, Olympus Scientific Business Solution Unit has launched a range of new products to elevate microscopy in Life Science Research to the next level. Introducing the ScanR-AI system, a modular microscope-based imaging platform designed for fully automate image acquisition, data analysis and employ deep learning AI to process increasingly challenging data required from biological samples. The IXplore SpinSR confocal system, which is designed for fast 3D super resolution imaging and prolonged cell viability in time-lapse experiments, the system offers XY resolution down to 120 nm without the need for dedicated labelling procedures. Next Olympus has developed the next-generation X Line objectives with ultra-thin lenses manufactured by a revolutionary polishing technique to simultaneously boost numerical aperture, flatness and chromatic correction – providing exceptional image quality for researchers.
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Welcome
The Macquarie Neurodegeneration Meeting is an annual event hosted by the Centre for Motor Neuron Disease Research, Macquarie University. The aim of this event is for Australian neuroscientists to showcase their research and to stimulate conversation and foster collaboration to develop treatments for diseases including motor neuron disease, Alzheimer’s disease, frontal temporal dementia, Parkinson’s disease and other degenerative brain disorders. We welcome your involvement and hope the day provides inspiration and assists in fostering collaboration and connections in the neurodegeneration research community. Yours Sincerely, The Programme Committee https://www.mq.edu.au/research/research-centres-groups-and-facilities/healthy-people/centres/macquarie-university-centre-for-motor-neuron-disease-research/conference
Committee Members: Professor Ian Blair – Centre Director and Group Leader Associate Professor Julie Atkin - Centre Executive Member and Group Leader Dr Marco Morsch - Team Leader Neurophysiology and Imaging Dr Bingyang Shi - Macquarie University Research Fellow Dr Shu Yang - Postdoctoral Research Fellow Dr Amanda Wright - Postdoctoral Research Fellow Christina Cassidy – Centre Administrator
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Connection to Wi Fi
Connect to Network Name: Macquarie Events Browse to www.mq.edu.au you will be redirected to insert the passcode: neuro
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Friday 19 July
SESSION 1 Venue: Australian Hearing Hub, Level 1 Theatre 16 University Ave – Macquarie University
8:45 9:10
REGISTRATION & REFRESHMENT Room Location: Level 1 Theatre Session Chair: Dr Lyndal Henden
9:10 9:15
Symposium Opening Welcome remarks by Professor Ian Blair from the Macquarie University Centre for Motor Neuron Disease Research.
9:15 9:45
Professor Ian Blair (30 min) The arbitrary genetic classification of MND; strategies to unravel missing heritability
9:45 10:15
Professor Michael Breakspear (30 min) Impulsivity and Gambling in Parkinson’s Disease after Subthalamic Deep Brain Stimulation Correlates with the Structural Connectivity of the Stimulation Field
10:15 10:30
Dr Gary Morris (15 min) Molecular Mechanisms of Neurodegeneration in Alzheimer’s Disease: The Role of Impaired GluA2 RNA Editing
10:30 10:45
Thomas Kavanagh (15 min) Splicing Dysregulation in Parkinson’s Disease Suggests RBP Dysregulation
10:45 11:15
Morning Tea Trade Displays from our sponsor – Olympus Posters
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Friday 19 July
SESSION 2 Venue: Australian Hearing Hub, Level 1 Theatre 16 University Ave – Macquarie University
Room Location: Level 1 Theatre
Session Chair: Dr Luan Luu
11:15 11:45
Associate Professor Kay Double (30 min) Shared proteinopathy pathways in Parkinson disease and amyotrophic lateral sclerosis
11:45 12:15 Senior Professor Mark R Wilson (30 min) Rapid flow cytometry screen to identify novel MND drug leads
12:15 12:30
Dr Shu Yang (15 min) Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
12:30 12:45
Dr Vanessa Tan (15 min) Neurotoxin BMAA is a contributor to Wallerian-like degeneration, and its transcellular transmission
12.45 12.50
Sian Genoud (5 min) Copper-deficient SOD1 aggregation in Parkinson’s disease and Amyotrophic lateral sclerosis
12.50 12.55
Stephanie Raynor (5 min) Rapid, unbiased identification of protein inclusion components from patient post-mortem brain tissue using Biotinylation by Antibody Recognition (BAR)
12:55 1:55
Lunch Poster Session – Meet our poster presenters Trade Displays from our sponsor Olympus
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Friday 19 July
SESSION 3 Venue: Australian Hearing Hub, Level 1 Theatre 16 University Ave – Macquarie University
Room Location: Level 1 Theatre
Session Chair: Dr Sandy Stayte
1:55 2:25
Associate Professor Antony Cooper (30min) Parkinson’s Disease: From disease mechanisms to biomarkers and clinical trials
2:25 2:55
Dr Angela Laird (30min) Treatment with sodium valproate is protective for models of spinocerebellar ataxia-3
2:55 3:10
Dr Amanda Wright (15min) A Nanoparticle Based Strategy for Treating Inflammation in Motor Neuron Disease
3:10 3:15
Dr Alison Hogan (5min) In vivo analysis of variant pathogenicity for the validation of novel motor neuron disease linked genes
3:15 3:45
Afternoon Tea Trade Displays from our sponsor Olympus Posters
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Friday 19 July
SESSION 4 Venue: Australian Hearing Hub, Level 1 Theatre 16 University Ave – Macquarie University
Room Location: Level 1 Theatre
Session Chair: Professor Roger Chung
3:45 4:15
Professor Tim Karl (30min) New strategies for preclinical research into amyotrophic lateral sclerosis - using copper-zinc superoxide dismutase 1 (SOD1) transgenic mice as an example
4:15 4:45
Professor Thomas Fath (30min) Targeting the actin cytoskeleton to modulate the function of CNS neurons
4:45 5:00
Georgia Watt (15min) Novel behavioural characteristics of male human P301S mutant tau transgenic mice - a model for tauopathy
5:00 5:05 Monokesh K. Sen (5min) Behavioural and histological changes in cuprizone-fed mice
5:05 5:10
Dr Wai Kuen Chow (5min) A feasibility study of an ambulatory non-invasive ventilation (NIV) set up model using intelligent volume assured pressure support mode in MND
5:10 5:15 Closing remarks by Professor Roger Chung from the Motor Neuron Disease Research Centre
5:15 5:40 Poster, Canapés & Social Drinks
5:40 5:50 Prize Presentation
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Invited Speakers PROFESSOR IAN BLAIR MOTOR NEURON DISEASE RESEARCH CENTRE - DIRECTOR AND GROUP LEADER Macquarie University
Prof Ian Blair leads a team at the Macquarie University Centre for Motor Neuron Disease Research, that seeks to unravel the molecular genetic basis of MND and frontotemporal dementia (FTD). In the past 10 years, his group has played a key role in most MND gene discoveries worldwide. His team also uses these genes to develop cell and animal models that express the mutant proteins as tools to study the molecular basis of disease. These discoveries have been used to develop new diagnostic tests, predict disease onset and progression, and establish new models for therapeutic development.
PROFESSOR MICHAEL BREAKSPEAR
SYSTEMS NEUROSCIENCE - SCHOOL OF PSYCHOLOGY The University of Newcastle
Michael Breakspear is Professor of Systems Neuroscience at the University of Newcastle. He has made important contributions to our understanding of the basic principles underlying activity in large-scale circuits and networks of the brain. The work of his group combines mathematical modelling with the novel analysis of neuroimaging data. As a psychiatrist, he has translated this knowledge into new perspectives on dementia, schizophrenia, bipolar disorder, major depression and epilepsy.
ASSOCIATE PROFESSOR ANTONY COOPER
HEAD – NEURODEGENERATION AND NEUROGENOMICS PROGRAM & DIRECTOR OF THE AUSTRALIAN PARKINSON’S MISSION Garvan Institute of Medical Research
Associate Professor Antony Cooper did an undergraduate Honours degree at Otago University (New Zealand) and completed his PhD at McGill University before undertaking post-doctoral studies at the University of Oregon where as a cell and molecular biologist he investigated protein misfolding, cellular quality control, mitochondrial dysfunction and associated reactive oxygen species. These features coalesce prominently in Parkinson’s Disease and while a tenured faculty member at the University of Missouri he re-
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directed his research focus to identify the underlying molecular mechanisms of the disease. He returned to Australia in 2006 to join the Garvan Institute of Medical Research in Sydney where he has continued investigating the molecular mechanisms of the disease with emphasis on alpha-synuclein, and how it relates to known problems within the cells of patients. This work has expanded to extensive transcriptomics of relevant brain regions that has identified the contribution of both alternative splicing and non-coding RNAs to the disease. He is the Director of the Australian Parkinson’s Mission (www.TheAPM.org.au) whose $30m MRFF funding is to integrate genomics, biomarkers and patient iPS cell phenotyping into disease modifying clinical trials to identify therapeutics that slow or stop disease progression in people with PD.
ASSOCIATE PROFESSOR KAY DOUBLE
NEUROSCIENCE - BRAIN AND MIND CENTRE AND DISCIPLINE OF PHARMACOLOGY University of Sydney
Kay Double is a neurochemist and leads the Neurodegeneration Research Laboratory at the Brain and Mind Centre at the University of Sydney. She has a particular interest in mechanisms underlying neuronal vulnerability in neurodegenerative diseases with a focus on neurodegenerative disorders of movement, including Parkinson’s disease and amyotrophic lateral sclerosis (ALS). Her ultimate aim is to understand why neurodegenerative diseases lead to the death of specific neuronal groups in order to identify and test disease-modifying interventions. Her approach employs cutting-edge technologies, often applying methods rarely used with human brain tissues. In addition to investigating disease-associated changes in human post mortem tissues, she uses model systems to functionally test the effects of identified disease-linked pathways on neuronal survival. Her work on the uniquely human pigment neuromelanin demonstrated the protective and metal-binding properties of this
molecule in the healthy human brain but also demonstrated toxic changes to the pigment in Parkinson’s disease. More recently she discovered the first mechanistic link between Parkinson’s disease and ALS. Her group are now investigating the potential of this pathway for disease modification. Her work is highly collaborative and she leads a number of multidisciplinary, international research groups. A past member of the PNSW Board and Past-Secretary of the Australasian Neuroscience Society, she currently chairs the Parkinson’s New South Wales Expert Advisory group.
PROFESSOR THOMAS FATH
DEMENTIA RESEARCH CENTRE – DEPUTY DIRECTOR Macquarie University
Prof Fath is Deputy Director of the Dementia Research Centre in the Department of Biomedical Sciences at Macquarie University (Sydney, Australia). Previous to this, he was the Head of the Neurodegeneration and Repair Unit (NRU) and Head of the Neuronal Culture Core Facility (NCCF) at UNSW. He received his PhD in 2002 from the University of Heidelberg, where he worked on the functional role of Tau phosphorylation in Alzheimer's disease. He then moved for his postdoctoral research to The Scripps Research Institute at La Jolla (USA) and the Children's Hospital at Westmead, shifting his research focus to the study of the actin cytoskeleton in neuronal function and morphogenesis. In 2009, he took on a Research & Teaching position in the School of Medical Sciences at UNSW
Sydney, where he established an ARC and NHMRC-funded research program, investigating cytoskeleton-associated patho-mechanisms of Neurodegenerative diseases and mechanisms of neurite regeneration. He is now heading the Cellular Neurobiology Group in the Department of Biomedical Sciences at Macquarie University (Sydney, Australia). The primary research focus of his lab is on the regulation of the cytoskeleton in injury and diseases of the central nervous system. For this, his team employs cell and tissue cultures and genetically modified mice, combined with advanced imaging technologies. His research team investigates the actin cytoskeleton as potential drug target to provide protection and promote regeneration in the injured or diseased nervous system.
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PROFESSOR TIM KARL
BEHAVIOURAL NEUROSCIENCE - SCHOOL OF MEDICINE Western Sydney University
Tim Karl graduated from the Leipniz University of Hanover (Germany) in 2003 with a PhD in Zoology (Behavioural Neuroscience). Until 2008, he was a postdoctoral researcher at the Garvan Institute working on rodent models for anxiety and schizophrenia. In 2008, Tim established his own research team at Neuroscience Research Australia (NeuRA) before taking on an academic position at Western Sydney University in February 2016. His research focused on the neuro-behavioural consequences of gene-environment interactions in animal models for schizophrenia and the discovery of new therapeutic targets for dementia. Tim’s team also investigates the detrimental and potentially beneficial properties of cannabis constituents for brain disorders. As all of his group’s research is based on mouse model systems, Tim’s research also aims to enhance the validity of rodent models and the well-being of test animals in medical research by providing more stimulating
housing conditions and utilising more natural test system models. Prof Karl has published over 90 research articles, reviews and book chapters and is currently funded by the National Health and Medical Research Council (NHMRC Project Grants and Dementia Research Team Initiative), the ARC, and the Ainsworth Medical Research Innovation Fund.ssociate Professor John Kwok is a career scientist who was awarded a Bacholor of Science (Honours class I) from the University of Sydney, Australia in 1989. He was then awarded a PhD in 1994 from the University of Cambridge, England. He then worked as a post-doctoral scientist at the Garvan Institute from 1995. During this time, he was awarded two prestigious NHMRC fellowships. In 2005, he was made conjoint senior lecturer in recognition of his publication of scientific journals and students he had co-supervised from the University of New South Wales (UNSW). In 2006 he was recruited to Neuroscience Research Australia (NeuRA) to head a research laboratory focussing on the genetics and molecular biology of neurodegenerative diseases. In 2017, his team was recruited to the Brain and Mind Centre – University of Sydney to head a Neurogenetics and Epigenetics Laboratory, and this has allowed him to build a vibrant research team by recruiting junior and senior research officers.
DR ANGELA LAIRD
MOTOR NEURON DISEASE RESEARCH CENTRE - GROUP LEADER Macquarie University
Dr Laird studies the pathogenesis of movement disorders with a particular focus on identifying and testing potential disease treatments. Her group has been successful at producing and characterising the world’s first zebrafish model of spinocerebellar ataxia type-3 (also known as Machado Joseph Disease, MJD).
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PROFESSOR MARK WILSON
PROTEOSTASIS AND DISEASE RESEARCH CENTRE The University of Wollongong
Prof Wilson is a co-founding member of the ‘Proteostasis and Disease Research Centre’, a nucleus of biomedical scientists focussed on aspects of protein homeostasis and its relevance to human diseases (currently comprised of 6 team leaders within IHMRI, with external members at Uni Melb, ANU, Oxford and Cambridge). Wilson currently has 119 publications, mostly journal articles, but including 5 book chapters, 9 review articles and 1 AV work. His work has been published in some of the top journals in the field, including Nature Structural and Molecular Biology, Proceedings of the National Academy of Sciences USA, Annual Review of Biochemistry, Cell Reports, FASEB Journal, and Traffic.
He has studied chaperones for more than 25 years, their effects on protein folding/misfolding, and their roles in proteostasis. His group, based at the University of Wollongong, has pioneered discoveries of the first known secreted (extracellular) mammalian chaperones. The first identified and most extensively studied of these extracellular chaperones is clusterin (CLU), sometimes still called ApoJ. Together with Prof Heath Ecroyd and a number of student researchers, he recently developed a unique flow cytometry based technique that is able to rapidly and accurately quantify and characterise inclusions formed by essentially any aggregation-prone protein inside cells. This assay is being further developed for use as a drug-screening platform targetted to the treatment of protein aggregation diseases, such as motor neurone disease (MND).
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Invited Speaker Abstracts
Ian Blair
The arbitrary genetic classification of MND; strategies to unravel missing heritability
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Michael Breakspear
Impulsivity and Gambling in Parkinson’s Disease after Subthalamic Deep Brain Stimulation Correlates with the Structural Connectivity of the Stimulation Field
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Antony Cooper
Parkinson’s Disease: From disease mechanisms to biomarkers and clinical trials
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Kay Double
Shared proteinopathy pathways in Parkinson disease and amyotrophic lateral sclerosis
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Thomas Fath
Targeting the actin cytoskeleton to modulate the function of CNS neurons
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Tim Karl
New strategies for preclinical research into amyotrophic lateral sclerosis - using copper-zinc superoxide dismutase 1 (SOD1) transgenic mice as an example
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Angela Laird Treatment with sodium valproate is protective for models of spinocerebellar ataxia-3
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Mark Wilson
Rapid flow cytometry screen to identify novel MND drug leads
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Abstracts (15 min)
Thomas Kavanagh Splicing Dysregulation in Parkinson’s Disease Suggests RBP Dysregulation
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Gary Morris
Molecular Mechanisms of Neurodegeneration in Alzheimer’s Disease: The Role of Impaired GluA2 RNA Editing
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Vanessa Tan Neurotoxin BMAA is a contributor to Wallerian-like degeneration, and its transcellular transmission
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Georgia Watt Novel behavioural characteristics of male human P301S mutant tau transgenic mice - a model for tauopathy 31
Amanda Wright A Nanoparticle Based Strategy for Treating Inflammation in Motor Neuron Disease
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Dr Shu Yang
Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
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Abstracts (5 min)
Wai Kuen Chow
A feasibility study of an ambulatory non-invasive ventilation (NIV) set up model using intelligent volume assured pressure support mode in MND
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Sian Genoud Copper-deficient SOD1 aggregation in Parkinson’s disease and Amyotrophic lateral sclerosis
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Alison Hogan
In vivo analysis of variant pathogenicity for the validation of novel motor neuron disease linked genes
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Stephanie Raynor
Rapid, unbiased identification of protein inclusion components from patient post-mortem brain tissue using Biotinylation by Antibody Recognition (BAR)
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Monokesh K. Sen Behavioural and histological changes in cuprizone-fed mice
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Posters
Uptal Kumar Adhikari Predictive Selection of Non-Toxic and Non-Allergenic B-cell Epitopes for Alzheimer’s Disease Therapy
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Dalal Alali Effects of Neuromuscular Electrostimulation and exercise in the Treatment of Dysphagia in Adults with Multiple Sclerosis: A Randomized Controlled Trial.
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Rustam Asgarov Treatment with high bioavailable Longvida curcumin protects against chronic brain inflammation and behavioural deficits in the GFAP-IL6 mouse
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Katerina Christofides Exploring the relationship between neuroinflammation, excitatory neurotransmitters, and tryptophan metabolism: implications for mild cognitive impairment (MCI)
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Laura Dodds Associated social factors for cognition of older adults receiving community aged care services 48
Emily Don An Inducible Zebrafish Model of Sporadic Neurodegenerative Disease 49
Shal1ni Elangovan Neuroprotective Effects of Fecal Microbiota Transplantation in a mouse model of Alzheimer’s Disease
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Sandine Chan Moi Fat Novel disease gene discovery in a small amyotrophic lateral sclerosis kindred using in silico and in vitro analysis pipelines
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Michelle Garcez Minocycline reverses memory impairment induced by β-Amyloid (1-42) and reduces inflammatory parameters in the brain of mice via TLR-2 and NLRP3.
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Natalie Grima A functional pipeline for the validation of novel amyotrophic lateral sclerosis (ALS) candidate genes 53
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Stefan Guerra First behavioural evaluation of the novel CCNFem1ANU mouse model for amyotrophic lateral sclerosis frontotemporal spectrum disorder
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Umma Habiba Optical detection of Alzheimer’s disease in mice with single domain antibody fragments. 55
Lyndal Henden Identity by descent analysis links sporadic motor neuron disease cases to familial cases with identical SOD1 mutations and identifies five SOD1 founder events
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Quy-Susan Huynh Neural electrode scaffold development for cochlear implant function 57
Luan Luu Activation of Autophagy pathways as Potential Therapeutics for Machado Joseph Disease 58
Emily McCann Characterising the genetic architecture of sporadic motor neuron disease in Australia 59
Garry Niedermayer Characterisation of the transgenic IL6 overproducing mouse model of chronic gliosis 60
Ananda Staats Pires Exacerbated BH4 Metabolism in Experimental Colitis Pain 61
Anishchal Pratap Impaired neuronal adiponectin signalling in the 5XFAD mouse model of Alzheimer’s disease 62
Katherine Robinson Are motor neuron abnormalities correlated with impaired motor function in SOD1-expressing zebrafish?
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Natalie Scherer Investigating the role of oxidative stress in spinal motor neurons in a zebrafish model of ALS 64
Sina Shadfar Identifying novel roles for Protein Disulfide Isomerase (PDI) in Amyotrophic Lateral Sclerosis (ALS)
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Sandy Stayte Targeting kainate receptors to inhibit MPTP-induced degeneration in the mouse midbrain 66
Benjamin Trist SOD1 protein misfolding is associated with altered cellular copper handling and pathological TDP-43 and p62 in familial and sporadic ALS
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Andres Vidal-Itriago Pectoral fin axotomy model: in vivo study of microglial response to peripheral nerve injury in zebrafish
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Guoying Wang Intrinsically Fluorescent PAMAM Dendrimer as Drug carrier and Nanoprobe: Bioimaging and Neuron protection Study
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Maxinne Watchon Induction of the autophagy pathway improves the motor function of a transgenic zebrafish model of Spinocerebellar ataxia type 3
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Carol Ling-Yi Zhou-Zheng NGF-treated PC12 cells in Schwann cell cocultures have enhanced neuronal-like morphology 72
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Invited Speaker Abstracts
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PROFESSOR IAN BLAIR MOTOR NEURON DISEASE RESEARCH CENTRE - DIRECTOR AND GROUP LEADER Macquarie University
The arbitrary genetic classification of MND; strategies to unravel missing heritability Ian P. Blair
Centre for MND Research, Faculty of Medicine and Health Sciences, Macquarie University
Most of what we know about MND biology has stemmed from the study of the familial form of the
disease, made possible by a series of MND gene discoveries. Historically, around 10% of cases have
been classified as familial, with the remainder considered to be sporadic. This classification is
arbitrary and increasingly recognized as a “false dichotomy”. There is strong evidence that genetic
factors play a role in most MND cases, regardless of their classification. The genetic architecture
underlying MND, as a whole, is complex and this has limited our capacity to identify the genetic
and genomic factors underlying the onset and progression of the disease. The “penetrance” of the
genetic factors in MND is influenced by an individual’s genetic background together with unknown
environmental factors. This is often referred to as the “missing heritability” that underlies MND
susceptibility (in the general population) as well as wide variation in onset and progression in
families. Technological advances have begun to help, but more is required to unravel the genomic
complexity. Larger cohorts of well-characterised patients are also required. Coordinated
recruitment efforts are underway, including Australia’s SALSA consortium, to build cohorts with
sufficient power to identify the genetic, genomic and environmental factors that underlie the
disease. We are applying a range of traditional genetic approaches together with novel strategies to
identify the missing heritability seen among MND cohorts. To truly solve the molecular and
cellular basis of the disease will require a multidisciplinary approach that looks at the complex
interactions between genetics, genomics, biological systems and clinical measures. A coordinated
approach will also provide the best opportunity for targeted therapeutic studies.
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PROFESSOR MICHAEL BREAKSPEAR
SYSTEMS NEUROSCIENCE - SCHOOL OF PSYCHOLOGY The University of Newcastle
Impulsivity and Gambling in Parkinson’s Disease after Subthalamic Deep Brain Stimulation Correlates with the Structural Connectivity of the Stimulation Field Philip E. Mosley, Saee Paliwal , Katherine Robinson, Terry Coyne, Peter Silburn, Marc Tittgemeyer, Klaas E. Stephan, Alistair Perry, Michael Breakspear Subthalamic deep brain stimulation (STN-DBS) for Parkinson’s disease treats motor symptoms and improves quality of life, but can be complicated by adverse neuropsychiatric side-effects, typically characterised by impulsivity. It is unclear whether ‘at-risk’ persons can be identified prior to DBS, how much subthalamic stimulation accounts for the genesis of neuropsychiatric symptoms and which brain networks are responsible for their evolution. Using a comprehensive neuropsychological battery and a virtual casino, 55 persons with Parkinson’s disease (19 females, mean age 62, mean Hoehn and Yahr stage 2.6) were assessed prior to STN-DBS and 3-months postoperatively. Reward evaluation and response inhibition networks were reconstructed with probabilistic tractography using the subthalamic volume of activated tissue as a seed. Amongst the neuropsychological instruments, the greater the connectivity of the site of stimulation with these frontostriatal networks, the greater the impulsivity. This reversed the relationship between connectivity and impulsivity observed in these networks prior to DBS. In the virtual casino, larger bet sizes were associated with an increase in connectivity of the site of stimulation with right and left orbitofrontal cortex, right ventromedial prefrontal cortex and left ventral striatum. For all behaviours, connectivity of these networks at baseline was not associated with postoperative impulsivity, suggesting that the site and distribution of stimulation was a greater determinant of outcome. Notably, a distinction could be made amongst participants with clinically-significant, harmful changes in mood and behaviour attributable to DBS, based upon the interaction of their network connectivity with gambling behaviour. Additional analyses suggested that this distinction could be mediated by differential involvement of fibres in the limbic hyperdirect pathway and superolateral branch of the medial forebrain bundle. These findings identify a substrate of neuropsychiatric impairment after STN-DBS and suggest that tractography could be used to reduce the incidence of adverse neuropsychiatric effects as well as to guide motor outcomes.
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ASSOCIATE PROFESSOR ANTONY COOPER
HEAD – NEURODEGENERATION AND NEUROGENOMICS PROGRAM & DIRECTOR OF THE AUSTRALIAN PARKINSON’S MISSION Garvan Institute of Medical Research
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ASSOCIATE PROFESSOR KAY DOUBLE
NEUROSCIENCE - BRAIN AND MIND CENTRE AND DISCIPLINE OF PHARMACOLOGY University of Sydney
Shared proteinopathy pathways in Parkinson disease and amyotrophic lateral sclerosis
Kay L. Double Brain and Mind Center, ForeFront and Discipline of Pharmacology, The University of Sydney, Camperdown, NSW 2050, Australia Neurodegenerative diseases are traditionally categorised based their characteristic proteinopathies but recent data suggest greater overlap between these pathologies than previously recognised. We have reported superoxide dismutase 1 (SOD1) proteinopathy associated with neuronal death in some cases of familial amyotrophic lateral sclerosis (ALS) is recapitulated in degenerating brain regions in idiopathic Parkinson disease. We posit that misfolding and deposition of wild-type SOD1 in Parkinson disease occurs as a result of altered metalation of the protein, consistent with our findings of a marked and regional deficiency in copper in Parkinson disease brain. Other atypical post-translational modifications, such as oxidative changes, may also contribute to misfolding of this protein. Our data suggest these two phenotypically-distinct disorders share a yet unrecognised aetiological pathway which may offer tractable therapeutic target(s). To date, therapies aimed at modulating protein aggregation in neurodegenerative disease have met with limited success, however increasing our understanding of initial protein misfolding events may lead to the development of therapies which target biomolecular events upstream of protein deposition with beneficial effects across several disorders. References: Trist, B.G., Hare, D.J., Double, K.L. (2018) A proposed mechanism for neurodegeneration in movement disorders characterized by metal dyshomeostasis and oxidative stress. Cell Chem Bio. 25(7):807-816.
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PROFESSOR THOMAS FATH
DEMENTIA RESEARCH CENTRE – DEPUTY DIRECTOR Macquarie University
Targeting the actin cytoskeleton to modulate the function of CNS neurons The structural integrity and dynamic changes of the cell architecture of neurons are important for establishing neuronal circuits in the developing and proper information processing in the adult nervous system. One of the main building blocks of the neuronal cell architecture is the actin cytoskeleton. Remodelling of the actin cytoskeleton in subcellular compartments of neurons requires a large number of regulatory proteins. The function of many of these regulators are controlled by the association of tropomyosins with actin filaments. The decoration of actin filaments with different tropomyosin isoforms bestows the filaments with distinct structural and dynamic properties, which are essential to support neuronal function. Targeting specific tropomyosin isoforms thereby allows us to specifically manipulate the cellular architecture of neuronal subcellular compartments and to modulate the functional properties of neurons. Recent work from our group has identified the Tpm3 product Tpm3.1 as [1] a major regulator of neurite growth of primary hippocampal neurons grown on inhibitory substrates and [2] as integral component of the axon initial segment. We have identified both Tpm3.1 and the Tpm4 product Tpm4.2 as major constituents of the post-synaptic actin cytoskeleton. This presentation will discuss our findings on the common and distinct functional properties of the two major tropomyosin isoforms Tpm3.1 and Tpm4.2 and how these tropomyosins define distinct subcellular compartments in neurons.
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PROFESSOR TIM KARL
BEHAVIOURAL NEUROSCIENCE - SCHOOL OF MEDICINE Western Sydney University
New strategies for preclinical research into amyotrophic lateral sclerosis - using copper-zinc superoxide dismutase 1 (SOD1) transgenic mice as an example
Fabian Kreilaus1 *, Stefan Guerra1 *, Justin Yerbury2,3 Tim Karl1,4,5 #
1School of Medicine, Western Sydney University, NSW 2560, Australia
2 Illawarra Health and Medical Research Institute, Wollongong, NSW 2522 Australia
3School of Chemistry and Molecular Bioscience, Faculty of Science, Medicine and Health, University of Wollongong, NSW, Australia 2522
4Neuroscience Research Australia (NeuRA), NSW 2031, Australia
5School of Medical Sciences, University of New South Wales, NSW 2052, Australia
Mutations in copper-zinc superoxide dismutase 1 (SOD1) are a known genetic cause of amyotrophic lateral sclerosis (ALS) and the SOD1G93A mouse has been used extensively for preclinical research into ALS. Recent evidence suggests that ALS and frontotemporal dementia (FTD) form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1G93A mice in behavioural domains relevant to both ALS and FTD. We also assessed the impact of different cage systems (i.e. filter top versus individually ventilated cage systems) on the behavioural phenotype of SOD1G93A transgenic mice.
SOD1G93A males displayed reduced locomotion, exploration and increased anxiety-like behaviours compared to control males. Intermediate-term spatial memory was impaired in SOD1G93A females, while long-term spatial memory deficits as well as lower acoustic startle responses and prepulse inhibition were identified in SOD1G93A mice of both sexes compared to respective controls. The nature of changes found in social behaviours of male and female SOD1G93A were task-dependent. Cage systems had a moderate effect on particular behavioural characteristics of the SOD1G93A transgenic mice.
In conclusion, SOD1G93A mice exhibit a variety of sex-specific behavioural deficits beyond motor impairments supporting the notion of an ALS-frontotemporal spectrum disorder. Particular behavioural characteristics appear to be sensitive to the cage system used for housing. SOD1G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms relevant to ALS-frontotemporal spectrum disorder but attention should be given to their housing environment.
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DR ANGELA LAIRD
MOTOR NEURON DISEASE RESEARCH CENTRE - GROUP LEADER Macquarie University
Treatment with sodium valproate is protective for models of spinocerebellar ataxia-3 Angela S. Laird1, Maxinne Watchon1,2, Luan Luu1, Albert Lee1, Katherine J. Robinson, Isabella Lambert-Smith1, Maddy Tym1, Kristy Yuan1, Hannah Suddull1, Alana De Luca1, Caitlin Lucas1, Emily K. Don1, Roger S. Chung1, Garth A. Nicholson1,2 1. MQ Centre of MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2. ANZAC Research Institute, Concord Repartition Hospital, Sydney.
Spinocerebellar ataxia-3 (SCA-3, also known as Machado-Joseph disease, MJD) is a fatal neurodegenerative disease that impairs control and coordination of movement. SCA-3 is caused by expansion of a trinucleotide (CAG) repeat region within the ATXN3 gene, resulting in a long polyglutamine (polyQ) region within the ataxin-3 protein. Ataxin-3 has multiple reported functions, including regulation of transcription and histone acetylation; expansion of the polyQ tract within ataxin-3 is believed to impair these functions. Here we treated transgenic SCA-3 zebrafish larvae expressing human ataxin-3-84Q with the known histone deacetylase inhibitor sodium valproate from 1-6 days post-fertilisation (dpf). Treating the SCA-3 zebrafish with SV improved the distances swum by the zebrafish and also increased levels of acetylated histone 3 and 4 in the SCA-3 zebrafish, suggesting improved transcription regulation. Proteomic analysis of protein lysates generated from the treated and untreated SCA-3 zebrafish predicted that SV treatment had activated the sirtuin longevity signaling pathway. This was confirmed by a finding of increased SIRT1 protein levels in SV treated SCA-3 zebrafish. We also performed SV treatment of HEK293 cells expressing human ataxin-3 and found that the SV treatment also increased levels of SIRT1 in these cells. SV treatment of the HEK293 cells expressing ataxin-3-84Q also resulted in induction of the autophagy protein quality control pathway and a nucleus-to-cytoplasm shift of the autophagy substrate selector LC3. These results provide the first evidence of sodium valproate inducing activation of the sirtuin pathway and suggest that drugs that target the sirtuin pathway warrant further investigation for the development of treatments for SCA-3.
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PROFESSOR MARK WILSON
PROTEOSTASIS AND DISEASE RESEARCH CENTRE The University of Wollongong
Rapid flow cytometry screen to identify novel MND drug leads
Mark R Wilson 1, Heath Ecroyd 1, Angela Laird 2, Adam Walker 3
1. School of Chemistry and Biomolecular Science, Faculty of Science, Medicine & Health, Illawara Health & Medical Research Institute, University of Wollongong, NSW 2522, Australia. Email: [email protected] 2. Department of Biomedical Sciences, Centre for Motor Neuron Disease Research, Macquarie University, Sydney, NSW 2109, Australia. 3. Queensland Brain Institute, University of Queensland, St Lucia QLD 4072, Australia.
There are currently no effective treatments for the devastating consequences of MND. It is therefore imperative that new drugs are identified to provide effective therapies. If it were possible to identify small molecules that, by whatever mechanism, reduce the cytoplasmic accumulation and aggregation of TDP-43 and extend motor neuron cell viability, these could feasibly provide starting points for the development of new MND therapies. We developed a new flow cytometry-based approach to quantify TDP-43-containing inclusions in a motor neurone cell model, and have optimized this technique (called FloIT) as a high throughput drug-screening platform. This provides a primary screening tool to identify small molecules that reduce the cytoplasmic load of TDP-43 inclusions and preserve cell viability. Molecules to be screened will be drawn from the vast chemical libraries managed by Compounds Australia (Griffith Institute for Drug Discovery, Griffith University, Qld), which manages small molecule compound libraries (comprised of >670,000 compounds) for access by national and international life science research teams. Their exceptional robotics capabilities that will provide us with assay-ready microplates containing many compounds not available anywhere else in the world. Once hits are identified and confirmed, Compounds Australia will perform substructure searches and similarity matching so that additional focussed subsets can also be supplied for further testing. With the aid of collaborators, molecules identified as being of particular interest will subsequently be tested in zebrafish and mouse models of MND for their ability to reduce pathology.
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Abstracts (15 mins)
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DR THOMAS KAVANAGH
Centre for Neuroscience and Regenerative Medicine Garvan Institute of Medical Research
Splicing Dysregulation in Parkinson’s Disease Suggests RBP Dysregulation Tomas Kavanagh1, Boris Guennewig1,2,, Lee Marshall1,2,, Antony Cooper1,2
1. Parkinson’s Disease and Neurodegeneration Group, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia 2. St. Vincent’s Clinical School, Faculty of Medicine, UNSW Australia, Sydney, NSW 2010, Australia
Parkinson’s disease (PD) is a complex neurodegenerative disease for which there is no cure or disease-altering treatment. Most patients are idiopathic (~90%), demonstrating the need for greater understanding of the molecular mechanisms of PD so that new treatment targets, drugs and biomarkers may be discovered. We assayed post-mortem brain tissue of healthy and Parkinson’s disease patients across three tissues with an increasing degree of disease pathology. By utilising a combination of total- and targeted capture- RNA-seq, microarray and PCR technologies we demonstrate extensive dysregulation of alternative splicing (AS) in Parkinson’s disease. Genes dysregulated by AS include: multiple PD familial genes and GWAS linked candidate genes and notably, most genes identified as dysregulated by AS are not observed in differential expression analysis of the same dataset. The affected genes are strongly enriched in biological pathways of high relevance to PD. These mis-splicing events are predicted to impact many protein functional domains, are enriched in genes which interact with each other and include many neuronally regulated micro-exons and splicing factors. Importantly these AS events reveal enrichment for binding motifs of specific RNA binding proteins (RBPs) and share a significant number of AS events with RNA-Seq datasets of shRNA knockdowns of these RBPs in cells. This suggests that specific RBPs may be dysregulated in PD, a theme that is becoming more common in multiple neurodegenerative diseases, and results in the mis-splicing of genes observed. These AS changes open new insights into disease and could offer future avenues of treatment.
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DR GARY MORRIS
Centre for Neuroscience and Regenerative Medicine
University of Technology Sydney
Molecular Mechanisms of Neurodegeneration in Alzheimer’s Disease: The Role of Impaired GluA2 RNA Editing Gary P. Morris1,2, Amanda L. Wright3, Lyndsey M. Konen1,2, Benjamin K. Lau4, Raphael Zinn1,2, Patrick W. Seow4, David I. Finkelstein5, Gordon A. Royle6, Christopher W. Vaughan4, Bryce Vissel1,2 1. Centre for Neuroscience and Regenerative Medicine, Faculty of Science, University of Technology Sydney, 15 Broadway, 2007, Sydney, NSW, Australia 2. St. Vincent's Centre for Applied Medical Research (AMR), 405 Liverpool St, 2010, Sydney, NSW, Australia 3. Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW, 2109, Australia 4. Kolling Institute of Medical Research, Royal North Shore Hospital, The University of Sydney, Australia, 2065 5. Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Australia, 3010 6. Department of Haematology, Middlemore Hospital, Otahuhu, South Auckland, New Zealand, 1062 Alzheimer’s disease (AD) dementia is a complex molecular and multicellular disorder of unknown cause. Substantial evidence has implicated reduced Q/R site RNA editing of the AMPA receptor subunit GluA2 in the aetiology of AD, and several other neurological conditions including schizophrenia, Huntington’s disease, amyotrophic lateral sclerosis, astrocytoma, stroke and cocaine seeking behaviour in rats. In this study we investigated the connection between reduced Q/R site GluA2 RNA editing and neurodegenerative disease in two ways. First, we generated a mouse model of reduced Q/R site GluA2 RNA editing. This model exhibits synaptic signalling deficits, hippocampal neurodegeneration and motor and cognitive impairments, implicating the ‘unedited’ GluA2(Q) protein as an upstream driver of these phenotypes. Second, we investigated the contribution of ‘unedited’ GluA2(Q) to neurodegeneration, synapse pathology and memory and learning impairments in the hAPP-J20 mouse model of AD. Collectively, our studies suggest GluA2 RNA editing deficits are a novel therapeutic target for AD, and neurodegenerative disease more broadly.
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DR VANESSA TAN
Centre for Motor Neuron Disease Research
Macquarie University
Neurotoxin BMAA is a contributor to Wallerian-like degeneration, and its transcellular transmission Vanessa X. Tan, Say Hwa Tan, Adrian Teo, Jean-Michel Peyrin, Chai K. Lim, Gilles J Guillemin Background The cyanobacterial neurotoxin, β-N-methylamino-L-alanine (BMAA) has been associated with MND associated mechanisms such as excitotoxity and protein aggregation [1]. It causes neurotoxicity in whole cells, however, the specific effects of this neurotoxin on subcellular compartments such as axons, are not understood. Exposure to BMAA may contribute to an increased susceptibility in MND, however it is unclear how BMAA enters the central nervous system (CNS). Objectives Determine (1) the subcellular neurotoxicity of BMAA on primary murine and human neurons, and (2) if BMAA can be transmitted transcellularly retrograde and anterograde in these neuronal cells in a prion-like manner. Methods Murine and human primary tissue cultures have been approved by the Macquarie University and C2EA-05 Ethics Committee. A patented microfluidic chip with physical and fluidic isolation is used to determine axonal and somatic neurotoxicity in primary neurons through the administration of BMAA in either somatic or axonal compartments. Results were quantified by immunostaining, to determine axonal fragmentation, cellular death, and presence of BMAA; from images of at least 6 frames per chip, and at least three independent experiments in triplicates. Results Low levels (50µM) of somatic BMAA exposure causes significant axonal fragmentation, but limited somatic death only at 500µM (p<0.0001) BMAA can be transported anterograde to neurons (62%, p<0.0014) and astrocytes (23%, p<0.05) in a dose-dependant manner. A new, improved design of microfluidic chip has been synthesized to investigate retrograde transcellular transmission of BMAA, and is currently being tested for retrograde transmission Discussion These results indicate that BMAA can contribute to neuronal network collapse, and that BMAA can spread through neuronal pathways. Importantly, the low levels of BMAA required to cause significant axonal damage indicate that it can contribute to Wallerian-like degeneration, leading to neuronal network collapse and loss of function, even though the cell soma remains intact. From a clinical perspective, this presents an opportunity to rescue damaged phenotypes by encouraging axonal repair or regeneration to restore function. Transport of BMAA through neuronal cells may mimic the focal neuroanatomical spread of neurodegenerative pathologies in MND [2]. This is also a feasible method by which peripheral exposure to BMAA can be the entry point for the neurotoxin into the CNS. These both are mechanisms which can be targeted to halt or delay the spread of BMAA, and if causative to MND, the spread of pathology. References
1. Cox PA, Davis DA, Mash DC, et al Proc Biol Sci 2016 283:979-980
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2. Braak H, Braak E Acta Neuropathol 1991 82:239-259
Acknowledgements We thank the patients for tissue donations. Funding was provided by the MNDRIA from the Lady Fairfax MND Research Grant.
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GEORGIA WATT
Western Sydney University
Novel behavioural characteristics of male human P301S mutant tau transgenic mice - a model for tauopathy Georgia Watt1, Magdalena Przybyla2, Janet van Eersel2, Arne Ittner2, Lars M Ittner2 and Tim Karl1,3,#
1. School of Medicine, Western Sydney University, Campbelltown, Australia 2. Dementia Research Centre and Department of Biomedical Sciences, Faculty of Medicine
and Health Sciences, Macquarie University, Australia 3. Neuroscience Research Australia (NeuRA), Randwick, Australia
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline and the accumulation of two hallmark proteins, amyloid-beta (Aβ) and tau [1]. Traditionally, transgenic mouse models for Alzheimer’s disease (AD) have generally focused on Aβ pathology, however, in recent years a number of tauopathy transgenic mouse models have been developed, including the TAU58/2 mouse model. These mice develop tau pathology and neurofibrillary tangles from 2 months of age and show motor impairments and alterations in the behavioural response to elevated plus maze testing [2-4]. The cognitive and social phenotype of this model has not yet been assessed in greater detail. Furthermore, the behavioural changes seen in the elevated plus maze have previously been linked to both anxiety and disinhibitory phenotypes. Thus, this study assessed 4 months old TAU58/2 males for disinhibitory and social behaviours, social recognition memory and sensorimotor gating. TAU58/2 males demonstrated reduced exploration and anxiety-like behaviours but no changes to disinhibitory behaviours, reduced sociability in the social preference test and impaired acoustic startle and prepulse inhibition. Aggressive and socio-positive behaviours as well as social recognition memory were not affected. Our study identified new phenotypic characteristics of young adult male TAU58/2 transgenic mice and clarified the nature of behavioural changes seen in these mice during elevated plus maze testing. Social withdrawal and inappropriate social behaviours are common symptoms in both AD and FTD patients and impaired sensorimotor gating is seen in moderate-late stage AD, emphasising the relevance of the TAU58/2 model to these diseases [5].
References:
[1] P. F. Chapman, A. M. Falinska, S. G. Knevett,M. F. Ramsay, Genes, models and Alzheimer's disease, 17(5) (2001) 254-261. [2] M. Przybyla, C. H. Stevens, J. Van Der Hoven, A. Harasta, M. Bi, A. Ittner, A. Van Hummel, J. R. Hodges, O. Piguet, T. Karl, M. Kassiou, G. D. Housley, Y. D. Ke, L. M. Ittner,J. V. Eersel, Disinhibition-like behavior in a P301S mutant tau transgenic mouse model of frontotemporal dementia, 631 (2016) 24-29.
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[3] A. Van Der Jeugd, B. Vermaercke, G. M. Halliday, M. Staufenbiel,J. Götz, Impulsivity, decreased social exploration, and executive dysfunction in a mouse model of frontotemporal dementia, 130 (2016) 34-43. [4] J. van Ersel, C. H. Stevens, M. Przybyla, A. Gladbach, K. Stefanoska, C. Chan, K. Xin, W. Y. Ong, J. R. Hodges,G. T. Sutherland, Early-onset Axonal Pathology in a Novel P301S-Tau Transgenic Mouse Model of Frontotemporal Dementia, Neuropathology Appl Neurobiol. (2015). [5] A. s. Association, 2018 Alzheimer's disease facts and figures, Alzheimer's & Dementia 14(3) (2018) 367-429.
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DR AMANDA WRIGHT
Centre for Motor Neuron Disease Research
Macquarie University
A Nanoparticle Based Strategy for Treating Inflammation in Motor Neuron
Disease
Amanda Wright1, Anne Zou1, Paul Della Gatta2, Adam Walker3, Bingyang Shi1 Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2. Deakin University, Melbourne Burwood Campus, 221 Burwood Highway, Burwood VIC 3125; Queensland Brain Institute, The University of Queensland Building 79, Upland Rd Brisbane Qld 4072 Australia Motor neuron disease (MND) is associated with neuron loss in the brain and spinal cord. Marked neuroinflammation also occurs within the CNS, however its contribution to disease pathogenesis is still unresolved. We have discovered a myriad of upregulated cytokines and chemokines in a novel MND mouse model, known as the rNLS model (1). Interestingly, we have identified that a specific chemokine (hereby termed ChemA) is highly upregulated in bigenic rNLS mice at an early stage of disease, prior to the appearance of neuron loss or motor symptoms. In bigenic rNLS mice, ChemA showed a 130-fold increase of mRNA at 2 weeks and a 38-fold increase at 4 weeks as compared to age-matched littermate controls. To confirm this result at the protein level, multiplex ELISAs were performed, revealing an 11-fold increase in ChemA protein expression in the cortex at 2 weeks. We hypothesise that targeting ChemA overexpression may provide a novel therapeutic treatment for disease. To target ChemA overexpression, we have encapsulated an siRNA against ChemA within a novel nanoparticle (NP) that is able to cross the Blood brain barrier (siChemA-NP). To date, we have shown that ChemA expression is substantially increased in LPS-stimulated microglia in vitro, and this increase is significantly reduced in the presence of an siChemA-NP. We have now completed initial trials of intravenous injections of siChemA-NP in rNLS mice to determine the effects of ChemA reduction in its ability to reduce motor impairment in MND. There are currently no treatments to prevent or cure Motor Neuron Disease (MND). This is in part due to the complex nature of the brain and the difficulties faced with delivering pharmaceuticals to deep areas that are damaged in disease. To effectively cross the blood brain barrier (BBB), molecules must be very small, meaning many large molecule therapeutics are unable to be utilised. My team at Macquarie University have developed a nanoparticle (NPs) drug delivery system that can cross the BBB to deliver short interfering RNA’s (siRNAs) to disease-affected regions of the brain/spinal cord. References: (1) Walker et al. (2015) "Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43." Acta Neuropathol. 130(5):643-60.
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DR SHU YANG
Centre for Motor Neuron Disease Research
Macquarie University
Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
Shu Yang1*, Emily P. McCann1*, Jennifer A. Fifita1*, Natalie Grima1, Jasmin Galper1, Prachi Mehta1, Sarah Freckleton1, Katharine Y Zhang1, Alison Hogan1, Sandrine Chan Moi Fat1, Lyndal Henden1, Kelly L. Williams1, Natalie Twine1,2, Denis Bauer2, John Kwok3, Glenda Halliday3, Matthew Kiernan3, Julie Atkin1,5, Dominic B. Rowe1, Garth A. Nicholson1,5,6,7, Adam K Walker4, Ian P. Blair1
1 Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia; 2Commonwealth Scientific and Industrial Research Organization, Health & Biosecurity Flagship, Sydney, Australia; 3 Brain and Mind Centre, Sydney Medical School, The University of Sydney, Sydney, Australia; 4 Neurodegeneration Pathobiology Laboratory, Queensland Brain Institute, The University of Queensland, Queensland, Australia; 5 Northcott Neuroscience Laboratory, ANZAC Research Institute, Sydney, New South Wales, Australia; 6 Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia; 7 Molecular Medicine Laboratory, Concord Hospital, Concord, New South Wales, Australia
Since the first report of CHCHD10 mutations in ALS/FTD patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of CHCHD10 in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. Whole exome and genome sequencing data from familial and sporadic ALS and FTD cases, were parsed for genetic variation in CHCHD10. CHCHD10 expression was characterised via immunohistochemistry and western blotting in control, ALS and/or FTD post-mortem tissues and further in the TDP-43 rNLS transgenic mouse model of TDP-43 pathology. No causal, novel or bona fide risk variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human CNS tissues, CHCHD10 was specifically expressed in neurons. While no changes in CHCHD10 level were apparent in ALS and ALS/FTD patients, a significant decrease was observed in FTD patients frontal cortex tissues. In the rNLS TDP-43 mice, CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in the cortex in the mid-stage of disease. Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. We showed that in humans, CHCHD10 may play a specific role in neurons and a loss of CHCHD10 function may be linked to FTD but not to ALS or ALS/FTD. Our data in rNLS TDP-43 mice suggested that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis. References: BANNWARTH, S., et al 2014. A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement. Brain, 137, 2329-45.
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Abstracts (5 mins)
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DR WAI KUEN CHOW
Macquarie University Hospital
A feasibility study of an ambulatory non-invasive ventilation (NIV) set up model using intelligent volume assured pressure support mode in MND
Wai Kuen Chow1, Dominic Rowe1, Brendon Yee2, Amanda Piper2
1. Department of Clinical Medicine, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2. Royal Prince Alfred Hospital, Camperdown, NSW 2050 Australia
Background In Australia, non-invasive ventilation (NIV) initiation typically requires an in-hospital admission and/or a diagnostic polysomnogram (PSG) followed by a PSG-directed titration. However, delay in commencing NIV while awaiting PSG-titration results in poorer 12-month survival.1 Auto-titrating NIV modes such as intelligent volume assured pressure support (iVAPS, ResMed) are routinely available in portable ventilators, but data is limited regarding compliance in MND. Objectives To evaluate the feasibility and compliance of an ambulatory NIV set-up model using iVAPS mode in symptomatic MND patients. Methods An open label prospective study was conducted in symptomatic MND patients who presented with overnight pulse oximetry, SpO2 <90% for >5% of total sleep time. The initial acclimatisation phase to the mask and pressurised air (20-30 minutes) occurred during an outpatient consultation (90 minutes). A cloud-based patient management system (ResMed AirView) enabled compliance monitoring and ventilator settings adjustments in the home. Respiratory consultations occurred at one, three and six months following the initiation of therapy. Ventilator data were downloaded for analysis of compliance and therapy settings. Results Nine MND patients enrolled: [median(IQR)], age 58(54-69) years, BMI 28.70(24.20-30.45) kg/m2 and ALSFRS scores 37(33-41). At one-month, median(IQR) average iVAPS usage was 5.8(4.65-8.45) hours/night. Compliance at six-months increased for all patients: median average usage 7.9(6.40-9.70) hours/night. Therapy settings at six-months: [median(IQR)] expiratory positive airway pressure 8(6-9) cmH20, 95thpercentile inspiratory positive airway pressure 14(13-16) cmH20, targeted tidal volume (VT) 6.4(6.2-6.7) ml/kg IBW with delivered VT of 450(419-462) ml. Discussion NIV initiation using ambulatory iVAPS set up model was feasible and achieved good compliance to therapy. Reference: Sheers N, et al. Improved survival with an ambulatory model of non-invasive ventilation implementation in motor neuron disease. Amyotrophic Lateral Sclerosis Frontotemporal Degeneration. 2014;15(3-4):180-184.
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SIAN GENOUD
The University of Sydney
Copper-deficient SOD1 aggregation in Parkinson’s disease and Amyotrophic lateral sclerosis Sian Genoud1*, Michael WM Jones2, Sylvain Bohic3, Dominic J Hare4, Kay L Double1
1. Department of Pharmacology, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2050, Australia; 2. Central Analytical Research Facility, Institute of Future Environments, Queensland University of Technology, Brisbane, Australia; 3. European Synchrotron and Radiation Facility, Grenoble, France; 4. Atomic Pathology Laboratory, Melbourne Dementia Research Centre at the Florey Institute of Neuroscience and Mental Health and The University of Melbourne, Parkville, Vic. 3052, Australia * Presenting author correspondence; [email protected]
Recently we identified a marked reduction in Cu1, and alterations in the metalloprotein superoxide dismutase 1 (SOD1) in the Parkinson’s disease (PD) brain2. SOD1 normally binds Cu and Zn in a 1:1 ratio for antioxidant activity and structural stability, however a lack of Cu-binding is implicated in SOD1 misfolding and neurotoxicity in another disease amyotrophic lateral sclerosis (ALS)3. We therefore quantified Cu and Zn levels in both SOD1 microaggregates within PD and ALS, and in Lewy bodies of PD, to determine Cu:Zn ratios in deposited proteins in these neurodegenerative diseases. X-ray fluorescence microscopy at the European Synchrotron and radiation facility, Advanced Photon Source and Australian Synchrotron obtained precise measurements of numerous biometals associated with microaggregates in PD and ALS human post-mortem tissues. Cutting-edge ptychography was also employed to identify high resolution images of structural components and distinguishing characteristics of these microaggregates. Directly comparing SOD1 aggregates to Lewy bodies within the PD brain highlights distinguishing features of these microaggregates identified within the same tissues, while the comparison of SOD1 aggregates in PD to those identified in ALS, provides a greater understanding of the mechanisms underlying protein misfolding of these potentially neurotoxic microaggregates in these distinct neurodegenerative diseases. An altered Cu:Zn ratio associated with SOD1 aggregates in both the PD brain and ALS spinal cord indicates Cu-deficient SOD1 may underlying protein misfolding and dysfunction of this antioxidant protein within these vulnerable regions. This presents a viable therapeutic target in restoring Cu to Cu-deficient SOD1 in vulnerable regions of both PD and ALS. References:
1. Genoud S, Roberts BR, Gunn AP et al. (2017) Subcellular compartmentalisation of copper, iron, manganese and zinc in the Parkinson’s disease brain. Metallomics. 18;9(10); 1447-1455 2. Trist BG, Davies KM, Cottam V, Genoud, S et al. (2017). Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson’s disease brain. Acta neuropathologica, 134(1); 113-127. 3. Bruijn LI, Houseweart MK, Kato S, et al. (1998). Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science, 281(5384); 1851-1854
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DR ALISON HOGAN
Centre for Motor Neuron Disease Research
Macquarie University
In vivo analysis of variant pathogenicity for the validation of novel motor neuron disease linked genes Alison Hogan1, Jennifer Fifita1, Shu Yang1, Lyndal Henden1, Emily McCann1, Natalie Grima1, Emily McCann1, Ian Blair1
1. Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, North Ryde, NSW 2109, Australia.
Motor neuron disease (MND) has a strong genetic component. Identification of MND-linked mutations has provided great insight into pathological mechanisms of the disease. However, approximately 40% of familial MND patients carry an unidentified MND-linked mutation. Disease in these remaining families often does not follow true Mendelian inheritance patterns and are challenging to solve. Traditional genetic analysis typically identifies multiple variants that segregate with disease but cannot determine which variant is pathogenic. We have established a functional pipeline for in vitro and in vivo assessment of variant pathogenicity with the aim of identifying novel MND-linked genes. This pipeline has been applied to one family (MQ1) in which two potential variants, located within 1.2 mega base pairs of each other, have been identified. The in vivo component of the pipeline assesses variant pathogenicity through transient overexpression in zebrafish. Transient overexpression of MND-linked mutations in multiple genes (including TARDBP, FUS, SOD1, CCNF) induces MND-relevant pathology in zebrafish, including aberrant axonal morphology, increased cell death and reduced motor function. Overexpression of both MQ1 variants increased cell death. However, neither variant induced an axonal or motor phenotype. We hypothesise that due to the chromosomal proximity of the variants, there is a high probability that they are linked and that MND develops in this family due to a combined effect of the variants. Supporting this hypothesis, co-expression of both variants did induce both aberrant axonal morphology and impaired motor function in zebrafish. Validation of these findings in patient derived motor neurons is currently in progress.
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STEPHANIE RAYNER
Centre for Motor Neuron Disease Research
Macquarie University
Rapid, unbiased identification of protein inclusion components from patient post-mortem brain tissue using Biotinylation by Antibody Recognition (BAR)
Stephanie Rayner1, Rowan Radford1, Roger Chung1, Albert Lee1
1. Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia
Many neurodegenerative diseases are characterised by the formation of insoluble protein inclusions in the brains of affected patients [1]. The composition of these aggregates from patient post-mortem tissue has provided invaluable insight into the mechanisms that lead to disease however, the insolubility of aggregate components limits the use of standard antibody-based approaches frequently used to study protein-protein interactions. A recently developed proximity-ligation method enables identification of insoluble interactomes from fixed, post-mortem tissue [2]. Here, we apply Biotinylation by Antibody Recognition (BAR) followed by mass spectrometry to specifically identify the composition of cytoplasmic phosho-Tau aggregates found in Progressive supranuclear palsy (PSP) patients. BAR is a recently developed method, by which a primary antibody recognises the target of interest in fixed samples. A secondary antibody conjugated to horseradish peroxidase recognises the primary antibody, and, with the addition of biotin phenol and hydrogen peroxide, facilitates the rapid deposition of biotin onto proteins within the vicinity of the antibody complex [2]. Biotinylated proteins are subsequently identified following reverse cross-linking, homogenisation and a streptavidin-conjugated bead pull-down. To identify the isolated proteins, an on-bead trypsin digest is conducted before tryptic peptides are analysed by mass spectrometry. Using BAR in fixed, post-mortem brain tissue from PSP patients, we identified several known aggregate components found in Tauopathies. Our data also identified confidently assigned phosphorylation sites that have been reported in other Tauopathies but not PSP. Together these data validate our approach for rapidly revealing the aggregate components of Tauopathies whilst also identifying many novel components that may provide valuable insight into disease mechanisms upon careful validation. References: [1] Ross, C. A., Poirier, M. A., Protein aggregation and neurodegenerative disease. Nature medicine 2004, 10 Suppl, S10-17. [2] Bar, D. Z., Atkatsh, K., Tavarez, U., Erdos, M. R., et al., Biotinylation by antibody recognition-a method for proximity labeling. Nature methods 2018, 15, 127-133.
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MONOKESH KUMER SEN
Western Sydney University
Behavioural and histological changes in cuprizone-fed mice
Monokesh K Sen1, Mohammed SM Almuslehi1,2, Jens R Coorssen3, David A Mahns1 and Peter J Shortland3*
Affiliation: 1School of Medicine, and 4Science and Health, Western Sydney University, New South Wales, Australia. 2Department of Physiology, College of Veterinary Medicine, Diyala University, Diyala, Iraq.3Departments of Health Sciences and Biological Sciences, Faculties of Applied Health Sciences and Mathematics & Science, Brock University, Ontario, Canada. *Corresponding author A/Prof. Peter J Shortland Department of Medical Sciences, School of Science & Health, Western Sydney University, Locked Bag 1797, Penrith NSW 2751, Australia E-mail: [email protected] Ph +61 2 4620 3804 Abstract Feeding cuprizone (CPZ) to rodents causes demyelination and reactive gliosis in the CNS; these are hallmarks of some neurodegenerative diseases like multiple sclerosis. Clinically, these conditions cause sensory and motor symptoms, such as pain and loss of motor function. However, relatively little is known regarding the behavioural deficits associated with CPZ-feeding and much of what is known is contradictory. This study investigated whether over 5 weeks oral feeding of 0.2% CPZ to young adult mice triggers sensorimotor behavioural changes. Behavioural tests included measurement of nociceptive withdrawal reflex responses, exploratory behaviour and locomotion tests and these were compared to histological analysis of the relevant CNS regions by analysis of neuronal and glial cell components. Within 2-3 weeks of CPZ-feeding, mice showed hyperactivity using the grooming and rearing tests and crossed the ladder more quickly compared to control mice. On both the ladder and beam tests, CPZ mice exhibited more foot slips compared to controls. In contrast, no changes in nociceptive thresholds to thermal or mechanical stimuli were seen between groups. Histological analysis showed demyelination throughout the CNS, which was most prominent in white matter tracts in the cerebrum but also elevated in areas such as the hippocampus, basal ganglia and diencephalon. Profound demyelination and gliosis was seen in the deep cerebellar nuclei and brainstem regions associated with the vestibular system. However, in the spinal cord changes were minimal. No loss of neurons or motoneurons was found but a significant increase in astrocyte staining was seen throughout the white matter tracts of all levels of the spinal cord. The results suggest that CPZ induces subtle motor changes such as ataxia and that this is associated with deficits in CNS regions associated with motor and balance functions such as the cerebellum and brainstem. Key words: Cuprizone, behaviour, motor incoordination, oligodendrocytosis
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Poster Abstracts
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UTPAL KUMAR ADHIKARI
School of Medicine, Western Sydney University,
Predictive Selection of Non-Toxic and Non-Allergenic B-cell Epitopes for Alzheimer’s Disease Therapy Utpal K Adhikari, Sachin Kumar and Mourad Tayebi School of Medicine, Western Sydney University, Campbelltown, NSW, Australia
Abstract Alzheimer’s disease (AD) is a chronic neurodegenerative disorder clinically characterized by progressive impairment of memory, cognitive ability, and behavioral changes. AD is recognized as a significant health burden worldwide, but no effective treatment or preventive vaccine is available. To date, research in this area focused mainly on attempting to eliminate/neutralise Aβ peptides derived from amyloid precursor protein (APP) instead of stabilising the full-length APP to prevent Aβ peptide formation associated with this disorder. Our study aimed at discovering novel potential non-toxic and non-allergenic B-cell epitopes targeting located on the entire APP using immunoinformatic approaches. We selected the wild-type and 17 Alzheimer associated mutated APP and conducted extensive physicochemical analysis for the identification of non-toxic and non-allergenic B-cell epitopes from linear epitopes. Initially, we found a total of 192 linear B-cell epitopes derived from the 3D protein structure; but only seven unique non-toxic and non-allergenic B-cell epitopes were identified and found to be common to the A2V, English, Flemish, Italian, Florida, Lowa, Osaka, Swedish, and Tottori Alzheimer mutations. These epitopes are localized to the most conserved E2 domain of the APP. Since more than 90% of experimentally identified epitopes are conformational epitopes, we also predicted the conformational epitopes from the protein structure to validate our unique seven linear non-toxic and non-allergenic epitopes. Surprisingly, the same regions were found to be conformational epitopes which suggest these epitopes as potential target outside the Aβ regions. Consequently, these findings indicate that the E2 domain of APP might be an effective new target for the screening and development of novel therapeutic antibody or peptide-based active immunization strategy for the prevention of Alzheimer’s disease. References: Panza, F., Lozupone, M., Logroscino, G., Imbimbo, B.P., 2019. A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease. Nat. Rev. Neurol. 15, 73–88. Saha, S., Raghava, G.P.S., 2007. Prediction of neurotoxins based on their function and source. In Silico Biol. 7, 369–387. Potocnakova, L., Bhide, M., Pulzova, L.B., 2016. An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction. J. Immunol. Res. 2016, 6760830. Källberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., Xu, J., 2012. Template-based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511.
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DALAL ALALI
Department of Speech Pathology
University of Sydney
Effects of Neuromuscular Electrostimulation and exercise in the Treatment of Dysphagia in Adults with Multiple Sclerosis: A Randomized Controlled Trial. Dalal Alali1, Kirrie Ballard2, Hans Bogaardt3 Department of Speech Pathology, Faculty of Health Sciences, University of Sydney, NSW 2141, Australia
Background: There are limited treatment options for dysphagia in adults with Multiple Sclerosis. In previous studies, neuromuscular electrostimulation (NMES) showed positive therapeutic effects in treating dysphagia, however these studies had limitations. Objectives: The aim of this study was to determine the effectiveness of NMES paired with an effortful swallow in improving or restoring the swallowing function and the quality of life in adults with MS-related dysphagia with an explanatory parallel-group randomized controlled trial. Methods: Twelve adults with MS were randomly assigned to two groups: active electrical stimulation group (n=6) or sham electrical stimulation group (n=6). Randomisation was carried out by a computer system. Both groups were treated twice per week for three weeks. At baseline and two weeks post treatment, fiberoptic endoscopic evaluation of swallowing was conducted to evaluate the effectiveness of the treatment using outcome measures including bolus residue scale, penetration/aspiration scale, and functional oral intake scale and swallowing quality of life questionnaire. Results: After treatment, participants who received active stimulation showed no yoghurt residue in the bolus residue scale yoghurt subscale. They also showed a significant improvement in the Symptom Frequency domain in the swallowing quality of life questionnaire. Improvements were noted in all other measures; however those improvements were not significant. No adverse effects were reported during or after the treatment. Conclusion: The study suggests a potential benefit of NMES in eliminating and or reducing residue of thick fluids in dysphagic adults with mild MS and in improving their quality References: 1.Tassorelli, C., et al., Dysphagia in multiple sclerosis: from pathogenesis to
diagnosis. Neurological Sciences, 2008. 29(S4): p. 360-363. 2.Matsuo, K.D.D.S.P. and J.B.M.D. Palmer, Anatomy and Physiology of Feeding and Swallowing:
Normal and Abnormal. Physical Medicine and Rehabilitation Clinics of North America, 2008. 19(4): p. 691-707.
3.Marchese-Ragona, R., et al., Evaluation of swallowing disorders in multiple sclerosis. Neurological Sciences, 2006. 27(S4): p. s335-s337.
4.Buchholz, D., Neurologic causes of dysphagia. Dysphagia, 1987. 1(3): p. 152-156. 5.Karagiannis, M.J.P., L. Chivers, and T.C. Karagiannis, Effects of oral intake of water in patients
with oropharyngeal dysphagia. BMC geriatrics, 2011. 11(1): p. 9-9. 6.Grigoriadis, N., V. Pesch, and M.S.G. Paradig, A basic overview of multiple sclerosis
immunopathology. European Journal of Neurology, 2015. 22(S2): p. 3-13. 7.Alali, D., K. Ballard, and H. Bogaardt, The Frequency of Dysphagia and its Impact on Adults
with Multiple Sclerosis Based on Patient-Reported Questionnaires. Multiple Sclerosis and Related Disorders, 2018.
8.De Pauw, A., et al., Dysphagia in multiple sclerosis. Clinical Neurology and Neurosurgery, 2002. 104(4): p. 345-351.
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9.Sura, L., et al., Dysphagia in the elderly: management and nutritional considerations. Clinical interventions in aging, 2012. 7: p. 287-298.
10.Giusti, A. and M. Giambuzzi, Management of dysphagia in patients affected by multiple sclerosis: state of the art. Neurological Sciences, 2008. 29(S4): p. 364-366.
11.Bogaardt, H., et al., Use of neuromuscular electrostimulation in the treatment of dysphagia in patients with multiple sclerosis. Annals of otology, rhinology, and laryngology, 2009. 118(4): p. 241-246.
12.Alali, D., K. Ballard, and H. Bogaardt, Treatment Effects for Dysphagia in Adults with Multiple Sclerosis: A Systematic Review. Dysphagia, 2016. 31(5): p. 610-618.
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RUSTAM ASGAROV
School of Medicine
Western Sydney University
Treatment with high bioavailable Longvida curcumin protects against chronic brain inflammation and behavioural deficits in the GFAP-IL6 mouse Rustam Asgarov1, Huazheng Liang1,2, Faheem Ullah1, Amanda Fernandez1, Garry Niedermayer5, David Harman1,2, Erika Gyengesi1,2, Tim Karl3, Gerald Muench1,2,4 1. Department of Pharmacology, School of Medicine, Western Sydney University, Campbelltown, Australia 2. Molecular Sciences Research Group, School of Medicine, Western Sydney University, Campbelltown, Australia 3. Department of Behavioural Neuroscience, School of Medicine, Western Sydney University, Campbelltown, Australia 4. National Institute of Complementary Medicine, Western Sydney University, Campbelltown, Australia 5. School of Science and Health, Western Sydney University, Campbelltown, Australia Introduction Patients affected by Alzheimer’s and other neurodegenerative diseases manifest common clinical symptoms which involve high level brain inflammation, oxidative stress and resultant progressive deterioration of cognitive capabilities. We used the GFAP-IL6 mouse which specifically expresses interleukin-6 cytokine in the brain. In this project, we investigated proteome profile of brain cerebellum region of control and GFAP-IL6 male mice to find significantly regulated proteins. We also studied treatment effects of curcumin, a natural compound which has multiple in-vivo anti-inflammatory (e.g. cytokine suppressing) and anti-oxidant activity and may potentially prevent brain inflammation and preserve neurological functions. Research aims include to examine whether 6-month long treatment with high bioavailability Longvida curcumin (500 mg/kg), downregulates microglial and astroglial activation and prevents behavioural deficits in the GFAP-IL6 mouse. Results Assessment of motor performance by elevated beam walking test showed significantly deteriorated ‘traverse time’ and ‘footslips’ scores of GFAP-IL6 mice in comparison to control mice. In addition, assessment by accelerod test revealed significant motor learning performance of mice across test trials. Female mice exhibited significantly better accelerod performance than male mice. Accelerod test data also displayed significant interaction effect of food and gender factors on the motor performance of mice. Memory assessment by Barnes maze showed significant spatial learning of mice in scores of primary latency, distance and errors (P=0.016, P=0.016 and P=0.045, respectively) as well as total latency, distance and errors (P>0.001, P=0.001 and P=0.003, respectively) across trials. Additionally, almost significant interaction effect of food and test trials was found on memory performance of mice in primary measures (P=0.055, P=0.055 and P=0.074, respectively). Significant positive correlation was found between TSPO levels in cerebellum brain region of mice and their average ‘traverse time’ and ‘footslips’ scores (P=0.009 and P=0.035, respectively). We found significantly downregulated cytosolic soluble proteins, peroxiredoxin-6, triosephosphate isomerase, heat shock cognate 71 kDa protein, haemoglobin subunit beta-1 and upregulated proteins, tubulin α-chain, phosphatidylethanolamine binding protein, stress-70 protein, clusterin, syntaxin, calbindin, transthyretin, superoxide dismutase in proteome analysis. Among membrane bound/hydrophobic proteins neurofilament medium polypeptide was upregulated and mitochondrial membrane protein ATP synthase subunit beta was downregulated.
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Discussion In conclusion, our data shows that GFAP-IL6 mice exhibit common phenotypic features of chronic brain inflammation including neurological impairment. Mice treated with Longvida curcumin in long term exhibited strong trend of spatial memory improvement. In addition, cerebelli of GFAP-IL6 mice also showed changes in abundance of proteins involved in oxidative stress response, glycose metabolism, neuronal cytoskeleton, synaptic activity, mitochondrial metabolism, intracellular Ca signalling etc. Therefore, GFAP-IL6 is highly suitable model for investigation of human brain inflammation disease. Behavioural, immunohistochemical and proteomics studies of GFAP-IL6 mice with higher doses and longer feeding times of Longvida curcumin will potentially lead to formulation of effective therapeutic treatment for brain inflammation diseases.
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KATERINA CHRISTOFIDES
Western Sydney University/ NICM
Exploring the relationship between neuroinflammation, excitatory neurotransmitters, and tryptophan metabolism: implications for mild cognitive impairment (MCI) K. Christofides1, C K. Lim2, G Z. Steiner1 1. NICM Health Research Institute, Western Sydney University, Penrith, NSW 2751, Australia k.christofides@westernsydney,edu.au 2. Centre for Motor Neuron Disease Research, Macquarie University, Macquarie Park, NSW 2109, Australia Neurodegenerative diseases such as Alzheimer’s Disease (AD) and the prodromal phase, mild cognitive impairment (MCI), are characterised by accelerated age-inappropriate cognitive decline.1 Currently, there are no early interventional strategies for MCI and it is not known what causes some patients to progress to AD. It is thought that neuroinflammation and dysregulation of key neurotransmitters contribute to this decline in memory and cognition.2 Recently, it has been shown that dysregulation in tryptophan metabolites in the Kynurenine Pathway (KP) is related to pro-inflammatory markers in patients with schizophrenia.3 Considering the prevalence of cognitive impairments and increased risk of dementia in those with schizophrenia4,5, this relationship between inflammation and KP dysregulation may be relevant for MCI. Furthermore, attention and cognition were found to be related to the Kynurenine to Tryptophan (KYN/TRP) ratio and was elevated in a subgroup of patients with high inflammation.3 We hypothesise that regulatory mechanisms within the KP may contribute to the dysregulation of neurotransmitters in the context of a pro-inflammatory environment. Our recent literature review suggests that further analysis of the contribution of neurotransmitters and other metabolites is warranted in MCI and specifically cognition. This study aims to delineate the complex link between biochemical imbalance, inflammation and cognitive function in MCI.
1. Petersen RC, Doody R, Kurz A, Mohs RC, Morris JC, Rabins PV, Ritchie K, Rossor M, Thal L, Winblad B. Current concepts in mild cognitive impairment. Archives of neurology. 2001 Dec 1;58(12):1985-92. 2. Zarrindast MR. Neurotransmitters and cognition. In Neurotransmitter interactions and cognitive function 2006 (pp. 5-39). Birkhäuser Basel. 3. Kindler J, Lim CK, Weickert CS, Boerrigter D, Galletly C, Liu D, Jacobs KR, Balzan R, Bruggemann J, O’Donnell M, Lenroot R. Dysregulation of kynurenine metabolism is related to proinflammatory cytokines, attention, and prefrontal cortex volume in schizophrenia. Molecular psychiatry. 2019 Apr 3:1. 4. Lyketsos CG, Peters ME. Dementia in patients with schizophrenia: evidence for heterogeneity. JAMA psychiatry. 2015 Nov 1;72(11):1075-6. 5. Ribe AR, Laursen TM, Charles M, Katon W, Fenger-Grøn M, Davydow D, Chwastiak L, Cerimele JM, Vestergaard M. Long-term risk of dementia in persons with schizophrenia: a Danish population-based cohort study. JAMA psychiatry. 2015 Nov 1;72(11):1095-101.
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LAURA DODDS
Australian Institute of Health Innovation
Macquarie University
Associated social factors for cognition of older adults receiving community aged care services
Laura Dodds1, Andrew Georgiou1, Johanna Westbrook1, Joyce Siette1
1. Centre for Health Systems and Safety, Australian Institute of Health Innovation, Macquarie University, Sydney, NSW 2109
Social networks play a crucial protective role in slowing the progression of cognitive impairment in older adults1-3. However, little knowledge exists about the impact of remaining socially connected amongst older individuals receiving home and community based aged care services. Hence, we aimed to investigate the association between cognitive function and interpersonal relationships in this population. A sample of older Australians (n=178) receiving community aged care services in NSW responded to questions about social networks, health-related quality of life and cognitive function. Data were also collected on community care service use and sociodemographic variables. Cognitive function was measured using the Telephone Interview for Cognitive Status-Modified (TICS-M). Using multiple regression analyses we ascertained the associations between quality of life, level of social support, relationship status, demographics and cognitive impairment. The sample was predominantly female (65.8%), with a mean age of 80.4± 6.7 years. A third had a cognitive impairment (37.6%) and reported a moderately high level of social networks (M=33.5, SD=11.8). Significant predictors of higher cognitive capacity included being male, having better social networks, requiring fewer service hours, and receiving a variety of service types. Our findings suggest that accessing a diverse range of aged care service types may enhance the quality of social networks, which may be important to better cognitive functioning. It also has important implications for community aged care services designed to reduce social isolation, particularly in community-dwelling males. Future studies could look at introducing more robust technologies to effectively assess cognitive function, and associated factors in this population. References: 1. Wang H, Karp A, Winblad B, et al. Late-life engagement in social and leisure activities is associated with a decreased risk of dementia: A longitudinal study from the kungsholmen project. Am J Epidemiol 2002;155(12):1081-87. doi: 10.1093/aje/155.12.1081 2. Holtzman RE, Rebok GW, Saczynski JS, et al. Social Network Characteristics and Cognition in Middle-Aged and Older Adults, 2004:P278-P84. 3. Barnes LL, Mendes De Leon FC, Wilson SR, et al. Social resources and cognitive decline in a population of older African Americans and whites. Neurology 2004;63(12):2322-26. doi: 10.1212/01.WNL.0000147473.04043.B3
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DR EMILY DON
Centre for Motor Neuron Disease Research
Macquarie University [email protected]
An Inducible Zebrafish Model of Sporadic Neurodegenerative Disease
Emily K Don1+, Elinor Hortle1+, Alison L Hogan1, Isabel Formella1, Marco Morsch1, Caitlin W Lucus1, Claire G Winnick1, Natalie Scherer1, Andres Vidal-Itriago1, Sharron Chow1, Roger S Chung1, Garth A Nicholson1,2, Nicholas J Cole1, Angela S Laird1
1. Centre for Motor Neuron Disease, Faculty of Health and Medical Sciences, Department of Biomedical Science, Macquarie University, Sydney, NSW, Australia
2. Concord Clinical School and ANZAC Research Institute, Concord Repatriation Hospital, Concord, NSW, Australia
Motor neuron disease (MND) is a fatal neurodegenerative disease that affects an increasing number of people each year. Treatment is limited, and the cause of disease is unknown. Historically, insights into the pathological trigger of MND have come from genetic studies of familial MND, which only account for approximately 10% of cases, leaving the remaining 90% of sporadic cases with no clear genetic link. Recent studies show that almost all cases of MND (including sporadic cases) share a common neuropathology characterized by the mislocalisation of TAR-DNA binding protein (TDP-43) to the cytoplasm of neurons, and the deposition of TDP-43-positive protein inclusions. To investigate the role of TDP-43 neuropathology in MND, we aimed to develop a novel transgenic model that would give insight into sporadic MND. Firstly, we found that transgenic expression of human wild type TDP-43 in a quasi-ubiquitous pattern was toxic, causing high levels of apoptosis and high levels of early mortality. We therefore established a spatial and temporal inducible transgenic zebrafish line expressing wild type human TDP-43 fused to EGFP (EGFP-Hsa.TDP-43). Induced larvae expressed full length EGFP-Hsa.TDP-43 for up to 48 hours post induction (hpi). However, by 96 hpi the full length EGFP-Hsa.TDP-43 was completely degraded leaving only smaller cleavage fragments of the protein, without any loss of GFP signal. Despite this degradation, the EGFP-Hsa.TDP-43 zebrafish had a reduced swimming phenotype at 96 hpi (6 days post fertilisation, dpf). Furthermore, constitutive expression of EGFP-Hsa.TDP-43 resulted in an increased occurrence of apoptotic cell death of cells expressing EGFP-Hsa.TDP-43 at 48 hpf. Taken together these findings demonstrate that the expression of human TDP-43 protein, even for only a short duration (days) is enough to produce apoptotic cell death and motor impairment in these in vivo models. Further, this manuscript demonstrates the benefits of using inducible transgenic models when studying proteins related to neurodegenerative disease.
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SHALINI ELANGOVAN
Laboratory of Molecular Neuroscience and Dementia, Brain and Mind Centre
The University of Sydney
Neuroprotective Effects of Fecal Microbiota Transplantation in a mouse model of Alzheimer’s Disease Shalini Elangovan1, Thomas J. Borody2, R. M. Damian Holsinger1,3 1Laboratory of Molecular Neuroscience and Dementia, Brain and Mind Centre, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2050 2Centre for Digestive Diseases, Level 1, 229 Great North Road, Five Dock, NSW 2046 3Discipline of Pathology, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney NSW 2006 Correspondence: [email protected] The intestinal microbiome has been shown to play a significant role in health and disease. Replenishing the gut microbiome with healthy donor fecal microbiota transplants has been established as a treatment for diseases such as Chron’s, ulcerative colitis and Clostridium difficile infection (CDI). The involvement of the intestinal microbial environment in neurodegenerative diseases such as Alzheimer’s and Parkinson’s has recently been demonstrated, prompting investigation into the restoration of gut microbiota in an attempt to reduce cognitive and behavioral effects associated with these diseases. Here we demonstrate the efficacy of fecal microbiota transplantation (FMT) in Alzheimer’s disease whereby treatment of old (30-32 weeks) transgenic 5xFAD Alzheimer’s mice resulted in significantly improved cognition. 5xFAD mice were gavaged a fecal slurry prepared from age-matched, wild-type littermate donors for a period of 7 days. Following a 21-day incubation period, animals were tested on spatial and recognition memory tasks. FMT treated mice demonstrated significant improvements of spatial learning and memory as well as enhanced recognition memory compared to transgenic littermates receiving saline. We demonstrate for the first time, the benefits of FMT in improving cognition in an aged mouse model of Alzheimer’s.
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SANDRINE CHAN MOI FAT
Centre for Motor Neuron Disease Research
Macquarie University [email protected]
Novel disease gene discovery in a small amyotrophic lateral sclerosis kindred using in silico and in vitro analysis pipelines
Sandrine Chan Moi Fat1, Jennifer A Fifita1, Shu Yang1, Emily P McCann1, Kelly L Williams1, Natalie Twine1,2, Denis Bauer2, Garth A Nicholson1,3, Ian P Blair1
1Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia 2 Commonwealth Scientific and Industrial Research Organization, Health & Biosecurity Flagship, Sydney, New South Wales, Australia 3ANZAC Research Institute, University of Sydney, Concord Hospital, Sydney, New South Wales, Australia
Amyotrophic lateral sclerosis (ALS), is a paralytic disorder caused by the death of motor neurons. About 10% of ALS cases are hereditary (familial ALS), of which one third still have an unknown genetic cause. The goal of this project is to find the causative gene mutation in a small ALS kindred using a mix of next generation sequencing (NGS) data, in silico, and in vitro tools. NGS data was generated from two affected siblings and their “married-in” parent. Shared variant analysis, including filtering to nonsynonymous and novel variants, resulted in 23 candidate variants. In silico analyses, namely protein prediction programs, conservation, genic tolerance, expression in brain/spinal cord, and gene function were used to rank each candidate variant. The top three variants after in silico analysis were then selected for evaluation through a novel in vitro analysis pipeline. Overexpression of wild-type and mutant constructs of the top three candidate genes in HEK293T cells did not cause significant cell toxicity. However, the mutant version of one candidate gene showed differences in cellular localisation as compared to its wild type. Genetics, in silico and in vitro pipelines have potential to elucidate the remaining causative gene mutations in small ALS kindreds. Further work should continue with the in vitro pipeline to continue with the assessment of all candidate genes identified from NGS analysis. This is vital to identify the disease cause in this family, which will help improve our understanding of the disease mechanisms and contribute to the development of future treatments.
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MICHELLE LIMA GARCEZ
Neuroinflammation Research Group, Faculty of Medicine and Health Sciences, Department of Biomedical Sciences Macquarie University [email protected]
Minocycline reverses memory impairment induced by β-Amyloid (1-42) and reduces inflammatory parameters in the brain of mice via TLR-2 and NLRP3.
Garcez ML1, Bellettini-Santos T2, Mina F2, Schiavo GL2, Luz AP2, Medeiros EB2, Budni J2, Guillemin GJ1.
1. Neuroinflammation Research Group, Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, Macquarie University, Sydney, NSW, 2109, Australia. 2. Laboratory of Experimental Neurology, Graduate Program in Health Sciences, University of Southern Santa Catarina, Criciúma, SC, 88806-000, Brazil.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss, accumulation of β-amyloid peptide (Aβ) (mainly Aβ1-42), and neuroinflammation, which plays a central role in the development and progress of the AD. In this study, 100-day-old Balb/c mice were administered with Aβ1-42 oligomers (400 pmol/site), into the left cerebral ventricle (ICV), to induce the AD-like dementia model. Twenty-four hours after ICV administration, the animals were treated with minocycline (50 mg/kg, via oral gavage) for 17 days. The spatial memory was assessed by radial arm-maze task and Y-maze task. The treatment with minocycline reversed the spatial memory impairment caused by Aβ1-42. In addition, minocycline reversed the increased levels of interleukin 1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and, IL-10 caused by Aβ (1-42) in the hippocampus and, in the cortex, the minocycline treatment reversed the increased levels of IL-1β, TNF-α and, IL-4. In the serum, Aβ1-42 increased the levels of IL-1β and IL-4, and minocycline was able to reverse this. In addition, minocycline reduced the levels of Aβ1-42 and microglial activation in the animals administrated with Aβ1-42, which was accompanied by the reduction of toll-like receptors 2 (TLR2) content, and its adapter protein MyD88, as well as a reduction in the levels of the protein NLRP3, protein indispensable in the assembly of the inflammasome. These results were evaluated by immunohistochemistry and confirmed using the western blot analysis. Thus, the anti-inflammatory effect of minocycline involves TLR2 receptors and NLRP3, besides being beneficial by ameliorating memory impairment.
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NATALIE GRIMA
Centre for Motor Neuron Disease Research
Macquarie University [email protected]
A functional pipeline for the validation of novel amyotrophic lateral sclerosis (ALS) candidate genes Natalie Grima1, Sharlynn Wu1, Jennifer Fifita1, Emily McCann1, Sandrine Chan Moi Fat1, Jasmin Galper1, Sarah Freckleton1, Katharine Zhang1, Shu Yang1 and Ian Blair1 1 Macquarie University Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia Email: [email protected] Background: Disease gene discoveries continue to drive our understanding of the molecular and cellular mechanisms underlying amyotrophic lateral sclerosis (ALS). While the causative gene mutation has been identified in around 60% of ALS families, the remaining families often do not follow true Mendelian inheritance patterns making them difficult to solve by traditional genetic analysis alone. In this study, we aim to develop an in vitro functional pipeline for the assessment and validation of novel ALS candidate genes. Methods: A panel of cell-based assays including immunofluorescence, flow cytometry and Western blot analysis were used to evaluate the effect of candidate gene mutants on known ALS pathologies. Examined ALS pathologies for each candidate included neuronal inclusion formation, interaction with TDP-43, cellular toxicity and accumulation in detergent-insoluble cellular fractions. Immunohistochemical and immunofluorescent staining of ALS patient spinal cord tissues were also used to determine whether candidates were present in neuronal inclusions. Results: The pipeline was applied to five candidate gene mutations identified in an ALS family negative for known ALS gene mutations. 2/5 mutants caused significantly higher toxicity and protein degradation defects compared to the wild-type, formed detergent insoluble inclusions and co-aggregated with TDP-43 in transfected neuronal cell lines. One mutant also co-localised with pathological TDP-43-positive neuronal inclusions in sporadic ALS patient spinal cord. Conclusion: We have successfully prioritised two novel candidate ALS genes using the proposed in vitro functional pipeline. These candidate gene variants will next be examined in animal models to garner further evidence for a role in ALS pathogenesis.
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STEFAN GUERRA
Western Sydney University
First behavioural evaluation of the novel CCNFem1ANU mouse model for amyotrophic lateral sclerosis frontotemporal spectrum disorder
Stefan Guerra1 *, Sandip Ghimire 1 *, Madilyn Coles 1 *, Ian Blair 2, Roger Chung 2 Gaetan Burgio 3 and Tim Karl.1,4,5
1School of Medicine, Western Sydney University, NSW 2560, Australia. 2Macquarie University Centre for Motor Neuron Disease Research, NSW 2109, Australia. 3John Curtin School of Medical Research (JCSMR), Australian National University, ACT 2601, Australia. 4Neuroscience Research Australia (NeuRA), NSW 2031, Australia 5School of Medical Sciences, University of New South Wales, NSW 2052, Australia *authors contributed equally Email contacts:
Recently, mutations in the gene encoding for Cyclin f (CCNF) have been identified in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and are believed to contribute to the ALS-Frontotemporal spectrum disorder. The potential impact of CCNF mutations on behavioural domains of a mammalian model has not been evaluated to date. Therefore, we developed a CCNFem1ANU point mutation mouse model using CRISPR/Cas9 gene editing technology carrying a substitution S621G and evaluated the effects of mutant CCNFem1ANU on motor behaviours and cognitive domains. As the onset and specifics of disease-relevant phenotypes of genetic mouse models are often age- and sex-dependent, both male and female CCNFem1ANU heterozygous, homozygous and wild type-like littermates at ages 10-12 months (adulthood) and 19-21 months (old age) were tested. Mice were assessed in a battery of disease-relevant behavioural tests for both ALS (weekly bodyweight recording, pole and accelerod testing as well as grip strength assessment) and FTD (testing social preference, social interaction, resident-intruder aggression, sensorimotor gating and fear-associated memory). Main parameters of the behavioural assessments of motor functions, social behaviours including aggression, sensorimotor gating, and cognitive domains of transgenic mice were unaffected by the CCNFem1ANU mutation compared to control mice at the ages tested. Mutant mice developed an overgrooming phenotype in their home cage at later age, which has to be investigated further. In conclusion, the first behavioural evaluation of a novel CCNFem1ANU mouse model revealed no gross behavioural abnormalities in the domains tested. Future studies will have to clarify the type of effect of the CCNFem1ANU mutation on the developing mouse physiology.
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UMMA HABIBA
Western Sydney University [email protected]
Optical detection of Alzheimer’s disease in mice with single domain antibody fragments.
Umma Habiba1 Dian Anggraini2 Utpal Kumar Adhikari3, Mourad Tayebi4 Neuro-immunology lab, School of Medicine, Western Sydney University, Sydney, NSW 2560, Australia Alzheimer’s disease (AD) is a progressive neurodegenerative disorder exhibiting gradual decline of cognitive function and memory loss. One of the principal neuropathological lesion observed in the brains of AD patients is the extracellular deposition of amyloid plaques. Aβ soluble oligomer (Aβo) is considered the most common neurotoxic form of Aβ species and is the intermediary conformation recognised in early pathogenesis. Early and accurate diagnosis of Alzheimer's disease (AD) is a major goal in order to reduce the impact of dementia and also represents an unmet medical need globally. The underlying molecular mechanisms of Alzheimer’s disease begins years before the clinical diagnostic confirmation. Therefore, researchers have been exploring ways to detect the disease at early stage before it affects the brain. Recently, a non-invasive method was developed, namely, optical coherence tomography angiography (OCTA) to examine retinal abnormalities associated with Alzheimer’s disease and other eye diseases. It was reported that post mortem eyes derived from patients with AD, showed signs of thinning in the centre of the retina and degeneration of retinal ganglion cells and the optic nerve. This suggests that possible retinal identification of Aβo may be an important pathological lesion that might identified early before neuropathology has ensued. Here, we used formalin-fixed paraffin-embedded (FFPE) retinal tissue derived from the 5xFAD mice model. This model displayed high levels of Aβo at early stages of the disease progression following assessment using immunohistochemistry (IHC) and immunofluorescence (IF) studies that were used to immune-detect retinal Aβo. Of importance, we show that Aβo was present in all retinal cell layers but more prominent in the ganglion layer. Finally, Aβo co-localised to late endosome within the cytosol of retinal cells, indicating that disturbance of the endocytic pathway leads to accumulation and aggregation of cellular Aβ that causes neurotoxicity, synaptic loss and amyloid plaque formation. In order to validate the above results, we have used another AD mouse model, namely, the APP/PS1 mice model to detect retinal Aβo but this time using our unique camelid anti-Aβo antibody. Here, fresh-frozen retinal sections derived from various APP/PS1 age groups were probed with tagged camelid anti-Aβo antibody. Similarly, we show that Aβo was detected very early in the disease process via IHC and IF. Moreover, these retinal Aβo were seen in all retinal cell layers and homed to late endosomes. Our data supports the hypothesis that the soluble Aβo are detected in the retina of AD animal models in early disease stage. The development of a diagnostic test screen for early AD using camelid anti-Aβo antibody for the detection of retinal oligomers is a major milestone towards the development of a human AD diagnosis. Keywords: Alzheimer’s disease; Aβo; Retina; Camelid antibodies; IHC, IF. References: [1] J.C. Blanks, S.Y. Schmidt, Y. Torigoe, K.V. Porrello, D.R. Hinton, and R.H. Blanks, Retinal
pathology in Alzheimer's disease. II. Regional neuron loss and glial changes in GCL. Neurobiology of aging 17 (1996) 385-95.
[2] A. Serrano-Pozo, M.P. Frosch, E. Masliah, and B.T. Hyman, Neuropathological alterations in Alzheimer disease. Cold Spring Harbor perspectives in medicine 1 (2011) a006189.
[3] U. Sengupta, A.N. Nilson, and R. Kayed, The Role of Amyloid-beta Oligomers in Toxicity, Propagation, and Immunotherapy. EBioMedicine 6 (2016) 42-49.
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DR LYNDAL HENDEN
Centre for Motor Neuron Disease Research
Macquarie University
Identity by descent analysis links sporadic motor neuron disease cases to familial cases with identical SOD1 mutations and identifies five SOD1 founder events
Lyndal Henden1, Natalie A. Twine2, Piotr Szul3, Emily P. McCann1, Garth A. Nicholson4, Dominic B. Rowe1,5, Matthew Kiernan6, Denis C. Bauer2, Ian P. Blair1 and Kelly L. Williams1 1. Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2. Transformational Bioinformatics, CSIRO, Sydney, NSW 2113, Australia; 3. Data61, CSIRO, Dutton Park, QLD 4102, Australia; 4. Concord Clinical School, ANZAC Research Institute, Concord Repatriation Hospital, Sydney, NSW 2139, Australia; 5. Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2019, Australia; 6. Brain and Mind Institute, The University of Sydney, Sydney, NSW 2050, Australia Email of presenting author: [email protected]
Approximately 10% of motor neuron disease (MND) cases have a family history of disease, while the remaining cases present as apparently sporadic. Heritability studies suggest a significant genetic component to sporadic MND, and although most sporadic cases have an unknown genetic etiology [1], mutations that are present in familial MND cases have also been found in sporadic cases [2]. This suggests that some sporadic cases may actually be unrecognised familial cases with reduced penetrance. Identifying a familial basis of disease in apparently sporadic MND cases has important genetic counselling implications for immediate relatives, including a 50% chance of inheriting the mutation and a significantly increased chance of developing MND. Identity-by-descent (IBD) analysis detects genomic regions that have been inherited from a common ancestor and is a powerful strategy to uncover a familial basis in apparently sporadic cases. We performed IBD analysis on 89 Australian familial MND cases from 24 families and four sporadic MND cases, each carrying one of three common SOD1 mutations in Australia (p.I114T, p.V149G and p.E101G). We identified five unique haplotypes carrying these mutations in our cohort, indicative of five founder events. This included two different haplotypes carrying SOD1 p.I114T, where one haplotype was present in two sporadic cases and 19 families and the second haplotype was found in the remaining two sporadic cases and one family, thus linking these familial and sporadic cases. Furthermore, we linked two families carrying SOD1 p.V149G and found that SOD1 p.E101G had arisen independently in both families carrying this mutation. References 1. Renton AE, Chio A and Traynor BJ (2014). State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci. 17(1): 17-23 2. Jones CT, Swingler RJ, Simpson SA and Brock DJH (1995). Superoxide dismutase mutations in an unselected cohort of Scottish amyotrophic lateral sclerosis patients. J Med Genet. 32: 290-292 3. Al-Chalabi A, van den Berg LH and Veldink J (2017). Gene discovery in amyotrophic lateral sclerosis: implications for clinical management. Nat Rev Neurol. 13: 96-104
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QUY-SUSAN HUYNH
The University of Sydney [email protected]
Neural electrode scaffold development for cochlear implant function Quy-Susan Huynh1,2, Philip Boughton3, Norbert Dommel4, Paul Carter4 and R. M. Damian Holsinger1,2 1. Laboratory of Molecular Neuroscience and Dementia, Brain and Mind Centre, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW 2. Discipline of Pathology, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW 3. Sydney Spine Institute, Burwood, NSW 4. Cochlear Ltd, Macquarie University NSW [email protected] Abstract Spiral ganglion neurons (SGNs) represent target cells for the electrode of the cochlear implant (CI). However, at time of insertion, degeneration of some SGNs occurrs while others are difficult to access as they are located behind the cochlea’s bony modiolar wall[1]. The CI provides suboptimal hearing as charge spreads from the electrode towards the surrounding tissue rather than solely to target tissue.[2] A deeper insertion depth of the electrode leads to an increase in scar tissue formation and the introduction of trophic factors into the cochlea for the regeneration of neural tissue requires a sustainable delivery method. [3,4] Our neural electrode scaffold system uses electrical stimulation (ES) to promote brain-derived neurotrophic factor (BDNF) expression, a potent trophic factor in the brain. ES can increase the size of the cell as well as extensions of neurites of SGNs and direct them towards the electrodes. Structural support for the cells and their neurites is provided through the addition of an electrospun nanofibre scaffold on the electrode. In a proof-of-concept experiment, the scaffold was electrospun onto wires and glass coverslips and tested in vitro using SH-SY5Y neuroblastoma and Schwann cells. The scaffolds and cell attachment to the scaffolds were analysed using scanning electron microscopy and tubulin immunostaining. We observed that not only did cells attach to the scaffold but also formed networks on the fibres. There was evidence of neurite extension aligning along the electrospun fibres and cell filopodia extending and interacting with the nanotopography. References [1] T. G. Landry, A. K. Wise, J. B. Fallon, and R. K. Shepherd, "Spiral ganglion neuron survival and function in the deafened cochlea following chronic neurotrophic treatment," Hearing research, vol. 282, pp. 303-313, 2011. [3] O. Adunka and J. Kiefer, "Impact of electrode insertion depth on intracochlear trauma," Otolaryngol Head Neck Surg, vol. 135, pp. 374-82, Sep 2006. [4] T. Lopatina, N. Kalinina, M. Karagyaur, D. Stambolsky, K. Rubina, A. Revischin, et al., "Adipose-Derived Stem Cells Stimulate Regeneration of Peripheral Nerves: BDNF Secreted by These Cells Promotes Nerve Healing and Axon Growth De Novo," PLOS ONE, vol. 6, 2011.
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DR LUAN LUU
Centre for Motor Neuron Disease Research
Macquarie University
Activation of Autophagy pathways as Potential Therapeutics for Machado Joseph Disease
Luan Luu 2, Maxinne Watchon1,2,3, Kristy Yuan2, Albert Lee2, Hannah Suddull2, Alana De Luca2, Nicholas J. Cole2, Roger S. Chung2, Garth A. Nicholson1,2,3, Angela S. Laird2. 1Sydney Medical School, University of Sydney 2Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University 3ANZAC Research Institute, Concord Repartition Hospital, Sydney Contact: [email protected], [email protected] Machado Joseph Disease (MJD) is a fatal neurodegenerative disease. MJD is caused by the expansion of the glutamine repeat in the Ataxin-3 protein. Normally there are 14-37 glutamine repeats, however in MJD patients there are 56-84 glutamine repeats which causes Ataxin-3 to aggregate and form neurotoxic inclusions. In animal models of MJD, there is an inhibition of Histone acetyl transferase (HAT) and hypoacetylation of histone 3 and 4 (1, 2). Sodium Butyrate (SB) is a histone deacetylase inhibitor and studies from our lab using a Zebrafish model of MJD have shown that SB rescues the hypoacteylayion of histone 3 and 4 in addition to a rescuing of the motor phenotype. Several of these findings were reproduced in our cell culture models of MJD. HEK293 cells stable expressing the Ataxin-3-84Q mutant were treated with SB resulted in an increase in histone acetylation and autophagy. Autophagy is a digestion process that encapsulates protein aggregates and damaged organelles into a double membrane structure known as autophagosomes. These autophagosomes fuse with lysosomes containing digestive enzymes that break down the contents of the autophagosomes. Western blotting showed an increase in LC3B and decreased p62. Immunostaining showed colocalization of LC3b and lysosomes. Taken together these indicates a flux in autophagy. Preliminary results also show that SB induces autophagy in SH-SY5Y stable expressing Ataxin-3-84Q mutant and decreases Ataxin-3 dimer protein levels. These studies elucidate potential therapeutics in the induction of autophagy and inhibition of histone deacetylase for the treatment of MJD. References: 1.Chou A-H, Chen S-Y, Yeh T-H, Weng Y-H, Wang H-L. HDAC inhibitor sodium butyrate reverses transcriptional downregulation and ameliorates ataxic symptoms in a transgenic mouse model of SCA3. Neurobiology of Disease. 2011;41(2):481-8. 2.Yi J, Zhang L, Tang B, Han W, Zhou Y, Chen Z, et al. Sodium Valproate Alleviates Neurodegeneration in SCA3/MJD via Suppressing Apoptosis and Rescuing the Hypoacetylation Levels of Histone H3 and H4. PLOS ONE. 2013;8(1):e54792.
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DR EMILY MCCANN
Centre for Motor Neuron Disease Research
Macquarie University
Characterising the genetic architecture of sporadic motor neuron disease in Australia Emily P. McCann1, Jennifer A. Fifita1, Lyndal Henden1, Natalie Grima1, Natalie A. Twine1,2, Denis C. Bauer2, Matthew Kiernan3,4, Garth A Nicholson1,5,6,7, Dominic B Rowe1,5, Kelly L. Williams1*, Ian P. Blair1* *Joint senior authorship 1. Macquarie University Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia 2. Transformational Bioinformatics, Commonwealth Scientific and Industrial Research Organisation, Sydney, New South Wales, Australia 3. Brain and Mind Centre, University of Sydney, Sydney, NSW, Australia 4. nstitute of Clinical Neurosciences, Royal Prince Alfred Hospital, Sydney, NSW, Australia 5. Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia 5. Northcott Neuroscience Laboratory, ANZAC Research Institute, University of Sydney, Concord, NSW, Australia 6. Sydney Medical School, University of Sydney, Sydney, NSW, Australia. 7. Molecular Medicine Laboratory, Concord Hospital, Concord, NSW, Australia. Email address of presenting author: [email protected] MND is a genetically heterogenous disease. More than 30 genes and hundreds of genetic variants have been implicated in MND to-date. We sought to characterise the genetic heterogeneity of MND among apparently sporadic Australian cases. To perform a comprehensive genetic screen, we compiled a list of 851 nucleotide variants and two repeat expansions from 31 genes that have been reported in MND as causal mutations or risk alleles. Forty-three different nucleotide variants were identified in whole genome sequencing data from 616 Australian sporadic MND cases, while 16 cases were previously known to carry a C9orf72 expansion. This equated to 31.98% of cases carrying at least one MND-implicated variant, while 4.71% of all cases carried multiple variants, indicating these cases may have an oligogenic underpinning to disease. Fisher’s Exact testing suggested two variants, SOD1 p.I114T and NEK1 p.S1036X, represented MND risk alleles in Australia. However, subsequent relatedness testing showed that the sporadic cases carrying SOD1 p.I114T are in fact low-penetrant familial cases. An additional 73 novel and 64 rare variants were identified among MND genes including SOD1 and FUS. Of these, 30 novel and 24 rare variants were assessed to have pathogenic potential using in silico tools and databases. The identification of genetic causes and risk factors among sporadic MND cases confirms that genetic variation plays a role in all forms of MND. Expansion of the genetic spectrum of MND aids the stratification of patient cohorts for more effective therapeutic trials, and eventual personalised medicine approaches to treating MND.
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DR GARRY NIEDERMAYER
Western Sydney University
Characterisation of the transgenic IL6 overproducing mouse model of chronic gliosis.
Garry Niedermayer1, Erika Gyengesi2, Gita Rahardjo3,4, Arvind Parmar3,4, Guo Jun Liu3,4, Gerald Muench2.
1. School of Science and Health, Western Sydney University, NSW 2560, Australia; 2. School of Medicine, Western Sydney University, NSW 2560, Australia; 3. The Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia 2234; 4. The Brain and Mind Centre, University of Sydney, Camperdown, NSW, Australia 2050.
Chronic microglial activation is a prominent feature of many chronic neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease. To investigate the effects of chronic microglial activation on cerebellar structure and motor function, transgenic GFAP-IL6 mice model was assessed for inflammatory markers throughout life span. In addition, we utilised 18F-PBR111 as a marker for the PET/CT imaging of neuroinflammation in vivo (1). In the cerebellum, increased numbers of Iba1+ microglia were observed as early as at 3 months of age. In addition, TNF‐α levels proved to be significantly higher in the GFAP-IL6 compared to WT mice at all time points. A difference in cerebellar volume between the GFAP-IL6 and WT mice was observed later in life, starting at 6 months and increasing to a loss of about 50% in aged (24 months old) GFAP-IL6 mice. PSD95 levels decreased in the aging GFAP-IL6 mice compared to their WT littermates from 14 months onward. The PET/CT study was performed in another subset of 5-month-old mice. 1 hour dynamic PET/CT scan was performed using 18F-PBR111. There was a statistically significant increase of in 18F-PBR111 binding in the cerebellum of the GFAP-IL6 mice group compared to the wild type mice group. This study has further validated the GFAP-IL6 mice model as a potential candidate to study gliosis and neuroinflammation both in vivo and in vitro. Furthermore, PET/CT imaging using 18F-PBR111 may be utilised to study the effect of potential drug candidates to treat or possibly prevent neuroinflammation in this mouse model.
1. Van Camp, N., et al., In vivo imaging of neuroinflammation: a comparative study between [18F]PBR111, [11C]CLINME and [11C]PK11195 in an acute rodent model. European Journal of Nuclear Medicine and Molecular Imaging, 2010. 37(5): p. 962-972.
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ANANDA CHRISTINA STAATS PIRES
Centre for Motor Neuron Disease Research
Macquarie University
Exacerbated BH4 Metabolism in Experimental Colitis Pain
1,2 Staats A.C., 1 Lenfers B., 1 Scheffer D L., 1 Bankhardt B., 1 Rizzatti S. M., ,2 Guillemin G., 1 Latini A. 1. Laboratório de Bioenergética e Estresse Oxidativo, Programa de Pós-graduação em Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil, 2. Neuroinflammation Group, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW, Australia. E-mail: [email protected]. INTRODUCTION: Chronic abdominal pain is a common symptom in inflammatory chronic diseases, including colitis (Nigg, Naumann, Kaser, & Vetter, 2008). There is evidence showing that increased levels of tetrahydrobiopterin (BH4), a mandatory cofactor for production of neurotransmitters and nitric oxide, intensify pain sensitivity (Latremoliere et al., 2015). OBJECTIVE: To investigate the participation of BH4 levels in pain hypersensitivity in an experimental model of colitis. METHODOLOGY: Colitis induction (2% dextran sodium sulfate (DSS) 6 days in drinking water) and a treatment with an inhibitor of BH4 synthesis (sulfasalazine (SSZ) 7 days) were performed in adult Swiss male mice. Twenty-four h later, abdominal mechanical pain sensitivity was determined, and biological samples were collected. The modulation of BH4 metabolism was assessed by measuring the levels of the neopterin and BH4 levels and the content of the regulatory enzyme of the BH4 pathway, GTP-cyclohydrolase (GTPCH). RESULTS: GTPCH content, the levels of BH4 and neopterin were significantly increased in colonic samples from DSS-receiving mice. Concomitant with BH4 pathway upregulation, abdominal mechanical hypersensitivity was increased in experimental colitis. Finally, SSZ administration induced analgesia (1 h after administration) suggesting the involvement of BH4 in colitis-induced abdominal hypersensitivity. RESULTS: GTPCH content, the levels of BH4 and neopterin were significantly increased in colonic samples from DSS-receiving mice. Concomitant with BH4 pathway upregulation, abdominal mechanical hypersensitivity was increased in experimental colitis. Finally, SSZ administration induced analgesia (1 h after administration) suggesting the involvement of BH4 in colitis-induced abdominal hypersensitivity CONCLUSION: To our knowledge, this is the first evidence showing the role of BH4 in colitis abdominal pain.
References:
1. Latremoliere, A., Latini, A., Andrews, N., Cronin, S. J., Fujita, M., Gorska, K., Hovius, R., Romero, C., Chuaiphichai, S., Painter, M., Miracca, G., Babaniyi, O., Remor, A. P., Duong, K., Riva, P., Barrett, L. B., Ferreirós, N., Naylor, A., Penninger, J. M., Tegeder, I., Zhong, J., Blagg, J., Channon, K. M., Johnsson, K., Costigan, M., & Woolf, C. J. 2015. Reduction of Neuropathic and Inflammatory Pain through Inhibition of the Tetrahydrobiopterin Pathway. Neuron, 86(6).
2. Nigg, C., Naumann, U. K., Kaser, L., & Vetter, W. 2008. [Ulcerative colitis. Main symptoms: bloody mucous diarrhea, tenesmus, abdominal pain]. Praxis (Bern 1994), 97(4): 167-173; quiz 174-165.
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ANISHCHAL PRATAP
The University of Sydney [email protected]
Impaired neuronal adiponectin signalling in the 5XFAD mouse model of Alzheimer’s disease Anishchal Pratap1,2, R. M. Damian Holsinger1,2
1. Laboratory of Molecular Neuroscience and Dementia, Brain and Mind Centre, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW
2. Discipline of Pathology, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW
Alzheimer’s disease (AD) is the most common form of dementia (60-80% of cases) affecting over 35 million people worldwide. The condition predominantly occurs late in life, affecting approximately 5% of the population over the age of 65. The aetiology of AD is currently unknown and therefore current therapies only target early symptomatic issues. Obesity and diabetes mellitus are metabolic conditions that have similar pathological characteristics as AD including cognitive impairment, inflammation and insulin resistance. Adiponectin is an adipokine that regulates energy metabolism in the body through its receptors, AdipoR1 and AdipoR2. To determine energy distribution in the AD brain, we examined the expression pattern of AdipoR1 and R2 in the 5XFAD mouse model of AD. Mice were sacrificed at different stages of disease progression and expression of AdipoR1 and R2 was examined using immunofluorescence staining. Results show AdipoR1 and R2 were expressed throughout the brain. Neuronal expression of both AdipoR1 and AdipoR2 were decreased in the 5XFAD mouse compared to wild-type controls. Additionally, endothelial expression of AdipoR1 and astrocytic expression of AdipoR2 was also observed. Reduced neuronal expression of AdipoR1 and AdipoR2 as the mice age support metabolic disturbance as a concomitant feature of AD.
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KATHERINE ROBINSON
Centre for Motor Neuron Disease Research
Macquarie University [email protected]
Are motor neuron abnormalities correlated with impaired motor function in SOD1-expressing zebrafish? Robinson KJ1, Yuan KC2, Don EK2, Hogan AL2, Winnick CG2, Tym MC2, Lucas CW2, Shahheydari H2, Watchon M2,3, Blair IP2, Atkin JD2, Nicholson GA2,4, Cole NJ2, Laird AS2
1. Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia; 2. Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Centre for Motor Neuron Disease Research, Macquarie University, Sydney, Australia; 3. Sydney Medical School, University of Sydney, Sydney, Australia; 4. Concord Clinical School and ANZAC Research Institute, Concord Repatriation Hospital, Concord, Australia. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modelled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 mRNA. At 48 hours post-fertilization, larvae movement (distance travelled during behavioural testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and travelled a significantly shorter distance during behavioural testing (p < 0.001) when compared with WT SOD1 and non-injected larvae. Furthermore, there was a positive correlation between distance travelled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS
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NATALIE SCHERER
Centre for Motor Neuron Disease Research
Macquarie University
Investigating the role of oxidative stress in spinal motor neurons in a zebrafish model of ALS
Natalie M. Scherer1, Isabel Formella1, Andrés Vidal-Itriago1, Emily K. Don1, Adam J. Svahn1, Manuel B. Graeber2, Roger S Chung1, Marco Morsch1
1. Centre for Motor Neuron Disease Research, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2. Brain and Mind Centre, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
Reactive oxygen species (ROS) are naturally occurring by-products of energy production and play important roles in many physiological processes. Increased ROS levels lead to an imbalance of the cell’s redox state resulting in oxidative stress (OS) and cell damage. OS is one pathological process that has been linked to motor neuron (MN) degeneration in ALS. What remains unclear is the exact mechanism of ROS-induced OS leading to neurodegeneration and whether MNs with ALS aggregates are more susceptible to OS. The goal of this project is to assess the tolerance and response of MNs to increased ROS levels. We are especially interested in understanding whether MNs expressing TDP-43 show an increased vulnerability to ROS level changes compared to healthy MNs in the zebrafish spinal cord. We are using a stable transgenic fish line [1] expressing the genetically encoded photosensitiser KillerRed (KR) in MNs. Upon green-light excitation, KR generates high levels of ROS associated with photo-bleaching of the fluorophore [2]. This optogenetic approach provides the unique opportunity to spatially and temporally control ROS levels and allows side by side comparison of stressed vs non-stressed MNs. In vivo time-lapse imaging showed a time and intensity dependent increase in ROS production after light-illumination resulting in MN deterioration with characteristic signs of apoptosis. Light-activation of TDP-43-WT/MUT co-expressing cells led to MN degeneration restricted to the light-illuminated area. The question of whether an increase in ROS levels results in ALS protein mislocalization is currently being addressed. References:
1. Formella I, Svahn AJ, Radford R, et al. Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo. Redox Biol. 2018; 19:226-234
2. Bulina ME, Chudakov DM, Britanova O V., et al. A genetically encoded photosensitizer. Nat Biotechnol. 2006; 24(1):95-99
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SINA SHADFAR
Centre for Motor Neuron Disease Research
Macquarie University
Identifying novel roles for Protein Disulfide Isomerase (PDI) in Amyotrophic Lateral Sclerosis (ALS)
Sina Shadfar1, Hamideh Shahheydari1, Sonam Parakh1, Angela S. Laird1 and Julie D. Atkin1,2 1.Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.2. La Trobe Institute for Molecular Science, La Trobe University, Victoria, Abstract: Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration and death of motor neurons in the brain, brainstem and spinal cord. Previous studies in our group have established that protein disulphide isomerase (PDI) is protective against dysfunction to proteostasis induced by multiple mutant proteins in vitro in ALS. However, it remains unclear if PDI is also protective in vivo, or against other cellular mechanisms associated with ALS, including DNA damage. In this study, we aimed to examine whether PDI is protective against DNA damage both in vivo and in vitro, thus further defining the protective properties of PDI in ALS. Expression of wild-type (WT) PDI rescued both motor impairment and axonopathy in zebrafish expressing mutant superoxide dismutase (SOD1) A4V. Hence, these data reveal that PDI is protective against ALS-like phenotypes in vivo. Furthermore, PDI was also protective against DNA damage, a mechanism that is becoming increasingly being implicated in ALS, in NSC-34 cells. In cells treated with etoposide, significantly fewer p53 binding protein (53 BP1) and H2A histone X (γH2AX) foci were formed in cells expressing PDI compared to controls. Moreover, in the zebrafish model, transient expression of PDI mRNA downregulated γH2AX levels induced by H2O2. These results demonstrate the protective role of PDI against DNA damage both in vivo and in vitro in ALS models. They also reveal that PDI has a broader protective role than previously realised. This study therefore has implications for future therapeutic studies based on PDI. References Parakh S, Jagaraj CJ, Vidal M, et al., Human Molecular Genetics, 2018.15;27(8):1311-1331. Walker AK, Farg MA, Bye CR, et al., Brain, 2010. 133(1): 105-116.
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SANDY STAYTE
University of Technology Sydney [email protected]
Targeting kainate receptors to inhibit MPTP-induced degeneration in the mouse midbrain
Sandy Stayte1, Kathryn Laloli1, Peggy Rentch1, Aimee Lowth2, Kong Li3, Russell Pickford4, Bryce VIssel1
1. Centre for Neuroscience and Regenerative Medicine, Faculty of Science, University of Technology Sydney, Ultimo, NSW 2007, Australia;
2. Department of Neuroscience, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia;
3. Bosch Institute, Sydney Medical School, The University of Sydney, Camperdown, NSW 2050, Australia;
4. Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South Wales, Kensington, NSW 2033
Despite decades of research, treatments for Parkinson’s disease (PD) are wholly symptomatic and do not address the underlying degeneration that occurs. Previous research suggest glutamate receptor antagonists offer promise through reducing excitotoxicity and aberrant signaling within the basal ganglia motor circuit. Unlike NMDA and AMPA receptors, the kainate receptor (KAR) is not critical to most fast excitatory synaptic transmission and instead is primarily a modulating influence. Furthermore, the KAR subunits GluK1-GluK3 are expressed in multiple PD-associated brain regions, suggesting a KAR antagonist may offer a novel neuroprotective therapy for PD. 12-week-old male C57BL/6, GluK1-/-, GluK2-/-, or GluK3-/- mice (and their WT littermate controls) were implanted with osmotic micro-pumps, containing 2.5μM UBP310 or vehicle control and then lesioned with MPTP the following day. Stereological quantification revealed administration of UBP310 significantly increased survival of dopaminergic and total neuron populations in the substantia nigra pars compacta. In contrast, UBP310 was unable to rescue MPTP-induced loss of dopamine levels or dopamine transporter expression in the striatum, suggesting neuroprotection is localised to the midbrain. Furthermore, deletion of GluK1, GluK2 or GluK3 had no effect on MPTP or UBP310-mediated effects across all measures. These results indicate UBP310 provides neuroprotection in the midbrain against MPTP neurotoxicity that is not dependent on specific KAR subunits and provide a foundation for further investigation of KARs as an alternative target for blocking cell death in PD.
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DR BENJAMIN TRIST
Brain and Mind Centre and The University of Sydney [email protected]
SOD1 protein misfolding is associated with altered cellular copper handling and pathological TDP-43 and p62 in familial and sporadic ALS
Benjamin Trist1*, Veronica Cottam1, Sian Genoud1, Stéphane Roudeau2,3, Jennifer A Fifita4, Asuncion Carmona2,3, Ian P Blair4, Richard Ortega2,3, Dominic J Hare5,6, Kay L Double1
1. Discipline of Biomedical Science and Forefront Research Team, Brain and Mind Centre, The University of Sydney, Sydney, NSW 2006, Australia. 2. University of Bordeaux, CENBG, UMR 5797, F-33170 Gradignan, France 3. CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan, France. 4. Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, Macquarie University, Sydney, New South Wales, Australia. 5. Elemental Bio-imaging Facility, University of Technology Sydney, Broadway, New South Wales 2007, Australia 6. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3052, Australia.
Presenting Author: Mr Benjamin Trist, [email protected]
Misfolded mutant SOD1 protein is strongly implicated in spinal cord motor neuron degeneration in SOD1-linked familial amyotrophic lateral sclerosis (SOD1-fALS).1 Mutations are believed to trigger SOD1 misfolding by reducing the binding of stabilizing copper or zinc metal co-factors, which compromises physiological SOD1 structure and function. Misfolded wild-type SOD1 may also contribute to the aetiology of the more common sporadic form of (s)ALS,2-4 highlighting the importance of non-genetic factors in SOD1 misfolding. Identifying specific biochemical factors which underlie the misfolding and deposition of SOD1 within select cellular populations in ALS will advance our understanding of the aetiology of sporadic ALS. We characterised SOD1 pathology in post mortem spinal cord tissues from SOD1-fALS (n=3), C9ORF-fALS (n=3) and sALS (n=9) patients, and age-matched controls (n=10), and identified misfolded soluble and misfolded insoluble SOD1 in the ventral spinal cord in all ALS cases. In contrast, misfolded SOD1 was not identified in either the soluble nor insoluble forms in the same tissues from age-matched controls. SOD1 misfolding was associated with alterations to cellular copper handling within the ventral spinal cord in ALS tissues, consistent with impaired copper delivery to SOD1. The presence of substantial TDP-43 and/or p62 pathology within the ventral spinal cord in all fALS and sALS cases suggests interactions between misfolded SOD1 and pathological TDP-43 and p62, previously identified in vitro,5,6 may occur in ALS patients. Future research should focus on characterizing the potential contribution of these interactions, as well as altered copper delivery to SOD1, to motor neuron death in ALS. References:
1. Rosen, D. R. et al. Mutations in Cu/Zn Superoxide-dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62, doi:10.1038/362059a0 (1993).
2. Bosco, D. A. et al. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nature Neuroscience 13, 1396-U1133, doi:10.1038/nn.2660 (2010).
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3. Guareschi, S. et al. An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1. Proc Natl Acad Sci U S A 109, 5074-5079, doi:10.1073/pnas.1115402109 (2012).
4. Pare, B. et al. Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis. Sci Rep 8, 14223, doi:10.1038/s41598-018-31773-z (2018).
5. Jeon, G. S. et al. Pathological Modification of TDP-43 in Amyotrophic Lateral Sclerosis with SOD1 Mutations. Molecular neurobiology 56, 2007-2021, doi:10.1007/s12035-018-1218-2 (2019).
6. Gal, J. et al. Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism. J Neurochem 111, 1062-1073, doi:10.1111/j.1471-4159.2009.06388.x (2009).
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ANDRES VIDAL-ITRIAGO
Centre for Motor Neuron Disease Research
Macquarie University
Pectoral fin axotomy model: in vivo study of microglial response to peripheral nerve injury in zebrafish Andres Vidal-Itriago1, Natalie Scherer1, Jason Aramideh2, Rowan Radford1, Emily K. Don1, Adam J Svahn1, Roger Chung1, Manuel B Graeber2 & Marco Morsch1
1. Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2. Department of Clinical Medicine, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia 2. Brain and Mind Centre, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia Microglia are phagocytic cells responsible for the innate immune response, the elimination of cell debris and the refinement of the synaptic network in the central nervous system (CNS). It is well known that microglia play a key role in the elimination of synapses during the early stages of development. Also, it has been shown in rodent models that microglia mediate synaptic changes in response to peripheral nerve injury and neurodegeneration [1]. However, the structural and functional features of microglia-neuron interactions governing those synaptic modifications remain elusive. Our aim is to describe in detail how microglia mediate synaptic changes upon neuronal degeneration and characterize microglial central response to peripheral nerve injury. Taking advantage of the suitability of performing in vivo long-term imaging in zebrafish, we have established a new model of peripheral nerve axotomy that allow us to characterize in real-time the microglia-neuron interactions after the injury of the zebrafish pectoral fin innervation. To that end, we use transgenic zebrafish lines expressing fluorescent proteins in motor neurons and microglia as well as fluorescent axonal tracers to label the neurons innervating the pectoral fin muscle. Subsequently we perform the pectoral fin nerve avulsion and we monitor the dynamics of microglial response using confocal microscopy. We further characterize the functional and morphological changes of microglia in the CNS using post-imaging analysis and 3D rendering. References:
1. Moran, L.B. & Graeber, M.B. (2004). The facial nerve axotomy model.” Brain Research Reviews, 44(2-3), 154-178.
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GUOYING WANG
Centre for Motor Neuron Disease Research
Macquarie University [email protected]
Intrinsically Fluorescent PAMAM Dendrimer as Drug carrier and Nanoprobe: Bioimaging and Neuron protection Study
Guoying Wang1, Afshin Babazadeh1, Bingyang Shi1 Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia
PAMAM dendrimers have been described as one of the most tunable and therefore potentially applicable nanoparticles both for diagnostics and therapy. In recent years, intrinsically fluorescent PAMAM dendrimers have attracted extensive attention in the fields of biological imaging and drug/gene delivery due to their excellent intrinsic fluorescence properties, functional surfaces and highly biocompatibility. Recently, we have developed a new green intrinsic fluorescent PAMAM through simply modification with acetaldehyde. We found that the fluorescence intensity of the prepared PAMAM can be increased by 45% after heating at 70 oC, and the fluorescence spectrum is narrowed, which is beneficial to its fluorescence performance. The novel intrinsically fluorescence PAMAM (IF-PAMAM) showed excellent biocompatibility with NSC 34 (Motor neuron like) cells, demonstrating great potential for motor neuron based biological imaging and drug delivery. In this study, we fabricated Edaravone (EDA) loaded IF-PAMAM nano-complexes and surface functionalized with transferrin (Tf), the resulting IF-PAMAM/EDV-Tf showed enhanced blood brain barrier (BBB) transportation and elevated neuron protection function. The biological imaging performance of IF-PAMAM was evaluated by zebrafish model to provide guidance for the in-vivo application of IF-PAMAM system.
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MAXINNE WATCHON
Centre for Motor Neuron Disease Research
Macquarie University [email protected]
Induction of the autophagy pathway improves the motor function of a transgenic zebrafish model of Spinocerebellar ataxia type 3 Maxinne Watchon1, Kristy Yuan1, Luan Luu1, Nicholas J. Cole1, Roger S. Chung1, Garth A. Nicholson1,2, Angela S. Laird2 1Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University 2ANZAC Research Institute, Concord Repartition Hospital, Sydney Spinocerbellar ataxia-3 (SCA3) is an autosomal dominant neurodegenerative disease caused by the ATXN3 gene with an increased number of trinucleotide repeats (CAG), with patient numbers >40 repeats as opposed to the healthy population (10-40 repeats). These repeats encode for a polyglutamine region within the ataxin-3 protein. Growing evidence suggests several neurodegenerative diseases share a common feature: accumulation or aggregation of disease causing proteins possibly due to impairment of protein quality control pathway, autophagy [1][2][3]. Thus, we aimed to investigate whether autophagy inducer compounds may provide a beneficial effect on our transgenic SCA3 zebrafish. Using a candidate-based screening approach by identifying autophagy compounds from the literature, we exposed our mutant ataxin-3 zebrafish between 1-6 days of age. ‘Drug Z’ was able to rescue a previously described motor phenotype of our mutant ataxin-3 zebrafish [4]. Immunoblotting analysis of 6-day-old SCA3 zebrafish samples revealed that Drug Z decreased human ataxin-3 levels (full-length and cleaved ataxin-3). These results are supported by decreased levels in p62 and lamp2a (autophagy substrates) from Drug Z exposure. From these results, autophagy-inducing compounds, like Drug Z, improve the movement of mutant ataxin-3 zebrafish. This may provide a new avenue of treatment not only for SCA3, but also for other neurodegenerative diseases.
References:
[1] Banerjee R., Beal M.F., and Thomas B. 2010. Trends Neurosci. 33(12):541-549. [2] Lee A., Rayner S.L., Gwee S.S.L., et al. 2017. Cellular and Molecular Life Sciences. https://doi-org.ezproxy1.library.usyd.edu.au/10.1007/s00018-017-2632-8 [3] Onofre I., Mendonca N., Lopes S., et al 2016. Nature Scientific Reports. 6:28220. [4] Watchon M.,Yuan K.C., Mackovski N., et al 2017. Journal of Neuroscience. 37(32):7782-7794.
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CAROL L-Y ZHOU-ZHENG
Laboratory of Molecular Neuroscience and Dementia, Brain and Mind Centre, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney
NGF-treated PC12 cells in Schwann cell cocultures have enhanced neuronal-like morphology Carol L-Y Zhou-Zheng1,2, Yogambha Ramaswamy2, Hala Zreiqat2 and R. M. Damian Holsinger1,3
1. Laboratory of Molecular Neuroscience and Dementia, Brain and Mind Centre, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW
2. Tissue Engineering & Biomaterials Research Unit, School of Aerospace, Mechanical & Mechatronic Engineering, Faculty of Engineering and Information Technologies, The University of Sydney, NSW
3. Discipline of Pathology, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW
Abstract Peripheral nerve injuries (PNIs) involve a loss of axonal function in the peripheral nervous system (PNS), interrupting the transmission of information [1]. PNIs may result in dysfunction in the sensory organs and effectors [2-3]. PNIs are potentially debilitating and may significantly reduce quality of life in an estimated 2-3% of all patients globally [3-4]. Schwann cells instigate numerous important processes in peripheral nerve regeneration [5]. In particular, the Schwann cells release trophic factors and other proteins during repair, which
stimulate the growth of the injured axons [1]. As part of the last stage of nerve regeneration, the Schwann cells also remyelinate the newly repaired axons. Thus, axonal growth and remyelination become important markers of peripheral nerve regeneration. Current approach in research for PNI treatment is focused on the direct injection of Schwann cells or Schwann cell-like differentiated stem cells into the nerve lesion to boost Schwann cell population in the area [6]. PC12 cells are rat pheochromocytoma cells of neural crest origin [8]. PC12 cells have been studied for their well-documented differentiation into neuron-like cells via exposure to nerve growth factor (NGF) [9]. The aim of our study was to investigate whether the presence of Schwann cells would enhance the NGF-induced differentiation, proliferation and myelination of PC12 cells in a mixed culture. In comparison to the NGF-treated monocultures, PC12 cells in NGF-treated Schwann cell cocultures had significantly improved cell proliferation and morphology, increased number of neurites per cell and increased neurite length. These findings demonstrate the cell-protective and reparative ability of Schwann cells, which may impact future PNI treatment and direction of peripheral nerve regeneration research. Although the peripheral and central nervous system (CNS) share similarities, yet differ in neuronal repair, it is
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hoped that this research may shed light on diseases of the CNS, including neurodegenerative diseases. References [1] M. G. Burnett and E. L. Zager, "Pathophysiology of peripheral nerve injury: a brief review," Neurosurg Focus, vol. 16, no. 5, p. E1, May 15 2004. [2] R. Deumens et al., "Repairing injured peripheral nerves: Bridging the gap," Prog Neurobiol, vol. 92, no. 3, pp. 245-276, 2010. [3] D. Grinsell and C. P. Keating, “Peripheral Nerve Reconstruction after Injury: A Review of Clinical and Experimental Therapies,” BioMed Res Int vol. 2014, Article ID 698256, pp. 1-13, 2014. [4] J.A. Kouyoumdjian, C.R. Graça and V.F. Ferreira, “Peripheral nerve injuries: A retrospective survey of 1124 cases,” Neurol India, vol. 65, pp. 551-555, 2017. [6] R. M. G. Menorca et al., "Peripheral Nerve Trauma: Mechanisms of Injury and Recovery," Hand Clinics, vol. 29, no. 3, pp. 317-330, 2013. [7] R. S. Tubbs et al., Nerves and Nerve Injuries: Vol 2: Pain, Treatment, Injury, Disease and Future Directions. Elsevier Science, 2015. [8] G. Guroff, "PC12 Cells as a Model of Neuronal Differentiation," in Cell Culture in the Neurosciences, J. E. Bottenstein and G. Sato, Eds. Boston, MA: Springer US, 1985, pp. 245-272. [9] L. A. Greene and A. S. Tischler, "Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor," Proc. Natl. Acad. Sci., vol. 73, no. 7, pp. 2424-2428, 1976.
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