Motor Neuron Disease

Motor Neuron Disease

Project 1: Characterisation of the biological processes affected by a new ALS gene, CCNF, in motor neuron proteostasis.

Introduction

This project will investigate the signalling pathways and biological processes affected by a new ALS/FTD gene discovered by our researchers at the Macquarie University Motor Neurone Disease (MND) Centre. Mutations in this new ALS/FTD gene, CCNF, which encodes the protein Cyclin F, is involved in maintaining cellular health by tagging unwanted proteins (ubiquitylation) for breakdown and recycling within the cell. Mutant versions of Cyclin F, found in some ALS patients, are defective in that they lack the necessary features needed to regulate proper function, which ultimately leads to improper function, accumulation of proteins, and effects on downstream signalling pathways and biological processes. This project will use quantitative proteomics to identify changes to the phosphoproteome and ubiquitome, and validate these biological changes in primary neurons and/or iPS-derived motor neurons.

Hypothesis:

Hypothesis 1:  Only six substrates of cyclin F have been characterised to date, all of which are involved with cell cycle processes. We predict that cyclin F in neurons (post-mitotic) plays a vastly different role in maintaining proteostasis.

Hypothesis 2:  We predict that wild-type cyclin F and ALS-causing mutant cyclin F bind to different protein substrates to direct them for degradation by the ubiquitin-proteasome system.

Hypothesis 3:  The different forms of cyclin F cause target common and unique signalling pathways and biological processes downstream, and these targets will be relevant to the biology of motor neurons.

Aims:

Aim 1: Establish inducible stable neuronal cell lines expressing cyclin F and examine neuronal markers for dividing and differentiated status.

Aim 2:  Identify protein interacting partners of cyclin F from neuronal cells by immunprecipitation and liquid chromatography mass spectrometry distinguishing between binding partner and substrate.

Aim 3:  Characterise the phosphoproteome and ubiquitome affected by the different forms of cyclin F to identify common and unique signalling pathways. These pathways will be further validated in primary neurons and/or iPS-derived motor neurons using standard biochemistry techniques.

Enquires:

Professor Roger Chung, roger.chung@mq.edu.au

Dr Marco Morsch, marco.morsch@mq.edu.au

Dr Albert Lee, albert.lee@mq.edu.au

Dr Bingyang Shi, bingyang.shi@mq.edu.au

Project 2: Does the transfer of ALS protein aggregates between motor neurons trigger neurodegeneration?

Introduction

Accumulation of proteins into insoluble aggregates in neurons and glia is now recognized as a common pathological hallmark of many neurodegenerative diseases (e.g. in Alzheimer’s, and Parkinson’s disease). In Amyotrophic Lateral Sclerosis (ALS), the intracellular accumulation of proteins in neurons is also well established. Importantly, clinical evidence indicates the transmissibility or spread of these aggregates in patients from a focal onset to other regions over time. This spread of aggregation is beginning to substantiate but is entirely limited to studies using cultured nerve cells (in-vitro studies).

This project will investigate this potential pathogenic mechanism using an animal model (in-vivo). Our team has established comprehensive preliminary data that establishes the release, survival and spread of aggregated ALS-proteins from neurons into other cells in the zebrafish spinal cord. We will use an innovative series of experiments to selectively trigger the death of a single neuron containing these aggregates and investigate their fate after being   released, and if they are incorporated into neighbouring cells.

Hypothesis:

This project will investigate the hypothesis that ALS proteins have propagating characteristics, such that insoluble aggregates can transfer between cells and seed aggregation and degeneration in non-affected cells.

Aims:

Aim 1:  Observe the fate of TDP-43 and SOD1 released from a single dying motor neuron and the impact upon the viability of surrounding motor neurons.

Aim 2:  Assess aggregation of ALS proteins released from dying motor neurons in vivo

Aim 3:  Histological verification of the intercellular transfer of ALS proteins

Outcome:

We predict that we will be able to track ALS aggregates and visualize their disintegration or survival in the living organism. This will provide important insights into the pathogenic mechanisms underlying ALS-mediated neurodegeneration

Enquires:

Professor Roger Chung, roger.chung@mq.edu.au

Dr Marco Morsch, marco.morsch@mq.edu.au

Dr Albert Lee, albert.lee@mq.edu.au

Dr Bingyang Shi, bingyang.shi@mq.edu.au

Project 3: Investigating the regulatory and functional roles of Cyclin F in the development of Amyotrophic Lateral Sclerosis (ALS)

Introduction

This project will investigate the cellular and functional roles of a new ALS/FTD gene discovered by researchers at the Macquarie University Motor Neurone Disease (MND) Centre. Mutations in this new ALS/FTD gene, CCNF, which encodes the protein Cyclin F, is involved in maintaining cellular health by tagging unwanted proteins (ubiquitylation) for breakdown and recycling within the cell. Mutant versions of Cyclin F, found in some ALS patients, are defective in that they lack the necessary features needed to regulate proper function, which ultimately leads to impaired ubiquitylation and accumulation of proteins. This project will systemically investigate the regulatory and functional role of each mapped phosphorylation site of Cyclin F focusing on those that have been mapped to ALS mutations, and determine whether upstream kinases can be modulated to promote survival responses in ALS cell models. Moreover, this project will investigate the role Cyclin F phosphorylation on its nuclear and cytoplasmic translocation and degradation.

Hypothesis:

Hypothesis 1:  Cyclin F contains >80 predicted phosphorylation sites some of which are hypothesised to be involved in nuclear/cytoplasmic shuttling.

Hypothesis 2:  What is the effect of mutations to cyclin F to its E3 ligase activity? And consequently how does this affect the ubiquitylation of substrates and formation of protein inclusions

Hypothesis 3:  Does cyclin F (and its ALS mutants) influence upstream kinases through a feedback mechanism?

Aims:

Aim 1: Determine whether phosphorylation plays a role in nuclear/cytoplasmic shuttling through dephosphorylation treatments and artificial cyclin F constructs.

Aim 2:  Measure the E3 ligase activity using our customised ELISA and other biochemical techniques and determine to effect does mutated versions of cyclin F influence protein inclusion formation.

Aim 3:  Generate phosphomimetic versions of cyclin F and monitor the effect of upstream kinase activity that are predicted to phosphorylate cyclin F.

Enquires:

Professor Roger Chung, roger.chung@mq.edu.au

Dr Marco Morsch, marco.morsch@mq.edu.au

Dr Albert Lee, albert.lee@mq.edu.au

Dr Bingyang Shi, bingyang.shi@mq.edu.au

Project 4: Why are neurons selectively vulnerable in MND? Optogentic approaches to understand the role of oxidative stress in ALS.

Introduction

Motor neurons are selectively vulnerable to oxidative stress in comparison to other neurons, and mutations in the anti-oxidant enzyme SOD1 are associated with 20% of all inherited cases of ALS. We have generated experimental zebrafish models that allow us to selectively induce oxidative stress within a single spinal motor neuron, in the presence or absence of co-expression of ALS genes (SOD1, TDP-43). 

The aim of this project is to investigate how sub-lethal and lethal levels of oxidative stress, delivered specifically to motor neuron subpopulations, contribute to the etiology of ALS. Our newly designed transgenic zebrafish allow us to induce different levels of oxidative stress in single spinal motor neurons and to visualize real-time responses of both the individually stressed neurons and surrounding cells such as neurons, microglia and astrocytes.

Our approach will determine the cellular mechanisms of stress induced motor neuron degeneration using a range of different techniques, including molecular biology, transgenic zebrafish lines, optogenetic techniques and confocal live-imaging protocols.

Hypothesis:

This project will demonstrate if oxidative stress is a primary instigator of the disease (e.g. if motor neurons in ALS patients are more vulnerable to oxidative stress than healthy motor neurons), and if oxidative stress can trigger secondary neurodegeneration in surrounding MNs.

Aims:

Aim 1:  Compare the susceptibility of individual spinal motor neurons expressing either ALS-wildtype or ALS-mutant genes to experimentally induced oxidative stress

Aim 2: Investigate the effect of oxidative stress induced degeneration of a single spinal MN upon surrounding motor neurons that express either ALS-wildtype or ALS-mutant genes

Outcome:

This approach will provide compelling in vivo evidence that oxidative stress could be involved in the propagation of neurodegeneration in ALS, and will provide critical insights into potential therapeutic interventions that could halt the progression of neurodegeneration in ALS.

Enquires:

Professor Roger Chung, roger.chung@mq.edu.au

Dr Marco Morsch, marco.morsch@mq.edu.au

Dr Albert Lee, albert.lee@mq.edu.au

Dr Bingyang Shi, bingyang.shi@mq.edu.au

Project 5: New approaches to plasma biomarker studies in MND

Introduction

There is an urgent need to identify a series of biomarkers that can be used to improve the speed of diagnosis, and predict more accurately prognosis and other clinical parameters in MND.  This project will utilize a new proteomic technology to identify potential protein biomarkers in plasma samples from MND patients. This will include identification of maps of proteins that can be used to distinguish between different clinical parameters, and evaluation of specific proteins biomarkers.  We predict that these biomarkers may be useful in future for improving diagnostic and prognostic clinical evaluations.  These protein biomarkers may identify also novel biological processes associated with disease pathogenesis, and this may lead to new insight into the causes of MND.

Importantly, this biomarker study will be undertaken using samples from two unique patient cohorts; i) identical twins with disease discordance (one with disease, the other without), and ii) multi-generational families with disease discordance.  This allows us to screen for disease-associated biomarkers with reduced variation across samples (ie: less genetic variation).  Identified biomarkers will subsequently be validated in a cohort of sporadic MND patients.  This provides a systematic approach towards identifying robust biomarkers of disease in MND.

Hypothesis:

We hypothesize that low-abundance plasma biomarkers are present that will be informative of disease pathogenesis.  We will use a new proteomic technique to screen for the presence of robust protein biomarkers that can be used in future for early diagnosis of MND and for tracking the prognosis of patients. New biomarkers may also add to our understanding of disease pathology and thereby could possibly highlight new avenues for research towards future therapies. 

Aims:

1. Unbiased proteomic profiling of plasma from cohorts of familial MND patients displaying disease discordance.

2. Validation of potential proteomic biomarkers in a cohort of sporadic MND patients.

Outcome:

We ultimately expect that a “toolbox” of biomarker parameters will be required to adequately address the clinical requirements for improved measures for diagnosis, prognosis and evaluation of disease progression and response to current and future therapeutic strategies.  The proteomic biomarkers identified through this project may become an important component of such a future “toolbox”, together with other existing biomarkers such as clinical examinations, genetic testing, electrophysiological recording and neuroimaging.  Such a biomarker toolbox is likely to be critical in improving the design of future clinical trials, as stratification of patients into subgroups and more sensitive predictors of disease progression and severity are essential for improving recruitment and analysis in clinical trials.

Enquires:

Professor Roger Chung, roger.chung@mq.edu.au

Dr Marco Morsch, marco.morsch@mq.edu.au

Dr Albert Lee, albert.lee@mq.edu.au

Dr Bingyang Shi, bingyang.shi@mq.edu.au

Project 6: Developing a proteogenomic insight into the molecular origins of MND

Introduction

Inherited forms of MND can be caused by mutations in one of a number of genes, which encode proteins that have quite different functions inside cells. For example, some of these proteins regulate DNA/RNA levels, and some are involved in pathways that regulate protein recycling.  However, it is not clear how mutations in these genes can all trigger MND.  We predict that there must be some common points of convergence in the actions of these disease-causing genes (and proteins).  We will use a combination of genetic and proteomic techniques to identify points of overlap that exist between different MND genes.

Hypothesis:

Hypothesis 1:  We predict that MND-causing genetic mutations will cause cells to display “signature” proteomic profiles that may demonstrate some common molecular links.  And non-replicated familial MND-causing genetic mutations will cause cells to display proteomic profiles that bear little similarity to genuine disease-causing genes.

Hypothesis 2:  That the proteomic profile of cells expressing genuine disease-causing genetic mutations can be used as a predictive tool for screening of new putative MND-causing genes.

Hypothesis 3:  That the proteomic profile of patient cells expressing genuine disease-causing genetic mutations can be compared to proteomic profiles from cells from sporadic MND patients, to identify common molecular mechanisms that represent the convergence points that link the etiology of both genetic and sporadic forms of MND.

Aims:

Aim 1:  MND proteomic signature discovery - develop a proteomic signature for cells expressing verified MND-causing mutations in selected genes (SOD1, TARDBP, FUS, UBQN-2, CCNF), and undertake bioinformatic pathway analysis to identify molecular pathways of uniqueness and convergence.

Aim 2:  MND gene validation – compare the proteomic profile of cells expressing “false-positive” familial MND-causing genetic mutations (profilin, angiogenin) with the proteomic signature of verified MND-causing mutations.

Aim 3:  MND gene discovery – screen the proteomic profile of cells expressing a series of candidate familial MND-causing genetic mutations from a multi-generational Australian family (in which the causative gene mutation has not yet been identified) against the proteomic signature of verified MND-causing mutations, and based upon convergent pathways identify “likely” candidate genes for further validation.

Aim 4:  Use a proteogenomic workflow to gain insight into origins of sporadic MND – develop a proteomic signature for induced pluripotent stem (IPS) cells generated from familial MND patients (SOD1, TARDBP, CCNF), and identify shared molecular signaling pathways with proteomic profiles generated from IPS cells derived from sporadic MND patients.

Outcome:

The potential long-term significance of this project is that the proteogenomic workflow will make an important contribution towards understanding the molecular etiology of individual MND patients, which could potentially lead towards personalized therapeutic intervention. As we understand in greater detail the similarities and differences in molecular signaling underlying various forms of MND (different familial forms and sporadic MND), this will inform the development of personalized therapies specifically targeting these pathways.

Enquires:

Professor Roger Chung, roger.chung@mq.edu.au, Dr Marco Morsch, marco.morsch@mq.edu.au

Dr Albert Lee, albert.lee@mq.edu.au, Dr Bingyang Shi, bingyang.shi@mq.edu.au

Project 7: Neuron disease and frontotemporal dementia

Introduction

Motor neuron disease (MND) is a neurodegenerative disease that causes progressive neuron loss, leading to gradual paralysis and death within ~3 years of symptom onset. Like MND, frontotemporal dementia (FTD) is also a progressive neurodegenerative disease, causing changes in personality, behaviour, and/or language abilities resulting in institutional care and ultimately leading to death. Both MND and FTD are devastating for patients and burdensome for society, and lack effective disease-modifying treatments.

The project is related to Dr Walker’s NHMRC Project grant (2017-2019) and will investigate mechanisms that lead to neurodegeneration in MND and FTD. The guiding hypothesis of these studies is that understanding the early mechanisms involved in the formation of pathology and neurodegeneration in MND and FTD will allow the rational design of effective therapeutics.

Aims:

The goal of these studies is to identify the key molecular drivers of disease and to test potential new therapeutics in pre-clinical mouse models.

1. To characterise new mouse models of MND and FTD, to investigate the biological basis of disease.

2. To determine if pharmacological and genetic modulation of newly identified and previously hypothesised key biochemical pathways can affect pathology, neurodegeneration and disease phenotype in mouse models of MND and FTD.

Research Plan:

This project will use transgenic mice, neuronal cell cultures and human brain and spinal cord tissues with a wide range of advanced molecular, biochemical, cell biology, microscopy and in vivo mouse behavioural and surgical techniques. Aspects of this project can be individualised to suit students’ interests in protein biochemistry, proteomics, cell culture, imaging and/or whole-animal studies.

Enquiry:

Dr Adam Walker Adam.walker@mq.edu.au

Project 8: Studying motor neuron disease, and its potential treatment, using transgenic zebrafish

Introduction

Motor neuron disease (MND) is a fatal neurodegenerative disease that causes progressive paralysis, eventually resulting in wheel-chair dependence and full-time care. Whilst most cases of MND are sporadic, without a known cause, some patients do carry a genetic mutation that causes the disease. Mutations in the human genes encoding TDP-43, FUS , SOD1 and CCNF are examples of these genetic causes. We have generated zebrafish models of MND that carry the different mutated human genes known to cause the disease.  Within this study we will compare the signs of disease that develop in these transgenic zebrafish (including impaired movement, neuropathology, proteinopathy and signs of cell death) and test ways to prevent the development of these signs of disease. We will take advantage of the fact that zebrafish can absorb drugs added to the water they live in to study the mechanisms of MND pathogenesis. We will examine the effects of inhibiting or inducing specific factors that may be involved in the pathogenesis of disease, as well as testing drugs we hypothesise to be protective.

Aims:

1. Examine the signs of MND that develop in our various zebrafish models of MND

2. Investigate different therapeutic approaches through drug testing studies on the MND zebrafish

Research Plan:

We will characterise the signs of MND that occur within the MND zebrafish, including examining the time-course of development of disease phenotypes through in-depth examination of motor behaviours, time-lapse imaging and immunoblot analysis. Following characterisation of the disease phenotypes developed by the zebrafish we will then explore the effect of treating the zebrafish with different drugs and small compounds that target relevant pathways hypothesised to be related to MND pathogenesis or neuroprotection. Any protective treatments identified will be followed up in our zebrafish studies, and may be examined further in cell culture and rodent models of MND.

Enquires:

Dr.Angela Laird, angela.laird@mq.edu.au

Project 9: The role of telomere dysfunction in Motor Neuron Disease (MND)/Amyotrophic Lateral Sclerosis (ALS)

Introduction

Our genome is under constant attack from both environmental agents and normal metabolic processes and every cell receives thousands of DNA injuries every day. These insults can generate mutations and compromise cellular viability, so safeguarding genetic integrity is of pivotal importance to human health. Cells have developed elaborate signalling systems to both detect and repair damage to DNA, termed the ‘DNA damage response’ (DDR). Depending on the extent of the damage, the cell either induces DNA repair pathways, ignores the lesion and risk mutation, or induces apoptosis to protect the organism. Motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS) is the third most common neurodegenerative disorder that targets motor neurons (upper/lower), leading to progressive paralysis.

The incidence of neurodegenerative diseases increases remarkably with age, and it is well established that DNA repair activities decrease during normal aging. DNA damage also accumulates during normal aging, particularly at ‘telomeres’. Telomeres are conserved DNA repeat sequences that protect the ends of chromosomes from degradation/damage. They are highly susceptible to DNA damage themselves, particularly during prolonged cellular stress, as implicated in MND/ALS. Telomeres normally shorten with aging but their length can be enhanced by the activity of telomerase. Telomeres are bound by a protein complex that protects the telomere from aberrant activation of the DDR, which contains TRF (telomere repeat-binding factor) 1 subunits that bind to DNA. If telomere function is compromised, they become deprotected, leading to activation of DDR and apoptosis, and neurons are particularly susceptible to telomere dysfunction. Hence, the presence of shortened telomeres triggers cell death and they are found in many age-related diseases, including MND/ALS. However it remains unclear how dysfunction to the telomere occurs in MND/ALS.

Hypothesis:

Impairment of the telomere triggers neurodegeneration in MND/ALS.

Aims:

This project will investigate how mutations that cause the most common forms of MND/ALS lead to dysfunction of the telomere.

1. Are telomeres the sites of DNA damage in cells expressing mutations associated with MND/ALS?

2. Are strategies to enhance telomerase activity protective in MND/ALS?

Research Plan:

Are telomeres the sites of DNA damage in cells expressing the C9ORF72 repeat expansion? We have previously identified that markers of DNA damage are up-regulated in motor neurons from MND/ALS patients, however the chromatin regions where this occurs are unknown. This project will examine whether the DNA damage occurs at the telomere and if so, the mechanisms involved. The formation of a focus containing DNA damage response proteins at a telomere is termed a ‘telomere dysfunction-induced focus (TIF). The presence of a DNA damage response at telomeres in MND/ALS will be examined using TIF assays in cells expressing MND/ALS mutants. Telomerase activity will also be examined in these cells using Telomeric Repeat Amplification Protocol (TRAP) assays, by which telomerase adds telomeric repeats to a forward primer, and the extended primer is amplified by qPCR. We will also examine whether telomere integrity is compromised, and also if mechanisms involved in maintaining telomere length are dysfunctional in cells expressing MND/ALS mutants, using biochemical and cell biology methods.

Are strategies to enhance telomerase activity protective? As telomeres are shorter in MND/ALS patients, strategies to enhance telomere function may be protective in ALS. This part of the project will involve overexpression of the catalytic subunit of telomerase; telomerase reverse transcriptase

(hTERT), in cells expressing MND/ALS mutations and controls to examine if hTERT is protective against apoptosis. If so, further studies will examine, (i) whether hTERT overexpresson is protective in vivo, in new mouse models of disease that more closely represent human pathology than previous models, and (ii) whether known compounds that enhance telomerase activity are protective in vivo.

Hence in summary, this project will investigate the basic molecular and cellular mechanisms underlying neurodegeneration in MND/ALS, and it will allow us to ascertain whether DNA damage at the telomere is a major contributor to this process.

Methods: Advanced confocal microscopy and live cell imaging, immunocytochemistry, immunohistochemistry, western blotting, cell culture, qPCR, telomere assays, molecular biology and in vivo work.

Enquiries:

A/Professor Julie Atkin, julie.atkin@mq.edu.au

Project 10: The role of DNA damage in frontotemporal dementia (FTD)

Introduction

Frontotemporal dementia (FTD) is the major cause of early-onset (<60 years of age) dementia, which variably causes deterioration in behaviour and personality, language disturbances, or alterations in muscle or motor functions. FTD is known to overlap genetically and pathologically with motor neuron disease (MND) but the mechanisms of neurodegeneration in FTD are unclear. Mutations in chromosome open reading frame 72 (C9ORF72) are the most common genetic cause of both FTD and MND and this discovery confirmed the molecular link between FTD and MND.

The genome receives thousands of DNA injuries every day. Hence maintaining genetic integrity is of pivotal importance to human health. Cells have developed the ‘DNA damage response’ (DDR) to both detect and repair damage to DNA. Chronic activation of the DDR leads to cell death and neurons are particularly vulnerable to DNA damage because they are post-mitotic, have a high metabolic rate and are susceptible to oxidative stress, a source of DNA damage. We have identified that in MND, C9ORF72 mutations induce DNA damage. However it is unknown if DNA damage is also involved in FTD.

Hypothesis:

DNA damage and impairment of the DDR are important mechanisms leading to neurodegeneration in FTD

Aims:

This project will investigate (i) whether DNA damage is present in FTD patients carrying the C9ORF72 mutation and (ii) the mechanisms by which this occurs.

Research Plan:

Do C9ORF72 mutations trigger DNA damage in FTD?

In this project, the DDR signalling pathway will be examined in frontal/temporal lobe brain tissues from FTD patients compared to controls. By examining typical markers of the DDR, gH2AX, 53BP1, ATM, ATR, and PARP-1, immunohistochemistry will be used to determine whether these markers are activated in C9ORF72-FTD affected tissues. This will allow us to determine if DNA damage is present in FTD patients.

Characterisation of the types of DNA damage triggered by C9ORF72 mutations in FTD.

It is important to characterise the form of DNA damage because different DNA repair pathways are activated in response to different types of damage, and enhancing DNA repair may be protective in FTD. Hence, the mechanisms of DNA damage induced by the C9ORF72 mutation will be examined. Both alkaline comet assays and neutral comet assays will be used in cells expressing C9ORF72, between 24-48 hr post-transfection, well before the onset of apoptosis, to identify the type of damage. This will allow us to determine if single-stranded or double stranded breaks are induced by the C9ORF72 mutation. Based on these findings, specific DNA repair pathways will next be examined to determine if they are compromised in FTD, and whether enhancing these pathways is protective both in vitro and in vivo.

Hence in summary, this project will investigate whether DNA damage is linked to neurodegeneration in FTD, and it will also characterise the type of damage involved, with the aim of enhancing the relevant DNA repair pathways as a novel therapeutic strategy.

Methods: Immunocytochemistry, immunohistochemistry, comet assays, DNA damage assays, western blotting, cell culture, advanced confocal microscopy and live cell imaging, western blotting, cell culture, molecular biology and protein chemistry.

Enquiries:

A/Professor Julie Atkin, julie.atkin@mq.edu.au

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