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