Investigating mechanistic causes of C9ORF72-related amyotrophic lateral sclerosis (ALS).

Cells constitute the basic building blocks of the human body converting food and oxygen into energy to produce proteins. The blueprint for making up proteins, the DNA, is housed in the nucleus, a cell centre separated from the surrounding compartment, the cytoplasm, where proteins are assembled into the machinery supporting life. Small messenger species (messenger RNAs) copied from the blueprint are able to pass from the nucleus into the cytoplasm where each guides the building of one protein. The quantity and function of proteins account for survival or death of cells including motor neurons. Amyotrophic lateral sclerosis (ALS) is a fatal adult disease caused by progressive death of nerve cells that connect muscles to the brain and the spinal cord. This provokes gradual paralysis and death usually 3-5 years from symptom onset. There is currently no cure and the only drug available, Riluzole, has modest effect prolonging life for only approximately 3 months. Expansion of a repeated sequence in the C9ORF72 gene is the commonest alteration found in the DNA of patients with ALS. This genetic alteration causes damage to thousands of messenger species resulting in the potential malfunction of multiple aspects of the cellular machinery. However, the actual culprit(s) causing motor neuron injury have not yet been found. This project aims to identify the contribution of each potential abnormal mechanism that causes motor neuron injury in C9ORF72-related ALS, their pathological mode of action, and will help us to find biomarkers useful in diagnosis and disease monitoring. We have used cutting-edge scientific methods to produce nerve cells from healthy and C9ORF72-ALS patients' skin cells and have generated other cell models engineered for studying individually the effects of one potential mechanism of disease at a time. Using these models, we will now be able to carry out experiments aimed at understanding how decreased electrical/survival properties of nerve cells, altered content of messenger species and abnormal passage of messengers from the nucleus into the cytoplasm, occur in disease. In the future, we expect that a better understanding of the basic biological mechanisms of C9ORF72-related ALS from this study will allow the development of novel strategies for neuroprotective therapy.

ALS is characterised by relentless motor neuron (MN) degeneration causing progressive paralysis and death usually within 3-5 years. Expansion of GGGGCC repeats in intron 1 of C9ORF72 is the commonest genetic cause of ALS, present in 10% of cases. Three potential pathophysiological mechanisms have been proposed: 1. Toxicity of the expanded c9orf72 pre-mRNA; 2. Aberrant synthesis of dipeptide repeat proteins (DPRs); 3. Haploinsufficiency due to decreased levels of C9ORF72 mRNA/protein. However, recent work has shown that C9ORF72 mRNA/protein levels are not significantly altered in C9ORF72-ALS patients. In contrast, a large body of evidence favours a toxic gain-of-function(s) of the expanded pre-mRNA via formation of RNA foci, sequestration of RNA-binding proteins and/or by repeat-associated non-ATG (RAN) translation and cytoplasmic accumulation of DPRs. This proposal aims to identify the contribution and molecular mechanisms of the toxic gain-of-function that causes C9ORF72-related neurodegeneration. To achieve this, we have generated induced pluripotent stem cells derived from control and C9ORF72-ALS patient fibroblasts which are further differentiated into MNs, or astrocytes recently shown to play a toxic role in MN injury. In addition, we have engineered inducible motor neuron-like and lentiviral transduction based cell models of primary MNs/astrocytes that allow controlled expression of either hexanucleotide repeat expansions (RNA toxic gain-of-function) independently of RAN translation, or production of DPRs (protein toxic gain-of function) independently of GGGGCC RNA repeats. These will allow an extensive and in depth examination of altered stress responses, cell survival, metabolic pathways, disease-related gene expression signatures and aberrant nuclear export of mRNAs. This innovative research work is likely to define novel therapeutic strategies for neuroprotection and to characterise much-needed biomarkers of disease progression and therapeutic response.

This work will have significant impact on scientific and clinical researchers, as well as patient populations. Current ALS treatment options are extremely limited and show poor efficacy. Novel therapeutic approaches will have significant impact on both society in general, and the population of patients afflicted by ALS and the related condition of fronto-temporal dementia. The C9ORF72 GGGGCC-intronic repeat expansion is the most common known genetic cause of ALS accounting for 10% of cases. The first step in developing new therapeutic strategies is to identify the pathophysiological mechanism(s) of motor neuron (MN) injury induced by the G4C2 repeat expansion. This project builds on 2 years of productive collaboration between high quality neurodegeneration research institutions in the UK and China. We have generated C9ORF72-ALS iPS-derived motor neurons and additional cell model systems designed to answer specific pathobiological questions. 1. iPS-derived MNs or astrocytes reproduce pathological RNA foci formation and repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs). 2. The inducible and lentiviral cell models we have engineered allow expression of the G4C2 expansion or of DPRs independently of the entire C9ORF72 gene. These tools will allow us to investigate the contribution of RNA foci with associated protein sequestration and the DPRs to MN injury and their mechanisms of action. Using transcriptomics, biochemical, cell biology and electrophysiological approaches, we will mechanistically study the molecular pathways relevant to the pathobiology of C9ORF72-ALS. We expect this novel approach to transform our understanding of the basic biology underlying the commonest subtype of ALS, to provide new insights into disease pathogenesis and to open the way for the development of new therapeutic approaches. Potential Beneficiaries Academic: The immediate beneficiaries of our work will be the academic community in which we conduct our research. We will ensure that our data is visible and recognised within the community by publication in top-ranking journals, verbal communication at national and international conferences and by liason with appropriate charitable organisations. It is through these communication activities that the collaborations underlying the current application were established. Clinical: We have major links with the ALS/MND patient community locally, nationally (through the DeNDroN MND Clinical Studies Group of which PJS is Chair), and internationally. PJS is clinically active and has already engaged the help of patients and family members in the donation of biosamples (fibroblasts and post-mortem CNS tissues) underpinning this project. C9ORF72 related ALS is a newly identified important molecular subtype of disease, accounting for approximately 10% of cases. Understanding the basic biology of C9ORF-related ALS is therefore likely to have a major impact on the evaluation and impact of new therapeutic clinical trials in this disease arena. All of our research proposals are reviewed by our PPI group, the Sheffield MND Research Advisory Group, and their views and suggestions are included in the development of our research proposals. Commercial: It is expected that the outcomes of this project will lead to future collaborative approaches between our academic laboratories and Pharma-Biotech partners. We already have effective partnerships in place with several industry partners in relation to both basic research and phase II and III clinical trials. We have a collaborative MRC MICA project with Astra-Zeneca for drug repurposing in the development of novel neuroprotective therapies for ALS. We are currently in advanced stages of discussion with Vertex Pharma and Biogen Idec in relation to the formation of formal academic-industrial partnerships for therapy development in ALS, including the C9ORF72 subtype. This project will enhance these partnership opportunities.