Identifying the early biochemical signature of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS, also known as motor neuron disease, MND) is a devastating disease in which motor neurons, nerve cells that control voluntary movement, die prematurely leading to progressive muscle weakness. In most cases, death occurs within three years from the onset of symptoms. There is currently no effective treatment. Accumulation of abnormal protein within affected nerve cells is a universal finding in people with ALS.

ALS can affect anyone, but 10% of cases carry an alteration in the code of one of a handful of genes meaning that they are very likely to develop the disease. Although the symptoms of ALS can start at any time, they usually begin in middle age, even in people who have carried a disease-causing genetic alteration from birth. This tells us that there must be compensatory mechanisms that allow motor neurons to cope with such changes. By the time people with ALS are diagnosed, these compensatory mechanisms are exhausted and motor neurons have already suffered irreparable damage. Although targeted treatment for the genetic forms of ALS is currently being tested, the irreversible damage already established by diagnosis means that treatment is likely to remain difficult unless we can identify signals of the disease before symptoms start.

Studying the cellular protein management processes in people carrying gene mutations before they have developed symptoms offers a unique opportunity to understand the early events that occur before damage occurs, the mechanisms that delay its onset and identify a signature of the disease before symptoms begin.

Cerebrospinal fluid (CSF) is the closest fluid to the cells affected by ALS. It contains proteins and other substances produced by cells of the nervous system and is therefore the best place to look for change. Within CSF, tiny fluid-filled bubbles released by nervous system cells called extracellular vesicles (EVs) are found. They carry proteins that open a window on the internal mechanics of nervous system cells. In this study, samples of CSF and blood will be collected from gene carriers before the onset of ALS, as well as from healthy non-genetic carriers and patients who have already developed ALS. Repeated samples will be collected from individuals at yearly intervals, to allow monitoring of changes over time.

This study will use proteomics - a state-of-the-art technique that allows the simultaneous measurement of thousands of proteins - to examine CSF and CSF EVs. Comparing the patterns of protein change in these different groups of people will reveal the compensatory pathways that occur before symptoms and shed light on the key events occurring before the onset of muscle weakness. The project will also study the role of these mechanisms in the disease by looking at cell models of the disease and examining nervous system and brain tissue of people who have died of ALS.

This work will allow better prediction of the risk and timing of the development of ALS in genetically-susceptible individuals allowing earlier treatment, but also identify the key pathways driving the development of ALS in patients more widely to enable new treatment development, as well as developing markers for the monitoring of the effectiveness of potential therapies.

Aims
- Characterise the pre-symptomatic cerebrospinal fluid (CSF) and CSF extracellular vesible (EV) proteome in ALS
- Identify key biochemical pathways reflecting pathological or compensatory mechanisms amenable to therapeutic intervention in both presymptomatic and symptomatic ALS

Objectives
- Establish a well-characterised longitudinal cohort of asymptomatic individuals carrying the highly-penetrant ALS-causing C9orf72 hexanucleotide repeat expansion
- Perform deep proteomic analysis of individual whole CSF and CSF EV samples from asymptomatic gene carriers, healthy controls and ALS patients
- Identify alterations in pathways implicated in ALS that vary longitudinally in gene carriers and predate clinical symptoms
- Explore mechanistic role of these pathways using in vitro models and post mortem tissue

Methodology
High-depth liquid chromatography tandem mass spectrometric analysis. Univariate and multivariate analysis to identify key proteins and pathways, including use of gene ontology, pathway and protein-protein interaction network analysis. Comparative studies of whole CSF and EV results. Subsequent validation using targeted proteomics of whole CSF, serum and urine as well as CSF and blood EVs. Further study of implicated pathways by quantitative immunohistochemistry of post mortem tissue and in C9orf72 iPSC-derived motor neuron model of ALS.

Scientific and medical opportunities
- Broadening the treatable window of ALS to include the period before the onset of symptoms
- Identification of previously unexplored markers of the earliest events in ALS pathogenesis and compensatory pathways might yield potential avenues for therapy
- Potential diagnostic value and as biochemical measure of response to intervention
- Broader value, through data-sharing, through contribution to global efforts to define the human CSF and EV proteome and validation of disease models

Patients with ALS and their families
The proposed project is highly translational and has the potential for major impact on those suffering from ALS and their families. Short term benefits, primarily the identification of early disease markers and identification of diagnostic or prognostic biomarkers, should be realised during the fellowship. The eventual goal of enabling earlier treatment of ALS will rely on additional work that is likely to take years and would not be expected to arise directly from this project; similarly, validation of biomarkers and incorporation for clinical use relies upon work by other researchers, likely to take time beyond the duration of the fellowship.
The scientific community and industry
There is great interest in the ALS field in understanding the cellular and neurochemical alterations occurring in carriers of ALS-causing gene mutations before the development of symptoms. This has the potential to advance our understanding of the disease and validate disease models. This information will be available within the 5 years of the fellowship, most likely in the 3rd-5th years.
The data will also be of interest to those involved in clinical proteomic analysis and study of cerebrospinal fluid (CSF) neurochemistry as well as extracellular vesicle (EV) biology and biomarker potential. This will also provide a large dataset of CSF and CSF EV proteomic data that could contribute to wider proteomic undertakings such as the Human Proteome Organisation's Human Proteome Project. This data will become available during the course of the fellowship, during years 3-4.
Depending on the outcome, the results of this work will benefit researchers developing novel treatments for sporadic ALS as well as gene-targeting treatments for genetic ALS, in both academic and industrial spheres. Demonstration of neurochemical alterations years prior to the onset of symptoms would suggest that targeted gene therapies will be useful in prevention of ALS in genetically predisposed individuals. These changes might also be useful as measures of efficacy or target engagement of potential treatments. Alterations indicative of compensation for gene mutations would provide a strong rationale for treatments aiming to enhance these mechanisms in both genetic and sporadic ALS. This benefit may be realised within the duration of the fellowship but will likely require external validation.
The project will also develop a cohort of subjects carrying ALS-causing genes who are likely to be motivated to take part in trials of targeted gene therapy and will be asked to consent to their details being shared for this purpose. This will be realised throughout the duration of the fellowship.
Economic benefits
I hope that the results of this work will be used to advance treatment of ALS and develop clinically useful assays in order to guide treatment. This has the potential for financial and economic benefits through commercialization. The timescale for these benefits is likely to be years beyond the end of the fellowship. Effective treatment or even prevention of ALS could reduce health and social care costs; any benefit brought about by this project is likely to occur years after its end.
Skills development for research staff
It is anticipated that the postdoctoral researcher and Dr Thompson will increase their experience in laboratory methods including liquid chromatography, immunohistochemistry and tissue culture. In particular, they will learn skills in shotgun and targeted proteomic analysis including bioinformatics and handling high-dimensional data. Beyond this, both Dr Thompson and the postdoctoral researcher will learn transferrable skills through the University's skills training including data management, communication and public engagement, statistics and computer programming, teaching and entrepreneurship as well as external courses such as the Wellcome Genome Campus proteomics bioinformatics course and bioinformatics summer school.