TDP-43 biomarker development in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS, the commonest form of motor neuron disease, MND) is a neurodegenerative disease with survival from symptom onset of around 30 months. There is currently no test or marker of disease activity and the diagnosis depends on the opinion of an experienced neurologist. Identification of specific disease markers ("biomarkers") are needed to facilitate effective therapy development. TDP-43 is a normal protein that abnormally accumulates in nerve cells of nearly all cases of ALS and half of those with the linked condition of frontotemporal dementia (FTD). Measuring the disease-relevant form of TDP-43 in the blood and spinal fluid of ALS patients has not been successful so far. Measuring TDP-43 in these fluids might reduce diagnostic delays, and offer a readout for use in future drug trials.

A study is proposed to enrich the disease-relevant form of TDP-43 across a range of already collected biofluids from well-characterised ALS patients and tissue donated after death. It aims to identify a specific disease-relevant form using a highly specialised technique of mass spectrometry, which can then be used to develop a test for TDP-43 from patient fluids.

Additionally the study will investigate proteins that interact with the disease-relevant form of TDP-43. These proteins may be just as important in the development of ALS and may provide the basis for developing novel treatments to alleviate the disease. We will study TDP-43 interacting proteins in two ALS disease models: A) spinal motor neurons from existing mouse embryonic stem cells that carry a mutation in the TDP-43 gene and B) human induced stem cells grown from an ALS patient skin biopsy with the same genetic defect. Isolation of TDP-43 from these different cellular disease models enables the identification, comparison and assessment of the most relevant TDP-43 interacting proteins.

This unique multimodal approach to the study of TDP-43 will provide much-needed markers of disease activity essential for the discovery and development of novel drug treatments.

The core aim is to identify the most relevant pathological form of TDP-43 in human biofluids and to establish the key pathological protein pathways involved in TDP-43 pathology for potential therapeutic targeting as well as biomarker development.

Objectives: A) enrich pathological forms of TDP-43 and identify candidate TDP-43 peptides to measure in patient samples; B) isolate pathological TDP-43 and its interacting proteins from novel ALS disease cellular models and identify interactors involved in pathogenesis.

Methodology: Cerebrospinal fluid, serum and case-matched post mortem tissue from ALS patients studied as part of the BioMOx cohort, and healthy controls, will be enriched for ubiquitinated and phosphorylated proteins and subsequently immunoprecipitated for TDP-43. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) samples will be analysed for disease relevant TDP-43 peptides and validated using multiple reaction monitoring (MRM) in biofluid samples from patients with ALS, mimic disorders (e.g. multifocal motor neuropathy), neurodegenerative disease controls, and healthy controls, followed by subsequent MRM validation in an independent cohort prior to immunoassay development. To study human TDP-43 and detect its interacting proteins, we will use mouse embryonic stem cell derived spinal motor neurons expressing human TDP-43 WT and TDP-43 M337V. TDP-43 will be immunoprecipitated and its interactors analysed using LC-MS/MS and tested for relevance in human disease by using human induced pluripotent stem cell derived motor neurons from an ALS M337V mutation carrier and human post mortem tissue.

The discovery of biomarkers reflecting the major pathology in ALS will profoundly influence the clinical and scientific implication for early identification of ALS patients and allow earlier access to therapeutic trials. Such biomarkers will facilitate monitoring of therapeutic response and has potential for the identification of novel drug targets.

Patients and Carers
The diagnosis of ALS is clinical, often made in tertiary references centres after significant referral delay because of the lack of objective biomarkers, so that at the time of diagnosis the disease is often in an advanced state. Patients frequently suffer from uncertainty, which is accompanied by efforts of further diagnostic procedures in mostly disabled patients. Phenotypes of ALS, which are not included in the recent clinical diagnostic certainty categories, may be even more challenging to diagnose and even more time consuming. ALS patients report that the delay in their diagnosis was distressing and meant a loss of their life quality remaining before the onset of severe disability. A specific disease-related marker for ALS would help facilitate diagnosis preferably soon after symptom onset thereby preserving quality of life. Being able to predict an individual rate of disease progression provides valuable information to both patients and carers for the planning the patient's remaining time. A new biomarker might allow earlier and more inclusive trial enrolment, to monitor the effectiveness of treatments and replace survival as outcome measure, resulting in shorter trial periods. In our group's experience many patients and carers wish to participate in biomarker studies with the hope to better understand the condition and move towards cure even if they will not benefit personally.

Commercial sector
Many pharmaceutical companies are attracted to therapeutics targeting pathological proteins found in neurodegenerative diseases. Most recently a trial has started targeting the Tau protein for a subgroup of FTD patients in which Tau aggregation is the dominant pathology. This is the first causal therapy approach in FTD as so far there have been only symptomatic treatments. The larger subgroup of FTD comprises cases with TDP-43 pathology common to ALS. Findings from studies in ALS patients therefore have translational potential for other diseases. Understanding TDP-43 pathology may open new insights to the pathological processing and trafficking of pathological altered and aggregated proteins in other neurodegenerative diseases.

A major advantage of a biomarker for industry is to evaluate disease severity prior to trial inclusion, in order to select those patients who are more likely to show a response to the drug, to provide reliable endpoints for clinical trials and a solid basis for power calculations. This includes drug repurposing. The opportunity to more quickly test a drug already on the market for another condition could potentially save years on the usual lengthy drug development process. The biotech industry also benefits through promotion and development of novel biomarker assays and diagnostic kits that may emerge from the work proposed.

UK economy and global science platform
ALS is a clinical diagnosis relaying on the exclusion of other diseases. The severity and impact of this disease on patients is often accompanied by extensive diagnostic investigations. Including a neurochemical marker in the diagnostic criteria of ALS increases the effectiveness of health services by making a diagnosis more accurately at an earlier stage, and avoiding unnecessary investigations, and occasionally surgery, known to be a significant problem in ALS. Our approach to develop a neurochemical biomarker on unique Oxford ALS disease models and in a large ALS biomarker cohort, including the long-term goal of therapeutic intervention in this disease, provides a very strong example of the UK leading the way in cutting-edge research. Global attention will be drawn to this model when informing about our research and providing information for medical professionals and scientists in ALS.