The impact of TDP-43 on translation and the response to axonal damage in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a devastating neurodegenerative disorder that causes progressive loss of MNs, leading to impaired muscle function and paralysis. ALS is incurable and leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of about 1 in 400.

Motor neurons (MNs) are the nerve cells that send signals from the spinal cord to our muscles. They are amongst the largest cells in the body, with their cell soma located in the spinal cord and its fiber (called 'axon') projecting outside to the muscles. All cell compartments, including axons, need a constant supply of newly synthesised proteins in order to function properly. On some occasions, for example when responding to an unpredicted event as cell damage, new and different proteins are absolutely needed to re-establish cell functionality and long term survival. This is particularly challenging for axons, as the RNA for these proteins, which is essential to make them, may need to be synthesized very far away in the nucleus.

Multiple lines of evidence indicate that one of the crucial players in the disease mechanism of ALS is a protein called TDP-43, which is important for the specific transport of RNA to different locations in the axons and in the response of cells to stress and damage.

In this Fellowship, I will test how TDP-43 impacts on the response of MNs to damage in the axons, and the relevance of this response pathway in ALS. To do so, I will combine novel mouse models of disease and patient cell lines with state-of-the-art biological tools that will allow the investigation of these very specific functions.

In particular, we have developed novel strains of mice that carry specific mutations in the mouse TDP-43 gene and, as a result, develop crucial features of ALS. These mouse models of ALS will allow us, by looking over time at pre-symptomatic mice and at mice with overt ALS symptoms, to identify the molecular and cellular changes occurring during disease progression. Using an innovative and fully integrated approach, we will analyse the disease phenotype of these mice with new molecular biology and microscopic techniques to identify which types of RNAs and proteins are present in different MN regions, including the axon, and how these change in response to damage.

I will therefore be able to investigate the cellular alterations triggered by TDP-43 in axonal damage response by studying: 1. MNs grown in special culture dishes where the axons are separated from the cell bodies; 2. The axons and their damage response in wild type and mutant mice; and 3. By validating our results in MNs derived from patient-induced stem cells and using post mortem brain samples donated to research by ALS patients.

In summary, this project will contribute to understand how changes in TDP-43 impacts on MN survival. This long-awaited information is essential to develop effective therapeutics for motor neuron disorders.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, which causes loss of motor neurons (MNs) leading to paralysis and ultimately death.
The RNA-binding protein TDP-43 is a central player in ALS pathogenesis, but how its mutations and malfunction lead to disease is unknown. Published work and our preliminary data show: 1) TDP-43 is involved in RNA splicing, transport and translation; 2) axonal RNA localisation and translation is crucial for neuronal response to injury; and 3) cellular responses to stress and axonal damage are impaired in ALS.
These observations converge to define my research question - I will investigate how mutations in TDP-43 impact on a MN process crucial for ALS: their response to damage.
In order to do so, I will use our novel mouse model which bears a single point mutation in endogenous TDP-43 gene and develops progressive MN loss, allowing us to investigate the consequences of mutations within a physiological in vivo setting.
I will combine novel experimental setups and more established approaches to address specific points:
a) What is the impact of mutant TDP-43 on axonal translation?
b) How does mutant TDP-43 alter the neuronal response to oxidative stress, and specifically to axonal stress?
c) Does mutant TDP-43 alter response to axonal damage in vivo?
d) Does the reversal of these changes impact on TDP-43 toxicity?
I will use novel mouse tools and refined culture and biochemical approaches to identify MN-specific and axonal translation changes both in vivo and in vitro. I will then use a novel set-up we have devised to study the MN response to axonal stress in vitro, and perform axotomies combined with laser capture microscopy to characterise the impact of TDP-43 on axonal injury in vivo.
Finally, I will use iPSC-derived MNs and post mortem material from patients to validate our findings, as this is paramount for the identification of novel pathways and potential therapeutic targets in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with a 1:400 lifetime risk, for which no effective treatment is available. The aim of this proposal is to generate new knowledge about the pathomechanisms underlying ALS, by using innovative biological tools and disease models; this data will have impact on researchers and clinician scientists operating in the ALS field and more in general in the study of neurodegenerative diseases, and will be important to enable drug discovery and therapy testing, resulting in a broad impact on patients and carers.

As outlined below, this work will have a significant impact on: 1) the neuroscience community; 2) ALS researchers; 3) neurodegeneration research; 4) biotech and pharma; and 5) the patients.

1) It has recently emerged that the local protein synthesis is crucial for axons and their response to damage. Our knowledge of axonal translation and axonal injury response is still very limited. In the first instance, this project will therefore provide novel biological information regarding axonal translation and neuronal response to axonal injury, and will therefore have a very broad impact in neuroscience research.

2) This project will provide insights in how disease-causing mutations impact on motor neuron viability and the capacity of motor neurons to respond to axonal damage. These areas of research are of high relevance to the understanding of ALS pathogenesis and therefore will have a significant impact on ALS research.

3) Axonal injury has been postulated to be involved in numerous neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease, therefore extending the relevance of these findings to the entire neurodegeneration field.

4) Our findings will be centred on a specific neuronal response and its alteration in pathological conditions. We will also test whether specific targets will modify the TDP-43 toxicity. Henceforth, the results generated during this fellowship will provide new insights for setting up new assays and identify new targets for the development of ALS therapeutics by pharma and biotech.

5) ALS patients will ultimately benefit from the development of drugs that can slow or halt their disease; this would directly contribute to improving the health and well-being of society. The development and commercialisation of an effective therapy for ALS would also lead to huge economic benefit.