Investigating deficits of axonal RNA metabolism and axonal signalling in amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS), also known as motor neuron disease (MND), is a devastating neurodegenerative disorder which causes progressive loss of 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.

The main cells affected in this disease are nerve cells called motor neurons (MNs), which progressively die during the course of ALS. MNs are amongst the largest cells of the body and connect the brain and the spinal cord to the muscles therefore making movement possible. In order to do this, MNs rely on one thin process, named the axon, which extends from the spinal cord out to each and every muscle of our body. In adults, a single axon can measure over a meter, running from the spinal cord to our fingers or toes, and needs sophisticated transport and communication systems to survive and function.

Importantly, research has shown that abnormalities in axons are found in the very early stages of ALS and other incurable human diseases. All cells in an individual's body, although very diverse from each other, contain the same genetic material called DNA, that gives instructions to each individual cell. Therefore the identity of each cell type (whether a MN or a heart cell, for example) is the result of which portions of DNA are active and produce another type of chemical called RNA. RNA carries all the necessary information for the cell to function. The sum of all the RNA in a cell, named the transcriptome, is the signature that characterises each cell type.

Knowing the transcriptome of a certain cell type provides insights into its biology and helps determine the causes of diseases. This is particularly relevant with MNs in ALS since there is good evidence showing that the biological processes linked to RNA 'metabolism' are primarily affected in ALS.

Further, RNA is transported in axons and this is essential for axon maintenance and its response to injuries.

The findings summarized above, highlighting that: 1) axons are involved in early stages of disease; and 2) ALS is caused by alterations of the RNA repertoire in MNs, alongside with novel preliminary data from my Sponsor's laboratory which shows that 3) key ALS molecules localise to cellular organelles which are involved in the communication system of axons, all converge to form my research questions.

I will use a novel animal model of ALS to investigate:

a) Which changes occur in the RNA of axons;

b) How these changes can play a role in ALS;

c) How the communication system between axons and the cell body is affected in ALS.

The feasibility of this project is ensured by the recent technological advances provided by my Sponsor's laboratory and collaborators. These cutting edge approaches will allow me to isolate and study RNA specifically found in MNs and their axons. Further, I will be able to isolate the small particles that contribute to transmitting survival signals in MN axons.

In summary this project will contribute to understand how axons function normally and what goes wrong in ALS. This will greatly help us to understand disease mechanisms and discover novel targets for effective therapies for ALS.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a relentlessly progressive neurodegenerative disorder, which causes loss of motor neurons (MN) leading to paralysis and ultimately death. It is currently untreatable, hence there is a desperate need for understanding its underlying mechanisms to develop novel and effective therapeutic strategies.

Published research and preliminary data show that: 1) RNA metabolism alterations play a role in ALS; 2) MN axons are affected in the early stages of disease; 3) axonal RNA transport is altered in ALS and deficits in axonal RNA localisation make axons more susceptible to noxious stimuli; 4) key ALS proteins associate with signalling endosomes (SEs), endosomal organelles which are responsible for axonal signalling and transport of survival messages.

These observations converge to define my research questions. I will investigate:

a) the axonal RNA changes occurring in ALS;

b) the mechanisms contributing to these changes, with a focus on RNA stress granules (cytoplasmic bodies, altered in ALS, where RNAs are protected during cell stress);

c) the novel link between key ALS proteins and axonal signalling.

In order to do so, I will combine the cutting edge tools and unique resources available in my Sponsor's laboratory and through collaborators. I will isolate MNs from a unique novel ALS mouse model which expresses an aggressive ALS-causative FUS mutation at physiological levels. I will then use microfluidic chambers and the UPRT RNA tagging technology in order to specifically isolate axonal RNA from MNs; I will then analyse SEs using magnetic isolation techniques and quantitative proteomics.

Finally I will be able to validate my results using differentiated MNs derived from human iPSC isolated from ALS patients. These findings will be further tested in patient tissue and will be 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 mechanisms underlying ALS, by using innovative biological tools and disease models; this data will have impact on researchers and clinician scientists and will be important to enable drug discovery and therapy testing in the future, with therefore a broad impact on patients and carers.

1) Although axons are a crucial compartment of neuronal cells, our knowledge of axonal RNA and signalling molecules, is still very limited. In the first instance, this project will provide novel biological information and data regarding the axonal transcriptome and will therefore have a very broad impact in neuroscience research.

2) This project will provide insights in axonal biology in ALS, and specifically in RNA metabolism and signalling alterations occurring in the disease and therefore have impact on ALS research.

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

4) Our findings may benefit biotech/pharma by providing new insights and new targets for the development of therapeutics for ALS, and possibly other RNA disorders.

5) Amyotrophic lateral sclerosis 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 a therapy for ALS would also lead to huge economic benefit.