RNA dysfunction in motor neuron disease: identification of novel changes in transcript processing and localisation through long-read RNA-seq

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Motor neurons (MNs) are the nerve cells that send signals from the spinal cord to our muscles to enable movement to happen. Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is a devastating neurodegenerative disorder which causes progressive loss of MNs, leading to 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. RNAs are essential molecules that carry the information for making individual proteins from DNA. The correct processing of RNAs is crucial for survival and one of the central players in the disease mechanism of ALS, a protein called TDP-43, is important for the processing of RNA.In order to study and understand ALS disease mechanisms, we have recently generated novel mouse models carrying TDP-43 mutations, which induce features of ALS, including the loss of MNs. These mice allow us, by looking over time, at presymptomatic mice and at mice with ALS, to identify what changes with the progression of disease. We have used RNA-sequencing (RNA-seq) to demonstrate that these mutations cause widespread and novel changes in cell RNA. RNAs can be very long molecules, they reach the size of thousands of base pairs, but the current technology to study RNAs, allows us to sequence small fragments of RNA of up to 150 base pairs. Then, powerful bioinformatics algorithms are used to analyse these results, but, although a lot of information and changes can be detected, the architecture of the long RNAs is reconstructed with a certain degree of uncertainty. One of the limitations in understanding how these changes impact on disease is that we cannot reconstruct the exact composition of the whole-RNA architecture.Very recently novel technologies have been made available to directly understand these long RNAs. Co-applicant Towfique Raj is pioneering the use of this technology to mammalian systems and has developed sophisticated tools for analysis.We here propose to apply this novel technology to our ALS mouse models in order to gain a better understanding of how RNA changes impact on disease. In summary, this project will contribute to understand how changes in TDP-43 impacts on MN RNA and survival. The identification of this information is essential to develop effective therapeutics for motor neuron disorders. Furthermore, this project will allow us to start a novel collaboration to apply this cutting-edge technology to numerous ALS mouse and human cell models, transforming our molecular understanding of ALS.