The role of AARS1 cryptic splicing in ALS: unravelling the mechanisms underlying neurodegeneration in TDP-43 proteinopathies

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterised by the loss of upper and lower motor neurons. The majority of ALS cases are sporadic and characterised by cytoplasmic mis-localisation of TDP-43, an RNA-binding protein that is predominantly nuclear under normal physiological conditions. The loss of nuclear TDP-43 results in the aberrant inclusion of intronic regions called “cryptic exons” (CEs) in mature transcripts. CEs typically lead to the loss of the protein they are encoding and the disease relevance of these events has been established, with therapies targeting them currently in clinical trials. Recently, we and others have described CE events that can be translated, therefore leading to the formation of proteins containing a novel “cryptic peptide”. Whilst the potential of such proteins as biomarkers is actively being pursued, whether they also drive motor neuron degeneration, and the molecular mechanisms by which this might occur, remain critical knowledge gaps.
Through RNA-seq data analysis from post-mortem tissues, we detected the expression of a CE in the alanyl-tRNA synthetase 1 (AARS1) mRNA specifically in the spinal cord and motor cortex from ALS patients with TDP-43 pathology. Intriguingly, the AARS1 CE is inserted in-frame, yielding a variant of the AARS1 protein that contains a cryptic peptide (AARS1Cryptic). Importantly, missense mutations in AARS1 have been linked to Charcot-Marie-Tooth disease type 2N, a hereditary peripheral neuropathy where motor axons are affected. Since AARS1 CE inclusion occurs specifically in regions where motor neurons degenerate in ALS and single amino acid changes in AARS1 can perturb motor neuron integrity, AARS1Cryptic, where a novel peptide is inserted and predicted to alter a crucial region of the protein, represents an intriguing candidate driver of ALS pathology.
In this proposal we present an experimental strategy to examine how expression of AARS1Cryptic...