In ALS patients, a rogue snippet of genetic code inserts itself into a key protein-building enzyme, AARS1, creating a flawed version that fails to reach the tips of motor neurons and disrupts sleep in fruit flies. This matters because ALS is a fatal, incurable disease where motor neurons die, and the underlying trigger in most cases is the misplacement of a protein called TDP-43. TDP-43 loss causes cells to accidentally include "cryptic exons" in their RNA, and while some of these errors are known to destroy proteins, the newly discovered AARS1 cryptic exon instead creates an altered protein. Whether this altered protein actively drives neuron death is unknown. If this research succeeds, it will determine whether the AARS1 cryptic protein is a direct cause of neurodegeneration. That would open a new class of therapeutic targets—drugs that block the cryptic exon or counteract the damaged enzyme—for a disease that currently has no cure. The work is fundamental science, but understanding exactly how a single cryptic protein disrupts motor neuron function could, like past discoveries in RNA biology, eventually translate into treatments that slow or halt disease progression.
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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 contributes to ALS. Utilising human iPSC-derived lower motor neurons (i3LMN) and the fruit fly, Drosophila melanogaster, we have generated unique in vitro and in vivo models expressing the AARS1 CE-containing protein (AARS1Cryptic). Our preliminary data indicate that AARS1Cryptic exhibits reduced protein translation/stability and a failure to localise to motor neuron axons. Furthermore, neuronal overexpression of cryptic (but not wild-type) AARS1 reduces night sleep in Drosophila, revealing that AARS1Cryptic disrupts a fundamental neurological process (sleep), recognised to be an important risk factor for neurodegenerative diseases. Using these models, we will test whether AARS1Cryptic expression can disrupt neurological function and modify neurodegenerative phenotypes caused by TDP-43 loss-of-function. Our research strategy promises to rigorously test whether AARS1Cryptic can drive neurodegeneration and/or modify TDP-43 pathology; define how this CE affects the enzymatic activity of AARS1 and its protein interactome; and gain further insights into the molecular mechanisms contributing to motor neuron loss downstream of TDP-43 mis-localisation. Our programme will thus advance the fundamental understanding of ALS biology and widen the range of potential therapeutic targets to treat this devastating disease.
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