Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease with severe symptoms and premature death in patients. This disease is characterised by the degeneration of upper and lower motor neurons in the brain and spinal cord, resulting in muscle denervation, weakness, and atrophy. ALS patients die within 3-5 years of symptom onset, usually due to respiratory failure after the death of muscles innervating the diaphragm. Since the 1990s, genome-wide association studies, next-generation sequencing and whole-genome sequencing identified over 20 ALS susceptibility genes. These genes appear to have a 5-10% familial origin, whereas the remaining 90-95% have a sporadic onset.Recent whole-exome sequencing in a cohort of 787 patients identified a novel ALS susceptibility gene, ARPP21, that accounted for approximately 1.7% of familial and 0.4% of sporadic cases. ARPP21 is a cytoplasmic RNA-binding protein with multiple isoforms, whereby the full-length protein harbours an N-terminus, putative RNA binding domains R3H and SUZ, and a C-terminus. We utilised in vitro and in vivo approaches to investigate the pathological consequences of ALS-linked mutations in the C-terminus of ARPP21: P563L, P563Q and P747L. Characterisation of the functional domains of ARPP21 revealed that the C-terminus, a low complexity region, formed granular structures in the cytosol and were insoluble, and mutations caused enhanced granule formation compared to ARPP21WT. Furthermore, the overexpression of mutant ARPP21 formed a higher number of larger cytosolic granules. Preliminary characterisation studies of endogenous immunostaining of wildtype ARPP21 in primary cortical neurons showed it is localised in the cytosol and synapses. Overexpression of ARPP21 and subsequent live monitoring identified a novel, consistent dynamic behaviour of ARPP21 whereby it formed pulsatile granules. Validation confirmed that ARPP21 granule formation depended on neuronal depolarisation. However, severe disease-causing mutants of ARPP21 did not have the same effect and were significantly less dynamic.
Since ARPP21 is an RNA-binding protein, we wanted to understand whether it was involved in translation. Therefore, we conducted a series of artificial assays to assess whether ARPP21 mutants could alter translation. While there was no indication of the reduction in local translation, the knockdown of ARPP21 in primary cortical neurons confirmed a reduction in translation, suggesting its function could have early implications in neuronal development. Finally, we generated a novel CRISPR knock-in mouse model of ARPP21 mutants to characterise early ARPP21 pathology: ARPP21P525L and ARPP21P709L. In addition, we assessed ARPP21 localisation and distribution in the motor cortex of 3–18-month-old mice and identified that these granules consistently arise and appear to be increased in homozygous animals. The knock-in mice revealed that these granules also exist endogenously, and more granules existed in (+/+) mutants through development. Arpp21P709L (+/+) mice exhibited reduced grip strength, and RNA sequencing (RNASeq) also revealed the downregulation of genes involved in RNA splicing, metabolism, and differentiation.
Further investigation in the lumbar spinal cord of these mice identified TDP-43 aggregates. In addition, RNA-Seq data suggested that there may be alterations in some genes associated with development and RNA processing. These novel findings around ARPP21 have supposed an exciting avenue to explore RNA granule biology, mechanisms around neuronal granules in disease and its relation to ALS pathology.
Together with the recent links of ARPP21 mutations in amyotrophic lateral sclerosis patients, these novel findings uncover a paradigm for ARPP21 modulating the balance between RNA granule formation, translation and RNA metabolism in health and disease.
| Date of Award | 1 Oct 2023 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Younbok Lee (Supervisor) & Christopher Shaw (Supervisor) |