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Therapeutic manipulation of SRSF1 mitigates genome-wide transcriptome alterations and neuronal hyperexcitability in C9ORF72-linked amyotrophic lateral sclerosis

ABSTRACT: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to relentless paralysis and death usually within a few years from symptom onset. Thousands of RNA molecules with roles in multiple cellular pathways are compromised in disease challenging the identification of alterations causing pathogenesis over downstream changes consequent to neurodegeneration. We recently showed that partial depletion of SR-rich splicing factor SRSF1 inhibits the nuclear export of pathological C9ORF72-repeat transcripts and subsequent translation of dipeptide-repeat proteins in patient-derived neurons and Drosophila, providing in turn a promising strategy of neuroprotection for the most common form of ALS. While the roles of SRSF1 remain poorly characterised in neurons, its therapeutic manipulation offers a rare opportunity to identify disease-modifying gene expression changes. Here, we report diseased and neuroprotected transcriptomes from human-derived neurons and Drosophila heads. Strikingly, while the depletion of SRSF1 has limited effects on genome-wide expression, splicing and nuclear export of RNAs, reversal of 90 disease-modifying transcripts were identified over 2,000 RNA changes in human disease. Functional validation further demonstrated that conserved potassium intermediate/small conductance calcium-activated KCNN channel inhibitors mitigate C9ORF72-ALS mediated hyperexcitability and death of human motor neurons as well as Drosophila motor deficits. Our data support efficacy and safety of SRSF1 manipulation as a therapeutic approach leading to rescue of multiple biological processes without disrupting neuronal transcriptomes. In addition to a potential new therapeutic target for pharmacological inhibition, we also identified in vitro and in vivo disease-modifying gene expression signatures to guide the development of new biomarkers and novel therapeutic approaches in C9ORF72-ALS/FTD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE139900 | GEO | 2021/07/19

REPOSITORIES: GEO

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Project description:Amyotrophic lateral sclerosis (ALS) is a deleterious neurodegenerative disease without effective treatment options. Recent studies have indicated the involvement of the dysregulation of RNA metabolism in the pathogenesis of ALS. Among the various RNA regulatory machineries, nonsense-mediated mRNA decay (NMD) is a stress responsive cellular surveillance system that degrades selected mRNA substrates to prevent the translation of defective or harmful proteins. Whether this pathway is affected in neurodegenerative diseases is unclear. Here we report that the NMD pathway is inhibited in ALS patients with C9orf72 hexanucleotide repeat expansion (HRE), the most common cause of familial ALS. Bioinformatic analysis of multiple transcriptome profiles revealed significant overlap of upregulated genes in NMD-defective cells with those in the brain tissues, micro-dissected motor neurons, or induced pluripotent stem cell-derived motor neurons from ALS patients carrying C9orf72 HRE, suggesting the suppression of NMD pathway in these patients. In contrast, there are few overlapping upregulated genes in brain tissues from sporadic ALS patients with those in NMD-defective cells. Using Drosophila models expressing various C9orf72 HRE products, we have validated the suppression of the NMD pathway by C9orf72 HRE and identified arginine-rich dipeptide repeats (DPRs) generated from HRE as the main culprits of NMD inhibition. Furthermore, in human SH-SY5Y neuroblastoma cells and in mouse brains, expression of arginine-rich DPRs was sufficient to cause NMD inhibition. Remarkably, expression of UPF1, a core gene in the NMD pathway, efficiently blocked neurotoxicity caused by arginine-rich DPRs in both cellular and Drosophila models. Although not as effective as UPF1, expression of another NMD gene UPF2 also ameliorated the degenerative phenotypes in DPR-expressing flies, indicating neuroprotection by genetically reactivating the NMD pathway. Finally, after validating Tranilast as an NMD-activating drug, we demonstrated its therapeutic potential in cellular and Drosophila models of C9orf72 DPR neurotoxicity. Therefore, our study has revealed the suppression of NMD in C9orf72 ALS and has suggested that the restoration of the NMD pathway by Tranilast, an asthma drug with solid safety record, could be a promising therapeutic strategy for ALS.

Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from lower motor neurons obtained by laser capture microdissection from autopsy material from neurologically healthy controls (n=6) and cases of sporadic ALS (n=3) and ALS due to C9ORF72 mutations (n=3).

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Therapeutic manipulation of SRSF1 mitigates genome-wide transcriptome alterations and neuronal hyperexcitability in C9ORF72-linked amyotrophic lateral sclerosis

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