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Single cell RNA sequencing of spinal motor neurons and interneurons differentiated from isogenic FUS and TARDBP human induced pluripotent stem cell lines harbouring ALS-causative mutations

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that can be inherited with genetic mutations in > 25 different genes including e.g. FUS, TARDBP, and C9ORF72. It is currently unclear how these mutations lead to motor neuron degeneration and whether different gene mutations converge on similar pathogenic pathways early on, or if each mutation leads to motor neuron death in a unique manner. To address these questions in depth, we generated a number of isogenic iPSC (induced pluripotent stem cell) lines harbouring point mutations in FUS (R495X and P525L) or TARDBP (M337V) as well as a knock-out of FUS (FUS-KO), to discern loss-versus gain-of-function mechanisms in FUS. The use of isogenic lines enables identification of subtle early disturbances in the transcriptome which may be detrimental but impossible to detect when lines of different backgrounds are used. We conducted single cell RNA sequencing of motor neurons and interneurons derived from these lines using Smart-seq2. We show that ALS-causative mutations elicit a much larger transcriptional response in motor neurons than in interneurons. A large number of transcripts were coregulated across FUS R495X and P525L motor neurons and the majority of these were part of gain-of-function of FUS. Cross-comparison with isogenic ALS-linked TARDBP M337V identified an early dysfunctions shared across ALS-causative mutations unique to motor neurons that could have major implications for motor neuron survival and that might be targeted therapeutically.

ORGANISM(S): Homo sapiens

PROVIDER: GSE226482 | GEO | 2025/04/30

REPOSITORIES: GEO

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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 fibroblasts grown from neurologically healthy controls (n=6) and 3 groups of patients with ALS: 1) sporadic cases (n=6); 2) cases due to mutations of SOD1 (n=4); 3) cases due to mutations of TARDBP (n=3). The three ALS groups were compared to the controls.

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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Single cell RNA sequencing of spinal motor neurons and interneurons differentiated from isogenic FUS and TARDBP human induced pluripotent stem cell lines harbouring ALS-causative mutations

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