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Human iPSC-derived motor neurons with TDP-43 nuclear loss present early ALS pathological phenotypes

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the degeneration of motor neurons and presents a critical need for effective treatment. By establishing and characterizing a human-induced pluripotent stem cell (iPSC)-derived motor neuron (iMN) model with dysfunctional TDP-43 nuclear localization signal (ΔNLS), this study investigates the impact of TAR DNA-binding protein 43 (TDP-43) nuclear depletion in ALS. The model successfully recapitulates functional deficits observed in ALS. Proteomic and metabolomic profiling revealed dysregulation in oxidative stress pathways and altered neurotransmitter release, shedding light on the underlying mechanisms of ALS pathophysiology. This work provides a comprehensive understanding of TDP-43 nuclear depletion in the context of ALS pathogenesis.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Generation Of Neurons

DISEASE(S): Amyotrophic Lateral Sclerosis

SUBMITTER: Tze Khee Chan

LAB HEAD: Aaron Zefrin Fernandis

PROVIDER: PXD062725 | Pride | 2026-07-31

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20250408_130940_dNLS-PRIDE.sne	Other
	DIA_iMN_WT_2uL_r4.mzML	Mzml
	DIA_iMN_WT_2uL_r5.mzML	Mzml
	DIA_iMN_WT_2uL_r6.mzML	Mzml
	DIA_iMN_dNLS_2uL_r1.mzML	Mzml

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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 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).

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.

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Human iPSC-derived motor neurons with TDP-43 nuclear loss present early ALS pathological phenotypes

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