Dataset Information

Maintenance of neuronal TDP-43 expression requires axonal lysosome transport

ABSTRACT: TDP-43 mislocalization and pathology occurs across a range of neurodegenerative diseases, but the pathways that modulate TDP-43 in neurons are not well understood. We generated a Halo-TDP-43 knock-in iPSC line and performed a genome-wide CRISPR interference FACS-based screen to identify modifiers of TDP-43 levels in neurons. A meta-analysis of our screen and publicly available screens identified both specific hits and pathways present across multiple screens, the latter likely responsible for generic protein level maintenance. We identified BORC, a complex required for anterograde lysosome transport, as a specific modifier of TDP-43 iP lysosome location is required for proper protein turnover. As such, lysosome location and function are crucial for maintaining TDP-43 protein levels in neurons.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yue Andy Qi

LAB HEAD: Veronica Ryan

PROVIDER: PXD063206 | Pride | 2025-08-01

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	032124_Bldg49_SL_D15_KD_H_1.raw	Raw
	032124_Bldg49_SL_D15_KD_L_1.raw	Raw
	032124_Bldg49_SL_D15_NT_H_1.raw	Raw
	032124_Bldg49_SL_D15_NT_L_1.raw	Raw
	032124_Bldg49_SL_D7_KD_H_1.raw	Raw

Items per page:

1 - 5 of 50

Publications

Maintenance of neuronal TDP-43 expression requires axonal lysosome transport.

Ryan Veronica H VH Lawton Sydney S Reyes Joel F JF Hawrot James J Frankenfield Ashley M AM Seddighi Sahba S Ramos Daniel M DM Epstein Jacob J Faghri Faraz F Johnson Nicholas L NL Zou Jizhong J Kampmann Martin M Replogle John J Qi Yue A YA Yuan Hebao H Johnson Kory R KR Maric Dragan D Hao Ling L Nalls Mike A MA Ward Michael E ME

bioRxiv : the preprint server for biology 20250630

TDP-43 mislocalization and pathology occurs across a range of neurodegenerative diseases, but the pathways that modulate TDP-43 in neurons are not well understood. We generated a Halo-TDP-43 knock-in iPSC line and performed a genome-wide CRISPR interference FACS-based screen to identify modifiers of TDP-43 levels in neurons. A meta-analysis of our screen and publicly available screens identified both specific hits and pathways present across multiple screens, the latter likely responsible for ge ...[more]

PMID: 39803527

Similar Datasets

Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of phosphoTDP-43, p-tau, alpha-synuclein and beta-amyloid pathology, were biochemically fractionated and analysed by immunoblot and MS for detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed synthesis of heavy isotope-labelled peptides for the absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson’s and 8 Alzheimer’s disease and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal Truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent TDP-43 pathology. In conclusion this is a novel, sensitive and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

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).