Dataset Information

Proteomics analysis of TDP-43 aggregates derived from HEK293A

ABSTRACT: 97% of sporadic ALS patients exhibit pathology and aggregation of a global RNA regulator protein, transactive response DNA binding protein of 43 kDa (TDP-43). The goal of this study was to optimize and characterize a novel immuno-purification platform for ALS-associated TDP-43 aggregates using a scalable HEK293A culture model. Proteomics analysis was used to profile disease-associated post-translational modifications and TDP-43 co-aggregating proteins. Our findings support use of this protocol to generate pathologically relevant TDP-43 aggregates suitable for mechanistic studies in biochemical and cell-based assays.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Laura Herring

LAB HEAD: Todd Cohen

PROVIDER: PXD035705 | Pride | 2023-07-20

REPOSITORIES: Pride

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Publications

Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates.

Evangelista Baggio A BA Cahalan Shannon R SR Ragusa Joey V JV Mordant Angie A Necarsulmer Julie C JC Perna Robert J RJ Ajit Tejazaditya T White Kristen K Barker Natalie K NK Tian Xu X Cohen Sarah S Meeker Rick R Herring Laura E LE Cohen Todd J TJ

iScience 20230411 5

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved, ubiquitously expressed nucleic acid-binding protein that regulates DNA/RNA metabolism. Genetics and neuropathology studies have linked TDP-43 to several neuromuscular and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under pathological conditions, TDP-43 mislocalizes to the cytoplasm where it forms insoluble, hyper-phosphorylated aggregates durin ...[more]

PMID: 37182104

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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.

Dataset Information

Proteomics analysis of TDP-43 aggregates derived from HEK293A

Publications

Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates.

Similar Datasets

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