Dataset Information

Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Perturb mRNA Splicing through Aberrant Interactions with SRSF2

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that impacts neurons in the brain and spinal cord. Although a substantial number of studies have been conducted, much remains to be learned about the cellular mechanisms underlying ALS. In this study, we employed an engineered ascorbate peroxidase (APEX)-based proximity biotinylation, together with affinity pull-down of the ensuing biotinylated peptides, to investigate the proximity proteomes of human SOD1 and its two ALS-linked mutants, G85R and G93A. We were able to identify 25 common proteins with preferential enrichment in the proximity proteomes of SOD1G85R and SOD1G93A over wild-type SOD1. Our co-immunoprecipitation and Western blot analyses showed stronger binding of the two SOD1 mutants toward one of these proteins, SRSF2, than the wild-type counterpart. We also observed aberrant RNA splicing of three neurodegeneration-associated and SRSF2-regulated genes, including EIF4A2, SETX, and MELK, in cells with ectopic expression of the two SOD1 mutants relative to cells expressing the wild-type protein. The differences in splicing elicited by the SOD1 variants were markedly attenuated upon knockdown of SRSF2. Collectively, we uncovered that ALS-liked SOD1G85R and SOD1G93A mutants interact more strongly with SRSF2, and the aberrant interactions perturbed mRNA splicing. Thus, our work offered novel mechanistic insights into the contribution of the ALS-linked SOD1 mutants to disease etiology.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Amyotrophic Lateral Sclerosis

SUBMITTER: Xingyuan Chen

LAB HEAD: Xingyuan Chen

PROVIDER: PXD050785 | Pride | 2025-06-16

REPOSITORIES: Pride

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Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Perturb mRNA Splicing through Aberrant Interactions with SRSF2.

Chen Xingyuan X Cao Zhongwen Z Wang Yinsheng Y

Analytical chemistry 20240525 23

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the degeneration of neurons in the brain and spinal cord. Although a substantial number of studies have been conducted, much remains to be learned about the cellular mechanisms underlying ALS. In this study, we employed an engineered ascorbate peroxidase (APEX)-based proximity biotinylation, together with affinity pull-down of the ensuing biotinylated peptides, to investigate the proximity proteomes of human SOD1 ...[more]

PMID: 38795036

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Project description:Familial amyotrophic lateral sclerosis (ALS) represents about 10% of ALS cases. In about 20% of familial ALS patients, a mutation in superoxide dismutase-1 (SOD1) can be found. The ubiquitous SOD1 protein converts superoxide radical anions to oxygen and hydrogen peroxide. Patients with familial ALS caused by mutations in SOD1 can show comorbidity with frontotemporal dementia and develop cognitive impairment, including apathy, inattention, verbal deficits, and hypersexuality. At the cellular level, pathological signs of ALS may include tau immunoreactive astrocytic and neuronal inclusions, suggesting that cognitive dysfunction in ALS may also reflect abnormal protein metabolism of the microtubule associated protein (MAP), tau. To identify cell-specific expression changes, we performed laser capture microdissection (LCM) to isolate anterior horn motor neurons and surrounding cells. To determine common pathways for the development of ALS due to dysfunction of SOD1 or TAU, we performed whole transcriptome analysis in SOD1 and TAU mouse models. Because ALS is a neurodegenerative disease that specifically affects motor neurons, these cells were the main investigative target. Global transcriptomes of glial cells surrounding the motor neurons were also assessed, because these cells have been implicated as triggers of neurodegeneration. Mouse experiments were performed at the presymptomatic stage, prior to the onset of cell loss, in order to reduce false positive signals due to tissue reactive changes. Lumbar anterior horn anterior horn motor neurons and surrounding cells (glia) were isolated from presymptomatic TAU-P301L and SOD1-G93A transgenic mice using laser capture microdissection (LCM; PixCellÂ® IIe LCM System, Arcturus, Molecular Devices, CA). On average, 500 motor neuron bodies or surrounding glial cells from 20 tissue sections per animal were collected. RNA isolation from LCM collected cells was performed using the Qiagen RNeasy Micro Kit. Only RNA with RNA Integrity Numbers (RIN) above 7.5 were taken for further analysis (Agilent Bioanalyzer 2100). A two-round T7-based amplification and labeling protocol (Agilent Low RNA Input Linear Amplification Kit PLUS) was used to generate high quality labeled cRNA. Agilent Whole Mouse Genome Microarray comparisons of motor neurons and surrounding glia were performed between transgenic (SOD1G93A and TAUP301L) and corresponding nontransgenic (control) littermate animals, producing four independent comparison groups: SOD1G93A motor neurons versus control motor neurons (SOD1mn), SOD1G93A motor neuron surrounding glia versus control glia (SOD1gl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Raw microarray data were acquired using the Agilent DNA Microarray scanner and processed with the accompanying Agilent Feature Extraction 10.5 Image Analysis software using default settings. Normalized signal intensities were used to identify gene expression changes in SOD1G93A and TAUP301L motor neurons and surrounding glial cells, generating four partially overlapping sets of data. For the identification of differential expression, the genes were required to pass two conservative criteria: a ratio beyond the 99.5% confidence interval observed in homotypic comparisons, which corresponded to an approximately 1.5-fold expression change, and a paired t-test (P<0.01) computed using 100 permutations of the data for each gene. Correction for multiple comparisons was performed using the adjusted Bonferroni test. The analysis was performed in the TM4: Microarray Software Suite. These techniques identified 251 transcripts representing 186 known genes for which expression was altered in at least one of the four comparisons. Four-condition experiment, SOD1G93A motor neurons versus control motor neurons (SODmn), SOD1G93A motor neuron surrounding glia versus control glia (SODgl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Biological replicates: 4 SOD1G93A motor neurons (SODmn), 4 non SOD littermate motor neurons (nonSODmn), 4 SOD1G93A motor neuron surrounding glia (SODgl), 4 non SOD littermate motor neuron surrounding glia (nonSODgl), 4 TAUP301L motor neurons (TAUmn), 4 non TAU littermate motor neurons (nonTAUmn), 4 TAUP301L motor neuron surrounding glia (TAUgl), 4 non TAU littermate motor neuron surrounding glia (nonTAUgl). Four independent comparison groups were generated: SOD1G93A motor neurons versus control nonSOD motor neurons, SOD1G93A motor neuron surrounding glia versus control nonSOD glia, TAUP301L motor neurons versus control nonTAU motor neurons and TAUP301L glia versus control nonTAU glia. Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Each replicate was repeated twice with dye flip to correct for unequal dye incorporation rates. Therefore, eight microarray hybridizations were performed for each biological comparison, for a total of 32 microarray slides and generating four groups of differentially expressed genes in SOD1G93A motor neurons (SODmn), SOD1G93A motor neuron surrounding glial cells (SODgl), TAUP301L motor neurons (TAUmn), and TAUP301L surrounding glial cells (TAUgl).

			Action	DRS
	bp-Ywhc-Sites.txt	Txt
	evidence.txt	Txt
	proteinGroups.txt	Txt
	sod1-G85R1-peptide.raw	Raw
	sod1-G85R2-peptide.raw	Raw