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Single-cell RNA-sequencing of stria vascularis cells in the adult Slc26a4-/- mouse

ABSTRACT: The primary pathological alternations of SLC26A4-related hearing loss are endolymphatic pH acidification and luminal enlargement of the inner ear. However, the molecular cell type-specific influences remain poorly characterized. In this study, we confirmed a method of isolating single cells in the cochlea's stria vascularis (SV) from wild-type (WT) animals. We then applied it to characterize the distinct transcriptomes from mutant (Slc26a4+/- and Slc26a4-/-) samples. Single-cell RNA-sequencing (scRNA-seq) identified transcriptional profiles in Slc26a4-expressing (spindle) cells and Kcnj10-expressing (intermediate) cells of the SV. By Gene Set Enrichment Analysis (GSEA), we found that spindle cells contain extrinsic cellular components, a factor that enables cell-to-cell communication. In addition, the gene expression profile informed signaling pathways associated with pH regulation in spindle cells. Compared to WT control, the transcriptional profiles in the SV cells of Slc26a4-/- mice showed downregulation of extracellular exosome-related genes in spindle cells and GPI-anchor-related genes in the intermediate cells. Immunofluorescence studies in spindle cells of Slc26a4-/- mice validated the increased expression of the clathrin-mediated endocytosis-related protein. Overall, cell isolation of SV from WT and mutant (Slc26a4+/- and Slc26a4-/-) samples combined with cell type-specific transcriptomic analyses revealed pH-dependent alternations in spindle cells and intermediate cells, inspiring further studies into the dysfunctional role of SV cells in SLC26A4-related hearing loss.

ORGANISM(S): Mus musculus

PROVIDER: GSE221146 | GEO | 2023/03/14

REPOSITORIES: GEO

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Project description:Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related disorders. Previously, we created mice expressing a proofreading-deficient version of the mtDNA polymerase gamma (Polg) which accumulate age-related mtDNA mutations and display premature aging. Here we performed microarray gene expression profiling to identify mtDNA mutation-responsive genes in the cochlea of aged mitochondrial mutator mice. Age-related accumulation of mtDNA mutations was associated with transcriptional alternations consistent with reduced inner ear function, mitochondrial dysfunction, neurodegeneration, and reduced cell structural modulation. Hearing assessment and histopathological results confirmed that aged PolgD257A/D257A (D257A) mice exhibited moderate hearing loss and severe cochlear degenerations. Age-related accumulation of mtDNA mutations also resulted in alternations in gene expression consistent with induction of apoptosis, proteolysis, stress response, and reduced DNA repair. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay confirmed that the cochleae from aged D257A mice showed significantly more TUNEL positive cells compared to wild-type (WT) mice. The levels of cleaved caspase-3 were also found to increase in the cochleae of aged D257A mice. These observations provide evidence that age-related accumulation of mtDNA mutations is associated with apoptotic cell death in aged cochlea. Our results provide the first global view of molecular events associated with mtDNA mutations in postmitotic tissue, and suggest that apoptosis is the major mechanism of mtDNA mediated cell death in the development of age-related hearing disorder. Experiment Overall Design: To determine the effects of age-related accumulation of mtDNA mutations, each WT sample (n = 5) was compared to each D257A sample (n = 5), generating a total of twenty-five pairwise comparisons. Genes with significantly altered expression levels were sorted into gene ontology biological process categories. Experiment Overall Design: Gene expression change was called statistically significant when at least one gene was called present in a group, the P value was < 0.0500, FC was > 1.1, and FDR was > 30.00 for identification of mtDNA mutations-induced genes. Experiment Overall Design: Affymetrix standard spike controls were used in all experiments (eukaryotic hybridization control kit). Quality control measures were not used. No replicates were done. Dye swap was not used.

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Single-cell RNA-sequencing of stria vascularis cells in the adult Slc26a4-/- mouse

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