RNA-seq analysis of gene expression profiles in isolated stria vascularis from wild-type and Alport mice reveals key pathways underling Alport strial pathogenesis

Abstract

Previous work demonstrates that the hearing loss in Alport mice is caused by defects in the stria vascularis. As the animals age, progressive thickening of strial capillary basement membranes (SCBMs) occurs associated with elevated levels of extracellular matrix expression and hypoxia-related gene and protein expression. These conditions render the animals susceptible to noise-induced hearing loss. In an effort to develop a more comprehensive understanding of how the underlying mutation in the COL4A3 gene influences homeostasis in the stria vascularis, we performed vascular permeability studies combined with RNA-seq analysis using isolated stria vascularis from 7-week old wild-type and Alport mice on the 129 Sv background. Alport SCBMs were found to be less permeable than wild-type littermates. RNA-seq and bioinformatics analysis revealed 68 genes were induced and 61 genes suppressed in the stria from Alport mice relative to wild-type using a cutoff of 2-fold. These included pathways involving transcription factors associated with the regulation of proinflammatory responses as well as cytokines, chemokines, and chemokine receptors that are up-or down-regulated. Canonical pathways included modulation of genes associated with glucose and glucose-1-PO4 degradation, NAD biosynthesis, histidine degradation, calcium signaling, and glutamate receptor signaling (among others). In all, the data point to the Alport stria being in an inflammatory state with disruption in numerous metabolic pathways indicative of metabolic stress, a likely cause for the susceptibility of Alport mice to noiseinduced hearing loss under conditions that do not cause permanent hearing loss in age/ strain-matched wild-type mice. The work lays the foundation for studies aimed at understanding the nature of strial pathology in Alport mice. The modulation of these genes under conditions of therapeutic intervention may provide important pre-clinical data to justify trials in humans afflicted with the disease.

Modeling and cleaning RNA-seq data significantly improve detection of differentially expressed genes

“…The first dataset is the result of work on the comparison of gene expression between Alport mice and wild mice [ 23 ]. Data (SRR11206238 and SRR11206239) were converted into mRNA counts using STAR and FeatureCounts on Galaxy [ 24 ].…”

“…DEGs were detected using EdgeR after application of different filters and annotated by DAVID as above. The criterion for DEGs was relaxed to |log2(FoldChange)|>1 to match the number of genes reported by the authors in Tables 1 , 2 , 3 and 4 [ 23 ] when using raw data (Table 2 , gene lists are at the project home page on GitHub).…”

“…RNAdeNoise does not introduce such a bias and shows an increase in number and statistical significance of functional DEGs (distributions of counts in DEGs is shown in Additional file 1 : Fig. S3) $ Genes from Tables 1 , 2 , 3 and 4 [ 23 ] *Criteria for DEGs: |log2(FoldChange)|>1.0, p -value < 0.0001 & DEGs with counts ≥ 30 at least in one sample…”

Hearing Function, Degeneration, and Disease: Spotlight on the Stria Vascularis

“…Notably, the COL4A4-knockout mouse has been used to demonstrate that SV function is impaired in the Alport syndrome model (Cosgrove et al, 1996). Specifically, SV capillary permeability is reduced in 8.5-week-old COL4A4 knockout mice when compared to age matched wild-type mice (demonstrated using intracardially injected Rhodamine dye and fluorescence spectrometry) (Dufek et al, 2020). In addition, RNA sequencing showed higher expression of inflammatory genes, such as nuclear factor kappa B inhibitor alpha (NFKBIA), in the SV of COL4A4 knockout mice when compared to wildtype controls.…”

“…In addition, RNA sequencing showed higher expression of inflammatory genes, such as nuclear factor kappa B inhibitor alpha (NFKBIA), in the SV of COL4A4 knockout mice when compared to wildtype controls. This would suggest that the host's inflammatory response damages the SV in those with Alport syndrome (Dufek et al, 2020). It remains to be seen what is causing this inflammation in the Alport syndromeaffected SV.…”