Abstract
Pathogenic mutations in the non-syndromic hearing loss and deafness 1 (DFNB1) locus are the primary cause of monogenic inheritance for prelingual hearing loss. To unravel molecular pathways involved in etiopathology and look for early degeneration biomarkers, we used a system biology approach to analyze Cx30−/− mice at an early cochlear post-natal developmental stage. These mice are a DFNB1 mouse model with severely reduced expression levels of two connexins in the inner ear, Cx30, and Cx26. Integrated analysis of miRNA and mRNA expression profiles in the cochleae of Cx30−/− mice at post-natal day 5 revealed the overexpression of five miRNAs (miR-34c, miR-29b, miR-29c, miR-141, and miR-181a) linked to apoptosis, oxidative stress, and cochlear degeneration, which have Sirt1 as a common target of transcriptional and/or post-transcriptional regulation. In young adult Cx30−/− mice (3 months of age), these alterations culminated with blood barrier disruption in the Stria vascularis (SV), which is known to have the highest aerobic metabolic rate of all cochlear structures and whose microvascular alterations contribute to age-related degeneration and progressive decline of auditory function. Our experimental validation of selected targets links hearing acquisition failure in Cx30−/− mice, early oxidative stress, and metabolic dysregulation to the activation of the Sirt1–p53 axis. This is the first integrated analysis of miRNA and mRNA in the cochlea of the Cx30−/− mouse model, providing evidence that connexin downregulation determines a miRNA-mediated response which leads to chronic exhaustion of cochlear antioxidant defense mechanisms and consequent SV dysfunction. Our analyses support the notion that connexin dysfunction intervenes early on during development, causing vascular damage later on in life. This study identifies also early miRNA-mediated biomarkers of hearing impairment, either inherited or age related.
Highlights
Cx26 and Cx30 are the prevailing gap junction proteins in the duct of the developing and mature mammalian cochlea (Mammano, 2019)
Deregulation of miRNAs and the protein– protein interaction (PPI) Network of Their Targets By comparing miRNA expression profiles at P5 across genotypes (Cx30−/− vs. Cx30+/+, see Materials and Methods and Supplementary Figures 1, 2), we identified a total of 16 deregulated miRNAs
Overexpression of miR-34a/c has been involved in drug-induced hippocampal neurodegeneration (Cao et al, 2015), whereas overexpression of miR-34a and miR-34c has been implicated in drug-induced hearing loss linked to dose-dependent apoptosis of inner ear cells (Yu et al, 2010)
Summary
Cx26 and Cx30 are the prevailing gap junction proteins in the duct of the developing and mature mammalian cochlea (Mammano, 2019). Pathogenic mutations in the DFNB1 locus, which contains both genes (GJB2/CX26 and GJB6/CX30) encoding these connexins, are the primary cause of monogenic inheritance for prelingual deafness. It remains unclear if their coordinated expression is due to digenic inheritance or mutations affecting cis-regulatory elements that in turn influence GJB2/CX26 expression (Del Castillo and Del Castillo, 2017). Cx30 homozygous knockout-LacZ mice (Gjb6tm1Kwi/Gjb6tm1Kwi; MGI:2447863; EM:00323), hereafter abbreviated as Cx30−/−, are a model for humans in which large deletions in the DFNB1 locus lead to downregulation of both GJB2/CX26 and GJB6/CX30 and profound deafness. Massive downregulation of Cx26, at both mRNA and protein levels, was reported in the developing OC of Cx30−/− mice at P5 in nonsensory cells located between outer hair cells and SV (Ortolano et al, 2008); both transcript and protein levels of Cx26 were reduced in the cochleae of Cx30−/− mice at P30 (Boulay et al, 2013)
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