miRNA and mRNA Profiling Links Connexin Deficiency to Deafness via Early Oxidative Damage in the Mouse Stria Vascularis

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.

Connexin 30 deletion exacerbates cochlear senescence and age-related hearing loss

“…On the basis of these findings, we wondered if Cx30 deletion, increasing cochlear susceptibility to aging processes, affects also Cx26 expression, involving oxidative stress mechanisms. In this study, we used a KO mouse model (Cx30 ΔΔ) in which, in contrast to the previous model (referred to as Cx30 −/− in the literature) ( Teubner, 2003 ; Gentile et al, 2021 ), Cx30 was removed without perturbing the surrounding sequences. Thus, the level of residual Cx26 protein is 5 times higher in Cx30 ΔΔ than in Cx30 −/− mice ( Boulay et al, 2013 ).…”

“…Looking for a molecular mechanism, we focused on the crossroad among oxidative stress, inflammation, and vascular dysfunction, considering that these damaging factors are common pathological markers of aging processes both in physiological cochlear-aging and in the genetic model of ARHL ( Someya et al, 2009 ; Fetoni et al, 2018 ; White et al, 2018 ; Wang and Puel, 2020 ; Fetoni et al, 2022 ). Moreover, at the same time, deletions of connexins have been related to oxidative-inflammatory processes ( Fetoni et al, 2018 ; Hua et al, 2021 ), as well as to vascular dysfunction and reduced endocochlear potential ( Cohen-Salmon et al, 2007 ; Gentile et al, 2021 ; Chen et al, 2022 ). Our data, demonstrating that the absence of Cx30 and the consequent downregulation of Cx26 are associated with increased ROS amount in Cx30 ΔΔ aged-cochleae, suggest that connexin hemichannels play a key role in protecting against oxidative stress during aging, probably allowing diffusion of antioxidant molecules to counteract redox imbalance.…”

“…The sections were then coverslipped with an antifade medium (FluorSave™ Reagent, Merk). To assess the integrity of the cochlear blood barrier, the stria vascularis was isolated after cochlear dissection as described by Gentile et al (2021) and the samples were incubated with Alexa Fluor ® 546 donkey antimouse IgG (ThermoFisher) for 2 h at RT. For all immunofluorescence analyses, control experiments were performed.…”

CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss

“…In contrast, the RNP-LNPs control group was mainly distributed into spiral ganglion cells. One of the pathogenic hearing loss mechanisms involved in progressive hearing loss is oxidative stress [38][39][40] .…”

“…In contrast, the RNP-LNPs control group was mainly distributed into spiral ganglion cells. One of the pathogenic hearing loss mechanisms involved in progressive hearing loss is oxidative stress 38-40 . Immunohistochemical evaluation of the oxidative stress biomarkers 3-Nitrotyrosine (3-NT) and 4-Hydroxy-2-nonenal (4-HNE) supported the functional restoration specifically from sensory hair cells after 6 months with μDES RNP EVs gene editing treatment in vivo .…”