Failure Of Hearing Acquisition in Mice With Reduced Expression of Connexin 26 Correlates With the Abnormal Phasing of Apoptosis Relative to Autophagy and Defective ATP-Dependent Ca2+ Signaling in Kölliker’s Organ

Abstract

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2loxP/loxP; ROSA26CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker’s organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker’s organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function.

Kölliker’s organ-supporting cells and cochlear auditory development

“…GJB2 mutations are mainly characterized by degeneration of outer hair cells and hypoplasia of the vascular stripe and are the main cause of deafness in 50% of pre-speech deafness (Hochman et al, 2010 ). In addition, GJB6 mutation in rodents was found to reduce Ca 2+ in Kölliker’s organ-supporting cells, leading to an increased hearing threshold (Rodriguez et al, 2012 ; Chen et al, 2021a ); while Cx26 cKD mice also showed reduced ATP release, downregulation of ATP-dependent calcium signaling, the disappearance of calcium waves and increased hearing threshold in Kölliker’s organ-supporting cells (Sun et al, 2022 ).…”

“…Therefore, the dynamic balance between autophagy and apoptosis regulates the normal differentiation and orderly organization of developing cochlear supporting cells, but the regulatory mechanism of the dynamic balance between autophagy and apoptosis is currently unknown (He et al, 2017 ; Zhou et al, 2020 ). Abnormal intracellular calcium signaling mediated by Gjb2 may be a key regulator of abnormal alterations in the autophagy-apoptosis signaling pathway and still requires in-depth study (Inoshita et al, 2014 ; Sun et al, 2022 ).…”

Pathological mechanisms of connexin26-related hearing loss: Potassium recycling, ATP-calcium signaling, or energy supply?

“…Connexin, as the core of ATP-triggered intercellular Ca 2+ signaling pathway, and its defect will lead to the disruption of calcium signal transmission. Both Gjb2 loxP/loxP ; Sox10-Cre mice and Gjb6 –/– mice showed that Ca 2+ waves failed to propagate in the Kölliker’s organ ( Ortolano et al, 2008 ; Crispino et al, 2011 ), and consequently failed to acquire normal hearing ( Sun et al, 2022 ). On the contrary, P2rx7 and Panx1, as alternative parts for ATP-triggered intercellular Ca 2+ signaling, P2rx7 –/– (MGI:3606250) and Panx 1 –/– (MGI:3606250) mice, showed that normal Ca 2+ waves spread in Kölliker’s organ and normal hearing phenotype ( Suadicani et al, 2006 ).…”

“…In conclusion, these results demonstrated that Cx-dependent ATP-triggered intercellular Ca 2+ signaling pathway plays a key role in postnatal auditory development. Some scholars proposed the hypothesis that the disrupted Ca 2+ signaling of developing cochlear epithelium prevents hearing acquisition in Cx26 deficiency mice ( Ceriani et al, 2016 ; Johnson et al, 2017 ; Sun et al, 2022 ). However, the disrupted Ca 2+ signaling hypothesis cannot explain the abnormal development of cochlear support cells, such as the failure of the tunnel of Corti to open and the failure of inner and outer column cells to differentiate and mature.…”

Early precision of radial patterning of the mouse cochlea is achieved by a linear BMP signaling gradient and is further refined by SOX2

“…JAG1 and SOX2 expression in the PSD becomes refined and is restricted to the sensory epithelium. Also, within phase two, beginning around E14.5 8 , a transient group of medial columnar cells emerges comprising Kölliker's organ (KO). It is a temporary structure essential for the cultivation of neural connections 9 , is a major signaling center during development [10][11][12] , and contains radially patterned cell types 13 .…”