Na/K-ATPase Gene Expression in the Human Cochlea: A Study Using mRNA in situ Hybridization and Super-Resolution Structured Illumination Microscopy

Abstract

BackgroundThe pervasive Na/K-ATPase pump is highly expressed in the human cochlea and is involved in the generation of the endocochlear potential as well as auditory nerve signaling and relay. Its distribution, molecular organization and gene regulation are essential to establish to better understand inner ear function and disease. Here, we analyzed the expression and distribution of the ATP1A1, ATP1B1, and ATP1A3 gene transcripts encoding the Na/K-ATPase α1, α3, and β1 isoforms in different domains of the human cochlea using RNA in situ hybridization.Materials and MethodsArchival paraformaldehyde-fixed sections derived from surgically obtained human cochleae were used to label single mRNA gene transcripts using the highly sensitive multiplex RNAscope® technique. Localization of gene transcripts was performed by super-resolution structured illumination microscopy (SR-SIM) using fluorescent-tagged probes. GJB6 encoding of the protein connexin30 served as an additional control.ResultsSingle mRNA gene transcripts were seen as brightly stained puncta. Positive and negative controls verified the specificity of the labeling. ATP1A1 and ATP1B1 gene transcripts were demonstrated in the organ of Corti, including the hair and supporting cells. In the stria vascularis, these transcripts were solely expressed in the marginal cells. A large number of ATP1B1 gene transcripts were found in the spiral ganglion cell soma, outer sulcus, root cells, and type II fibrocytes. The ATP1B1 and ATP1A3 gene transcripts were rarely detected in axons.DiscussionSurgically obtained inner ear tissue can be used to identify single mRNA gene transcripts using high-resolution fluorescence microscopy after prompt formaldehyde fixation and chelate decalcification. A large number of Na/K-ATPase gene transcripts were localized in selected areas of the cochlear wall epithelium, fibrocyte networks, and spiral ganglion, confirming the enzyme’s essential role for human cochlear function.

Wfs1E864K knock-in mice illuminate the fundamental role of Wfs1 in endocochlear potential production

“…The pump is involved in different functions in the inner ear including the ionic ux in the stria vascularis. Molecular studies showed that the β1 subunit is present at the basolateral membrane of the marginal cells of the stria vascularis in human 20,21 and murine cochleae 22,23 .…”

GJB2 and GJB6 gene transcripts in the human cochlea: A study using RNAscope, confocal, and super-resolution structured illumination microscopy

“…The specificity of the labeling was validated by using alternate gene biomarkers characterizing various cell domains in the SV. The KCNJ10 encodes the inward rectifying potassium channel 4.1 (Kir4.1) that was specifically located in the intermediate cells ( Wangemann et al, 1995 ; Hibino et al, 1997 ) while ATP1A1 encoding Na/K-ATPase α1 was restricted to the marginal cells ( Liu and Rask-Andersen, 2022 ). The basal cells expressed ATP1A2 encoding Na/K-ATPase α2 and could be used to validate the various gene transcripts’ locations.…”

“…The technique used for the RNAscope ® analyses of HC sections was recently described ( Liu and Rask-Andersen, 2022 ). In short, fixed-frozen human tissue sections underwent pretreatment with H 2 O 2 (10 min, RT) and protease III (30 min, 40°C).…”

“…It was combined with immunohistochemical analyses of Cx26 and Cx30 proteins. In an earlier investigation, we analyzed Na/K-ATPase (NKA) gene transcripts— ATP1A1 , ATP1A3 , and ATP1B1 —encoding the Na/K-ATPase α1, α3, and β1 isoforms together with GJB6 and fractalkine gene transcripts ( Liu et al, 2021a ; Liu and Rask-Andersen, 2022 ). In this study, we extended these analyses to include GJB2 , KCNJ10, and ATP1A2.…”

Transcriptome Analyses Provide Insights into the Auditory Function in Trachemys scripta elegans

“…Functional enrichment analysis showed that FGFR1 is involved in numerous biological functions, such as inner ear morphogenesis, protein binding, and calcium ion binding, which suggests that it plays a vital role in the function of the inner ear, specifically in receiving and processing sound, in T. scripta elegans . The ATP1A1 gene encodes Na/K-ATPase α1 isoforms [ 42 ], which are critical for maintaining hyperpolarized membrane potential by controlling cellular sodium and potassium concentrations [ 43 ]. This indicates that ATP1A1 is essential for mechanoelectrical transduction in the inner ear.…”