Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.

Selection of viral capsids and promoters affects the efficacy of rescue of Tmprss3-deficient cochlea.

Adeno-associated virus (AAV)-mediated gene transfer has shown promise in rescuing mouse models of genetic hearing loss, but how viral capsid and promoter selection affects efficacy is poorly characterized. Here, we tested combinations of AAVs and promoters to deliver Tmprss3, mutations in which are associated with hearing loss in humans. Tmprss3tm1/tm1 mice display severe cochlear hair cell degeneration, loss of auditory brainstem responses, and delayed loss of spiral ganglion neurons. Under the ubiquitous CAG promoter and AAV-KP1 capsid, Tmprss3 overexpression caused striking cytotoxicity in vitro and in vivo and failed to rescue degeneration or dysfunction of the Tmprss3tm1/tm1 cochlea. Reducing the dosage or using AAV-DJ-CAG-Tmprss3 diminished cytotoxicity without rescue of the Tmprss3tm1/tm1 cochlea. Finally, the combination of AAV-KP1 capsid and the EF1α promoter prevented cytotoxicity and reduced hair cell degeneration, loss of spiral ganglion neurons, and improved hearing thresholds in Tmprss3tm1/tm1 mice. Together, our study illustrates toxicity of exogenous genes and factors governing rescue efficiency, and suggests that cochlear gene therapy likely requires precisely targeted transgene expression.

Gpr125 Marks Distinct Cochlear Cell Types and Is Dispensable for Cochlear Development and Hearing.

The G protein-coupled receptor (GPR) family critically regulates development and homeostasis of multiple organs. As a member of the GPR adhesion family, Gpr125 (Adgra3) modulates Wnt/PCP signaling and convergent extension in developing zebrafish, but whether it is essential for cochlear development in mammals is unknown. Here, we examined the Gpr125 lacZ/+ knock-in mice and show that Gpr125 is dynamically expressed in the developing and mature cochleae. From embryonic day (E) 15.5 to postnatal day (P) 30, Gpr125-ß-Gal is consistently expressed in the lesser epithelial ridge and its presumed progenies, the supporting cell subtypes Claudius cells and Hensen's cells. In contrast, Gpr125-ß-Gal is expressed transiently in outer hair cells, epithelial cells in the lateral cochlear wall, interdental cells, and spiral ganglion neurons in the late embryonic and early postnatal cochlea. In situ hybridization for Gpr125 mRNA confirmed Gpr125 expression and validated loss of expression in Gpr125 lacZ/lacZ cochleae. Lastly, Gpr125 lacZ/+ and Gpr125 lacZ/ lacZ cochleae displayed no detectable loss or disorganization of either sensory or non-sensory cells in the embryonic and postnatal ages and exhibited normal auditory physiology. Together, our study reveals that Gpr125 is dynamically expressed in multiple cell types in the developing and mature cochlea and is dispensable for cochlear development and hearing.

Opposing effects of Wnt/ß-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea.

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via ß-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development.

Oxidation of retinol via retinaldehyde results in the formation of the essential morphogen all-trans-retinoic acid (ATRA). Previous studies have identified critical roles in the regulation of embryonic ATRA levels for retinol, retinaldehyde, and ATRA-oxidizing enzymes; however, the contribution of retinaldehyde reductases to ATRA metabolism is not completely understood. Herein, we investigate the role of the retinaldehyde reductase Dhrs3 in embryonic retinoid metabolism using a Dhrs3-deficient mouse. Lack of DHRS3 leads to a 40% increase in the levels of ATRA and a 60% and 55% decrease in the levels of retinol and retinyl esters, respectively, in Dhrs3(-/-) embryos compared to wild-type littermates. Furthermore, accumulation of excess ATRA is accompanied by a compensatory 30-50% reduction in the expression of ATRA synthetic genes and a 120% increase in the expression of the ATRA catabolic enzyme Cyp26a1 in Dhrs3(-/-) embryos vs. controls. Excess ATRA also leads to alterations (40-80%) in the expression of several developmentally important ATRA target genes. Consequently, Dhrs3(-/-) embryos die late in gestation and display defects in cardiac outflow tract formation, atrial and ventricular septation, skeletal development, and palatogenesis. These data demonstrate that the reduction of retinaldehyde by DHRS3 is critical for preventing formation of excess ATRA during embryonic development.

ELKS1 and Ca(2+) channel subunit ß4 interact and colocalize at cerebellar synapses.

The cytoskeletal matrix of the active zone and synaptic voltage-dependent calcium channels (VDCCs) are both necessary components for the organization and regulation of synaptic vesicle release. In this study, we report a novel interaction between the cytoskeletal matrix of the active zone protein, ELKS1b, and the VDCC subunit, ß4, in the molecular layer of the cerebellum. We found that the two proteins coimmunoprecipitated using antibodies against each protein. Using fluorescent immunohistochemistry, we observed colocalization between ELKS1b and VDCC ß4 in the molecular layer of the cerebellum, suggesting that these proteins are both present in molecular layer synapses. Analysis of a P/Q-type VDCC knockout mouse (Cacna1a(-/-)) revealed that the localization of the VDCC ß4 subunit to the molecular layer was disrupted, although ELKS1b protein localization was not affected. These results demonstrate that these two proteins interact in vitro and colocalize in the cerebellum, and suggest that their interaction may play a role at the molecular layer synapses of the cerebellum.

Calcium channels link the muscle-derived synapse organizer laminin ß2 to Bassoon and CAST/Erc2 to organize presynaptic active zones.

Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC ß1b or ß4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin ß2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin ß2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin ß2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin ß2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane.