The Cochlear Matrisome: Importance in Hearing and Deafness.

peer reviewedThe extracellular matrix (ECM) consists in a complex meshwork of collagens, glycoproteins, and proteoglycans, which serves a scaffolding function and provides viscoelastic properties to the tissues. ECM acts as a biomechanical support, and actively participates in cell signaling to induce tissular changes in response to environmental forces and soluble cues. Given the remarkable complexity of the inner ear architecture, its exquisite structure-function relationship, and the importance of vibration-induced stimulation of its sensory cells, ECM is instrumental to hearing. Many factors of the matrisome are involved in cochlea development, function and maintenance, as evidenced by the variety of ECM proteins associated with hereditary deafness. This review describes the structural and functional ECM components in the auditory organ and how they are modulated over time and following injury

Matrice extracellulaire et fonction auditive : Rôle de l'acide hyaluronique

Hyaluronic acid (HA), a non-sulfated glycosaminoglycan, is part of the ground substance, a gel-like structure that bathes all the ECM components. HA has been involved both in tissue biomechanics, as it awards visco-elastic properties, and in cell signaling, through cell-ECM receptors. Thus, HA takes part in several biological processes, including morphogenesis and inflammation. Multiple studies demonstrated the importance of ECM during inner ear development and in hearing and some HA-related genes have been associated with hearing impairments. As such, the gene Cemip (or KIAA1199), encoding a Hyaluronidase, has been reported to be mutated in deaf patients. We therefore aim at characterizing the role of HA in hearing function. By combining specific immunostainings, RNAscope and qRT-PCR assays, we first explored the spatio-temporal distribution of cochlear HA as well as the expression profile of enzymes responsible for its synthesis and degradation. In addition, we generated a mutant mouse model in which Cemip gene has been invalidated. We took advantage of this model to investigate the impact of HA accumulation on cochlear morphogenesis and hearing function, by performing morphological analyses and Auditory Brainstem Response (ABR) recordings. We found that HA is highly enriched in the basilar membrane (BM), for which visco-elastic properties are instrumental in sound wave decomposition, frequency discrimination and mechanical sound wave conversion. The main enzymes involved in HA metabolism are present at embryonic and postnatal stages in the cochlear duct and in the spiral ganglion. Despite an accumulation of HA in the BM region below hair cells, the global morphology of Cemip-deficient cochleae is preserved, suggesting that Cemip has no prominent role in cochlear development. However, we evidenced that hearing function of Cemip KO mice is slightly impaired, as ABR recordings revealed an increase in peak 1 amplitude at some frequencies. Although unexpected, this result suggests that either more sensory cells are stimulated by sound, or that spiral ganglion neurons are over-activated compared to control mice. Altogether, our data suggest that HA might be instrumental in cochlear biomechanics, by awarding visco-elastic properties to the BM. Cemip loss has no critical impact on cochlear development and hearing, although an overstimulation of the cochlear nerve has been observed. We are currently investigating further this phenotype to identify the cause of this neuronal over-stimulation and we particularly focus on BM morphology and cochlear perineuronal nets. In the future, we also plan to examine whether Cemip KO mice are more prone to noise-induced hearing loss due to neuron excitotoxicity, for example

Cochlear Organoids for the Study of Hair Cell Development and Regeneration

Cochlear hair cells (HC) are susceptible to noise exposure, ototoxic treatments, and age. Currently, no treatment exists to promote HC regeneration in the cochlea and deafness is therefore irreversible once HCs are lost. Many efforts are put on identifying genes, pathways and molecules that would make cochlear HC regeneration possible. However, the low number of cochlear HC as well as the size of the cochlea and its inaccessibility limit in vivo studies. Thus, there is a critical need for a reliable model to examine HC development and to screen for treatments that could stimulate hair cell regeneration. To develop such a model, we generated a mouse strain expressing mCherry under the Atoh1 promoter and combined it with a mouse strain expressing GFP under the Lgr5 promoter. We obtained mice in which a subset of supporting cells and all HCs were labeled in different colors. Application in this new mouse strain of a previously established protocol that allows for clonal expansion of HC progenitors and subsequent HC differentiation, results in labeled cochlear cells in organoids: Lgr5-expressing supporting cells which include hair cell progenitors are labeled in green and HCs in red. Using flow cytometry from dissociated organoids, we can track HC differentiation from Lgr5+ cells to Atoh1+ cells and assess both the number of HC progenitors and the number of HCs at progressive differentiation times. Finally, our model helps to track HC differentiation and to easily assess the number of HC progenitors and HC in the organoids. Additionally, we can sort these populations and assess gene expression (RNA-sequencing), protein expression (immunostaining), and protein-chromatin interactions (CUT&RUN). As the effect of treatments during proliferation or differentiation can be easily determined, the two-color model provides an easy-to-use and reliable tool for assessing the effect of treatments on HC generation, and we are currently using the model to screen small molecules and genes critical for HC development and regeneration

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth

International audienceVAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth