Academic literature on the topic 'Stereocilin'

La déficience auditive constitue un handicap fonctionnel majeur, affectant plus d'un demi-milliard de personnes dans le monde. Malgré sa prévalence élevée, aucun traitement curatif n'existe actuellement. Mon projet de thèse est translationnel et vise à établir la preuve de concept selon laquelle la thérapie génique virale peut restaurer l'audition dans le modèle préclinique de surdité DNFB16. La surdité DFNB16 est la deuxième cause de déficience auditive congénitale d'origine génétique. Elle est causée par des mutations du gène codant pour la stéréociline (STRC) et se caractérise par une surdité légère à modérée. La protéine STRC est principalement exprimée dans les cellules ciliées externes (CCE) de l'oreille interne, l'un des deux types de cellules sensorielles de la cochlée, responsables de l'amplification et la discrimination fréquentielle du signal sonore. La protéine STRC est cruciale pour le maintien de la morphologie des stéréocils des CCE. Les mutations du gène STRC sont responsables d'un dysfonctionnement des CCE conduisant à l'abolition de l'amplification cochléaire et donc à une augmentation des seuils auditifs. A ce jour, il n'existe aucun traitement curatif pour la surdité DFNB16.L'objectif principal de mon projet était de développer une thérapie génique basée sur les virus adéno-associés (AAV) pour remplacer le gène mutant par une copie fonctionnelle dans un modèle murin DFNB16. Compte tenu de la grande taille de la séquence codante du gène Strc, dépassant la capacité d'empaquetage de l'AAV, j'ai utilisé une stratégie hybride de double vecteur pour charger l'ADNc de la Strc dans les capsides de l'AAV. Sachant que les CCE sont intrinsèquement difficiles à transduire par les vecteurs AAV, j'ai tout d'abord effectué une analyse comparative du tropisme cellulaire de différents sérotypes d'AAV après administration dans l'oreille interne afin d'identifier le la capside la plus efficace pour cibler les CCE. Ensuite, j'ai utilisé le sérotype AAV le plus performant pour construire le vecteur thérapeutique qui a été administré dans les cochlées des souris DFNB16.Les résultats montrent que la thérapie génique a rétabli une expression robuste de la protéine STRC et ciblée dans les touffes ciliaires des CCE chez les souris traitées. Cette expression a entraîné la restauration de la morphostructure des touffes ciliaires et de l'amplification cochléaire, permettant une récupération stable et durable des seuils auditifs, similaires à ceux de souris sauvages. Par ailleurs, les mesures psychométriques de la perception des fréquences à l'aide d'une tâche de Go/NoGO ont montré que la discrimination fréquentielle du signal sonore chez les souris DFNB16 traitées étaient comparables à celles des souris sauvages. Ces résultats soulignent l'efficacité de la thérapie génique sur la restauration de la perception sonore dans un modèle préclinique de surdité DFNB16. Cette découverte jette les bases d'une thérapie génique translationnelle efficace pour les patients atteints de DFNB16<br>Hearing impairment stands as a significant contributor to disability, affecting over half a billion individuals throughout their lifespans. Despite its pervasive prevalence, no curative treatment currently exists. My Ph.D. project is translational, aiming to establish the proof of concept that viral gene therapy can restore hearing in a preclinical model for DFNB16 deafness. DFNB16, considered the second most common cause of hearing impairment, is caused by mutations in the stereocilin (STRC) gene and is characterized by mild-to-moderate deafness. The stereocilin (STRC) protein is predominantly expressed in outer hair cells (OHCs), one of the two types of cochlear sensory hair cells, responsible for sound amplification. STRC protein is crucial for the cohesion and maintenance of OHC bundles. Mutations in STRC result in defective OHCs, leading to abolished cochlear amplification and subsequent reduction in hearing sensitivity. As of now, there exists no cure for DFNB16.My main objective was to develop an adeno-associated virus (AAV)-based gene therapy to replace the mutant gene with its correct copy in a DFNB16 mouse model. Given the large size of the Strc coding sequence, exceeding AAV packaging capacity, I employed a hybrid dual-vector strategy to load Strc cDNA into AAV capsids. Since OHCs are inherently difficult to transduce with AAV vectors, we firstly conducted a comparative analysis of AAV cellular tropism within the inner ear to identify the most efficient AAV serotype for targeting OHCs. Secondly, I used the best performing AAV serotype to construct the therapeutic vector, which was administered into the cochleas of DFNB16 mice.Following the gene therapy, we found a robust restoration of STRC protein expression and its appropriate targeting at the tips of OHC stereocilia in treated mice. This process results in the restoration of the normal morphostructure of OHC bundles and cochlear amplification, ensuring stable and long-lasting restoration of hearing in the treated mice, similar to those of the wild-type mice. Notably, psychometric measurements of frequency perception using a Go/NoGo task demonstrated that frequency discrimination exhibited by the treated Strc-/- mice was comparable to those of wild-type mice, underscoring the efficacy of gene therapy in recovering essential features of natural sound perception associated with DFNB16. This finding lays the foundation for effective translational gene therapy for DFNB16 patients and facilitates the development of preclinical gene therapy studies for mouse models of human deafness

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 137-154).<br>Because there is little knowledge in the areas of stereocilia development, maintenance, and function in the hearing system, I decided to pursue a proteomics-based approach to discover proteins that play a role in stereocilia function. I employed a modified "twist-off" technique to isolate hair bundle proteins, and I developed a method to purify proteins and to process them for analysis using multi-dimensional protein identification technology (MudPIT). The MudPIT analysis yielded a substantial list of proteins. I verified the presence of 21 out of 34 (62%) existing proteins known to be present in stereocilia. This provided strong evidence that my proteomics approach was efficient in identifying hair bundle proteins. Next, I selected three proteins and localized them to murine cochlear stereocilia. StarD10, a putative phospholipid binding protein, was detectable along the shaft of stereocilia. Nebulin, a putative F-actin regulator, was located toward the base of stereocilia. Finally, twinfilin 2, a putative modulator of actin polymerization, was found at the tips of stereocilia. In order to determine the function of twinfilin 2, I localized the protein predominately to the tips of shorter stereocilia where it is up-regulated during the final phase of elongation. When overexpressed, I found that twinfilin 2 causes a shortening of microvilli in LLC-PK1/CL4 cells and in native cochlear stereocilia. The main result of this thesis was determining the sub-cellular localization of three interesting proteins and functionally characterizing one protein. My thesis also confirmed the proteomics screen I developed as an efficient method for identifying proteins in stereocilia.<br>by Anthony Wei Peng.<br>Ph.D.

Development of hair cell stereocilia bundles involves three stages: elongation, thickening, and supernumerary stereocilia retraction. Although Myo-XVa is known to be essential for stereocilia elongation, its role in retraction/thickening remains unknown. We quantified stereocilia numbers/diameters in shaker-2 mice (Myo15sh2) that have deficiencies in “long” and “short” isoforms of myosin-XVa, and in mice lacking only the “long” myosin-XVa isoform (Myo15ΔN). Our data showed that myosin-XVa is largely not involved in the developmental retraction of supernumerary stereocilia. In normal development, the diameters of the first (tallest)/second row stereocilia within a bundle are equal and grow simultaneously. The diameter of the third row stereocilia increases together with that of taller stereocilia until P1-2 and then either decreases almost two-fold in inner hair cells (IHCs) or stays the same in outer hair cells (OHCs), resulting in a prominent diameter gradation in IHCs and less prominent in OHCs. Sh2 mutation abolishes this gradation in IHCs/OHCs. Stereocilia of all rows grow in diameters nearly equally in Myo15sh2/sh2 IHCs and OHCs. Conversely, ΔN mutation does not affect normal stereocilia diameter gradation until ~P8. Therefore, myosin-XVa “short” isoform is essential for developmental thinning of third row stereocilia, which causes diameter gradation within a hair bundle.