Tesis sobre el tema "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
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.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 137-154).
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.
by Anthony Wei Peng.
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.

In the inner and outer hair cells (OHCs) of the inner ear, an unconventional myosin 15a localizes at the tips of mechanosensory stereocilia and plays an important role in forming and maintaining their normal structure. A missense mutation makes the motor domain of myosin 15a dysfunctional and is responsible for the congenital deafness DFNB3 in humans and deafness and vestibular defects in Shaker-2 (Sh2) mouse model. All hair cells of homozygous Shaker-2 mice (Myo15sh2/sh2) have abnormally short stereocilia, but, only stereocilia of Myo15sh2/sh2OHCs start to degenerate after the first few days of postnatal development and lose filamentous tip links between stereocilia that are crucial for mechanotransduction. The exact mechanisms of this degeneration are unknown even though they may underlie DFNB3 deafness in humans. We hypothesize that structural abnormalities in Myo15sh2/sh2 OHCs may alter the mechanical forces applied to the mechano-electrical transduction (MET) channels resulting in abnormal ionic homeostasis, which may lead to eventual degeneration of Myo15sh2/sh2 OHCs. Therefore, we investigated the ionic conductances and integrity of mechanotransduction apparatus in Myo15sh2/sh2 OHCs. Surprisingly, we found that myosin 15a-deficiency is associated not only with structural abnormalities of OHC stereocilia but also with alterations of voltage-gated ion conductances.

Le pilotage de véhicule blindé est rendu difficile par la faible visibilité offerte aux pilotes face aux environnements et aux situations complexes qu’ils doivent traverser.La protection des opérateurs de véhicules militaires et l’intégrité de ces véhicules sont des besoins primordiaux pour l’armée de terre.Afin de répondre à la problématique : sécuriser le pilotage des véhicules militaires avec comme périmètre la définition d’un système de perception d’environnement, nous avons procédé à l’étude au sens large de l’aide au pilotage dans le contexte militaire en environnement naturel et semi-structuré afin de mettre en exergue les moyens et les capteurs utilisables pour réaliser un système d’aide au pilotage.Ainsi, nous offrons une réponse technique pour la réalisation d’un tel système au travers premièrement d’une étude des méthodes et algorithmes existants applicables à notre cas d’application. Ensuite nous définissons les capteurs utilisables avec de telles méthodes. De cet état de l’art, nous définissonsune système répondant à notre problématique et nous expliquons sa mise en pratique au travers de la création d’une plateforme d’expérimentation.Cette plateforme se compose des solutions présentées et permet de valider le concept par l’évaluation des solutions d’acquisition de l’environnement afin d’offrir les données nécessaires à une aide au pilotage.Puis, l’étude des moyens d’analyse de cet environnement offre des pistes de réflexion sur le futur système d’aide au pilotage.Enfin, une l’étude d’un moyen alternatif de restitution de l’information à l’opérateur complète la solution présentée en offrant une piste de réflexion sur l’impact de la restitution dans les performances des opérateurs
Armored vehicule driving is difficult because of low visibility given to pilots in tough environnements conditions and complex situations they have to manage.Soldiers safety and vehicle integrity are part of main topics for French “Armée de Terre”. To answer the problem Make the driving of military vehicles safer by improving environnement perception through driver asssistance systems, we study driving assistance in unstructured environnemnt by looking for sensors and methods which are suitable to realize such a system.First, we study existing methods and algorithms which fit our application case. Conclusion of this study is the definition of our system.Second, thanks to the previous study we explain the creation of an experimentation platform allowing evaluation of our concept. Data obtained from reconstruction are then exploited through environment analysis to bring obstacle extraction methods.Third, study of an alternative display solution is exposed and complete this work in explaining impact of restitution in operating cycle

Les problèmes auditifs affectent 16% des Européens, dont la moitié a plus de 60 ans, faisant de la presbyacousie, la surdité liée à l’âge, un véritable problème de santé publique. Chez l’homme, la mutation du gène SLC17A8, codant pour le transporteur vésiculaire du glutamate (VGLUT3) est à l’origine d’une surdité progressive prédominante sur les fréquences aigues (DFNA25), présentant ainsi les signes d’une presbyacousie précoce. Pour déterminer les mécanismes responsables de la surdité DFNA25, nous avons étudié le phénotype de la souris dont le gène SLC17A8 porte la mutation ponctuelle humaine (mutation p.A224V correspondant à la mutation humaine p.A211V). Nos résultats montrent que la souris VGLUT3A224V/A224V développe une surdité progressive mimant DFNA25. L’observation en microscopie électronique démontre un effondrement précoce des faisceaux de stéréocils provoquant l’incapacité des cellules sensorielles auditives à détecter les signaux acoustiques. L’observation en microscopie à super-résolution révèle un remodelage tardif des synapses à rubans des cellules ciliées, à savoir un allongement des rubans associé à une augmentation de l’exocytose soutenue dans le temps. L’ensemble de ces résultats indique une atteinte des mécanismes de mécano-transduction et de transfert synaptique dans le modèle murin de la surdité DFNA25
DFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied the phenotype of the mouse harboring the p.A211V mutation in human (corresponding to p.A224V in mouse). Using auditory brainstem response and distortion products of otoacoustic emissions, we showed that VGLUT3A224V/A224V mouse replicates DFNA25 progressive hearing loss with intact cochlear amplification. Scanning electron microscopy examinations demonstrated fused stereocilia bundle of the inner hair cells (IHCs) as the primary cause for DFNA25. In addition, we observed a change in the structure-function of the IHC ribbon synapse at later stages. Using super-resolution microscopy, we noticed an elongation of the synaptic ribbon, associated with an increase in the rate of the sustained releasable pool of exocytosis. These results indicate that the primary defect in DFNA25 stems from a defective mechano-transduction followed by a change in the synaptic transfer

Indiana University-Purdue University Indianapolis (IUPUI)
Age-related hearing loss is an acute health problem affecting people worldwide, often arising due to defects in the proper functioning of sensory hair cells in the inner ear. The apical surface of sensory hair cells contains actin-based protrusions known as stereocilia, which detect sound and head movements. Since hair cells are not regenerated in mammals, it is important to maintain the functioning of stereocilia for the life of an organism to maintain hearing ability. The actin filaments within a stereocilium are extensively crosslinked by various actin crosslinking proteins, which are important for stereocilia development and maintenance. Multiple studies have shown that the stereocilia actin core is exceptionally stable whereas actin is dynamic only at the tips of stereocilia. However, whether the actin crosslinking proteins, which are nearly as abundant as actin itself, are similarly stable or can freely move in and out of the core remains unknown. Loss or mutation of crosslinkers like plastin-1, fascin-2, and XIRP2 causes progressive hearing loss along with stereocilia degeneration while loss of espin prevents stereocilia from even developing properly. Do these phenotypes stem from an unstable stereocilia core? Does crosslinking confer stability to the core? To address these questions, we generated novel transgenic reporter lines to monitor the dynamics of actin in mice carrying fascin-2R109H mutation and espin null mice and also to study the dynamics of actin crosslinkers, in vivo and ex-vivo. We established that actin crosslinkers readily exchange within the highly stable F-actin structure of the stereocilia core. In addition, we determined that stereocilia degeneration in mice carrying fascin-2R109H mutation and espin null mice could possibly occur due to a less stable actin core. These studies suggest that dynamic crosslinks stabilize the core to maintain proper stereocilia functioning. Future work warrants understanding the reason behind the importance of dynamic crosslinks within a stable stereocilia core. Actin stability not only depends on actin crosslinkers, but also on actin filament composition as evident from distinct stereocilia degeneration and progressive hearing loss patterns in hair-cell specific knockout of actin isoforms. Although beta- and gamma- actin polypeptide sequences differ by only 14 four amino acids, whether the latter determine the unique function of each cytoplasmic actin isoform was previously unknown. Here we determined that these four critical amino acids determine the unique functional importance of beta-actin isoform in sensory hair cells. Taken together, our study demonstrates that actin cytoskeletal proteins are important for the morphogenesis and maintenance of stereocilia.

Age-related hearing loss is an acute health problem affecting people worldwide, often arising due to defects in the proper functioning of sensory hair cells in the inner ear. The apical surface of sensory hair cells contains actin-based protrusions known as stereocilia, which detect sound and head movements. Since hair cells are not regenerated in mammals, it is important to maintain the functioning of stereocilia for the life of an organism to maintain hearing ability. The actin filaments within a stereocilium are extensively crosslinked by various actin crosslinking proteins, which are important for stereocilia development and maintenance. Multiple studies have shown that the stereocilia actin core is exceptionally stable whereas actin is dynamic only at the tips of stereocilia. However, whether the actin crosslinking proteins, which are nearly as abundant as actin itself, are similarly stable or can freely move in and out of the core remains unknown. Loss or mutation of crosslinkers like plastin-1, fascin-2, and XIRP2 causes progressive hearing loss along with stereocilia degeneration while loss of espin prevents stereocilia from even developing properly. Do these phenotypes stem from an unstable stereocilia core? Does crosslinking confer stability to the core? To address these questions, we generated novel transgenic reporter lines to monitor the dynamics of actin in mice carrying fascin-2R109H mutation and espin null mice and also to study the dynamics of actin crosslinkers, in vivo and ex-vivo. We established that actin crosslinkers readily exchange within the highly stable F-actin structure of the stereocilia core. In addition, we determined that stereocilia degeneration in mice carrying fascin-2R109H mutation and espin null mice could possibly occur due to a less stable actin core. These studies suggest that dynamic crosslinks stabilize the core to maintain proper stereocilia functioning. Future work warrants understanding the reason behind the importance of dynamic crosslinks within a stable stereocilia core. Actin stability not only depends on actin crosslinkers, but also on actin filament composition as evident from distinct stereocilia degeneration and progressive hearing loss patterns in hair-cell specific knockout of actin isoforms. Although beta- and gamma- actin polypeptide sequences differ by only 14 four amino acids, whether the latter determine the unique function of each cytoplasmic actin isoform was previously unknown. Here we determined that these four critical amino acids determine the unique functional importance of beta-actin isoform in sensory hair cells. Taken together, our study demonstrates that actin cytoskeletal proteins are important for the morphogenesis and

maintenance of stereocilia.

Indiana University-Purdue University Indianapolis (IUPUI)
Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.