Synaptopathies of auditory hair cells due to Ca2+-dependent exocytosis dysruption

Les signaux acoustiques sont encodés en impulsions électriques nerveuses au niveau des synapses à ruban des cellules ciliées internes (CCI) de la cochlée. L'exocytose des vésicules synaptiques glutamatergiques des CCIs est déclenchée par l'activation des canaux calciques voltage-dépendent CaV1.3 et requiert un senseur calcique particulier : l'otoferline. Ces canaux calciques CaV1.3 s’organisent de façon spatialement resserrée autour des rubans synaptiques en formant un complexe avec l’harmonine et la clarin-1 associées à l’actine-F. Dans la première partie de notre étude, nous avons étudié le rôle possible du nouvellement identifié paralogue de la clarine-1, la clarine-2, dans les synapses à ruban des CCI. Nous démontrons que la clarine-2 est impliquée dans la régulation du potentiel d'activation des canaux calciques des CCIs et en conséquence de l’exocytose synaptique. Dans une deuxième partie de notre travail nous avons étudié le rôle de la SNAP-25 dans l’exocytose des CCI. La SNAP-25 est une protéine essentielle du complexe SNARE qui permet l'amarrage des vésicules synaptiques à la membrane et leur fusion dans les synapses neuronales centrales mais dont le rôle au niveau des CCI est encore largement débattu. En utilisant des souris dont la SNAP-25 est sélectivement supprimée dans les cellules ciliées, nous montrons que la SNAP-25 est essentielle à la fonction auditive et vestibulaire, du fait d’une perte de l’exocytose rapide des cellules ciliées, suivi par une dégénérescence progressive des synapses et des cellules ciliées elles-mêmes. Dans une troisième partie, nous avons étudié la dégénérescence des synapses à ruban des CCI au cours du vieillissement. Nous observons chez les souris C57BL/6J âgées que les synapses à rubans des CCIs restantes, qui n’ont pas encore dégénérées, présentent des rubans agrandis, une augmentation des microdomaines calciques autour des rubans et une plus grande capacité à soutenir l’exocytose suggérant une potentialisation de ces synapses. Cette potentialisation des synapses à ruban âgées pourrait en partie expliquer l’hyperacousie paradoxalement observée dans la presbyacousie.Acoustic signals are encoded into electrical nerve impulses at the ribbon synapses of the cochlear inner hair cells (IHC). Exocytosis of the synaptic glutamatergic vesicles at these ribbon synapses is triggered by the activation of the voltage-dependent calcium channels CaV1.3 and required a specific calcium sensor: otoferlin. In the first part of my thesis, we studied the role of clarin-2, a newly identified tetraspan protein paralog of clarin-1, in these synapses. We know from previous studies that clarin-1, by forming a complex associating CaV1.3 channels, harmonin-b and actin-F, is essential for a compact spatial organization of the Ca1.3 channels at the active zone of the IHC ribbons. We show that clarin-2 is essential for a normal voltage-dependent activation of the IHC calcium channels and is likely part of the clarin-1 complex at the ribbon synapses. In the second part of my thesis, we studied the role of SNAP-25, a protein of the SNARE complex implicated in the docking, priming and fusion of the synaptic vesicles at neuronal central synapses but whose implication at the ribbon hair cell synapses remain still under debate. We show that mutant mice specifically deleted for SNAP-25 in hair cells are profoundly deaf and have a severe vestibular deficit. Electrophysiological patch-clamp recordings in IHCs and utricular hair cells indicated a severe defect in fast exocytosis. This synaptic functional defect was associated with a progressive degeneration of the ribbon synapses and the hair cells themselves, suggesting that SNAP-25 is also essential for the release of important neurotrophic factors. In the third part of my thesis, we investigated the functional changes of the aging ribbon synapses in C57BL/6J mice. We found that the aging synaptic IHC ribbons, while progressively degenerating, become larger with enlarged Ca2+ microdomains to produce a more sustained exocytosis of synaptic vesicles. The expression and the voltage dependence of the fast BK channels, essential for fast IHC repolarization, were also severely affected with aging. All these results suggested that the IHC ribbon synapses become potentiated with aging, a process that may partly explain the paradoxical hyperacusis phenomenon in presbyacusis

Synaptopathies des cellules ciliées auditives associées à un dysfonctionnement de l'exocytose Ca2+-dépendante

Acoustic signals are encoded into electrical nerve impulses at the ribbon synapses of the cochlear inner hair cells (IHC). Exocytosis of the synaptic glutamatergic vesicles at these ribbon synapses is triggered by the activation of the voltage-dependent calcium channels CaV1.3 and required a specific calcium sensor: otoferlin. In the first part of my thesis, we studied the role of clarin-2, a newly identified tetraspan protein paralog of clarin-1, in these synapses. We know from previous studies that clarin-1, by forming a complex associating CaV1.3 channels, harmonin-b and actin-F, is essential for a compact spatial organization of the Ca1.3 channels at the active zone of the IHC ribbons. We show that clarin-2 is essential for a normal voltage-dependent activation of the IHC calcium channels and is likely part of the clarin-1 complex at the ribbon synapses. In the second part of my thesis, we studied the role of SNAP-25, a protein of the SNARE complex implicated in the docking, priming and fusion of the synaptic vesicles at neuronal central synapses but whose implication at the ribbon hair cell synapses remain still under debate. We show that mutant mice specifically deleted for SNAP-25 in hair cells are profoundly deaf and have a severe vestibular deficit. Electrophysiological patch-clamp recordings in IHCs and utricular hair cells indicated a severe defect in fast exocytosis. This synaptic functional defect was associated with a progressive degeneration of the ribbon synapses and the hair cells themselves, suggesting that SNAP-25 is also essential for the release of important neurotrophic factors. In the third part of my thesis, we investigated the functional changes of the aging ribbon synapses in C57BL/6J mice. We found that the aging synaptic IHC ribbons, while progressively degenerating, become larger with enlarged Ca2+ microdomains to produce a more sustained exocytosis of synaptic vesicles. The expression and the voltage dependence of the fast BK channels, essential for fast IHC repolarization, were also severely affected with aging. All these results suggested that the IHC ribbon synapses become potentiated with aging, a process that may partly explain the paradoxical hyperacusis phenomenon in presbyacusis.Les signaux acoustiques sont encodés en impulsions électriques nerveuses au niveau des synapses à ruban des cellules ciliées internes (CCI) de la cochlée. L'exocytose des vésicules synaptiques glutamatergiques des CCIs est déclenchée par l'activation des canaux calciques voltage-dépendent CaV1.3 et requiert un senseur calcique particulier : l'otoferline. Ces canaux calciques CaV1.3 s’organisent de façon spatialement resserrée autour des rubans synaptiques en formant un complexe avec l’harmonine et la clarin-1 associées à l’actine-F. Dans la première partie de notre étude, nous avons étudié le rôle possible du nouvellement identifié paralogue de la clarine-1, la clarine-2, dans les synapses à ruban des CCI. Nous démontrons que la clarine-2 est impliquée dans la régulation du potentiel d'activation des canaux calciques des CCIs et en conséquence de l’exocytose synaptique. Dans une deuxième partie de notre travail nous avons étudié le rôle de la SNAP-25 dans l’exocytose des CCI. La SNAP-25 est une protéine essentielle du complexe SNARE qui permet l'amarrage des vésicules synaptiques à la membrane et leur fusion dans les synapses neuronales centrales mais dont le rôle au niveau des CCI est encore largement débattu. En utilisant des souris dont la SNAP-25 est sélectivement supprimée dans les cellules ciliées, nous montrons que la SNAP-25 est essentielle à la fonction auditive et vestibulaire, du fait d’une perte de l’exocytose rapide des cellules ciliées, suivi par une dégénérescence progressive des synapses et des cellules ciliées elles-mêmes. Dans une troisième partie, nous avons étudié la dégénérescence des synapses à ruban des CCI au cours du vieillissement. Nous observons chez les souris C57BL/6J âgées que les synapses à rubans des CCIs restantes, qui n’ont pas encore dégénérées, présentent des rubans agrandis, une augmentation des microdomaines calciques autour des rubans et une plus grande capacité à soutenir l’exocytose suggérant une potentialisation de ces synapses. Cette potentialisation des synapses à ruban âgées pourrait en partie expliquer l’hyperacousie paradoxalement observée dans la presbyacousie

Synaptopathies des cellules ciliées auditives associées à un dysfonctionnement de l'exocytose Ca2+-dépendante

Acoustic signals are encoded into electrical nerve impulses at the ribbon synapses of the cochlear inner hair cells (IHC). Exocytosis of the synaptic glutamatergic vesicles at these ribbon synapses is triggered by the activation of the voltage-dependent calcium channels CaV1.3 and required a specific calcium sensor: otoferlin. In the first part of my thesis, we studied the role of clarin-2, a newly identified tetraspan protein paralog of clarin-1, in these synapses. We know from previous studies that clarin-1, by forming a complex associating CaV1.3 channels, harmonin-b and actin-F, is essential for a compact spatial organization of the Ca1.3 channels at the active zone of the IHC ribbons. We show that clarin-2 is essential for a normal voltage-dependent activation of the IHC calcium channels and is likely part of the clarin-1 complex at the ribbon synapses. In the second part of my thesis, we studied the role of SNAP-25, a protein of the SNARE complex implicated in the docking, priming and fusion of the synaptic vesicles at neuronal central synapses but whose implication at the ribbon hair cell synapses remain still under debate. We show that mutant mice specifically deleted for SNAP-25 in hair cells are profoundly deaf and have a severe vestibular deficit. Electrophysiological patch-clamp recordings in IHCs and utricular hair cells indicated a severe defect in fast exocytosis. This synaptic functional defect was associated with a progressive degeneration of the ribbon synapses and the hair cells themselves, suggesting that SNAP-25 is also essential for the release of important neurotrophic factors. In the third part of my thesis, we investigated the functional changes of the aging ribbon synapses in C57BL/6J mice. We found that the aging synaptic IHC ribbons, while progressively degenerating, become larger with enlarged Ca2+ microdomains to produce a more sustained exocytosis of synaptic vesicles. The expression and the voltage dependence of the fast BK channels, essential for fast IHC repolarization, were also severely affected with aging. All these results suggested that the IHC ribbon synapses become potentiated with aging, a process that may partly explain the paradoxical hyperacusis phenomenon in presbyacusis.Les signaux acoustiques sont encodés en impulsions électriques nerveuses au niveau des synapses à ruban des cellules ciliées internes (CCI) de la cochlée. L'exocytose des vésicules synaptiques glutamatergiques des CCIs est déclenchée par l'activation des canaux calciques voltage-dépendent CaV1.3 et requiert un senseur calcique particulier : l'otoferline. Ces canaux calciques CaV1.3 s’organisent de façon spatialement resserrée autour des rubans synaptiques en formant un complexe avec l’harmonine et la clarin-1 associées à l’actine-F. Dans la première partie de notre étude, nous avons étudié le rôle possible du nouvellement identifié paralogue de la clarine-1, la clarine-2, dans les synapses à ruban des CCI. Nous démontrons que la clarine-2 est impliquée dans la régulation du potentiel d'activation des canaux calciques des CCIs et en conséquence de l’exocytose synaptique. Dans une deuxième partie de notre travail nous avons étudié le rôle de la SNAP-25 dans l’exocytose des CCI. La SNAP-25 est une protéine essentielle du complexe SNARE qui permet l'amarrage des vésicules synaptiques à la membrane et leur fusion dans les synapses neuronales centrales mais dont le rôle au niveau des CCI est encore largement débattu. En utilisant des souris dont la SNAP-25 est sélectivement supprimée dans les cellules ciliées, nous montrons que la SNAP-25 est essentielle à la fonction auditive et vestibulaire, du fait d’une perte de l’exocytose rapide des cellules ciliées, suivi par une dégénérescence progressive des synapses et des cellules ciliées elles-mêmes. Dans une troisième partie, nous avons étudié la dégénérescence des synapses à ruban des CCI au cours du vieillissement. Nous observons chez les souris C57BL/6J âgées que les synapses à rubans des CCIs restantes, qui n’ont pas encore dégénérées, présentent des rubans agrandis, une augmentation des microdomaines calciques autour des rubans et une plus grande capacité à soutenir l’exocytose suggérant une potentialisation de ces synapses. Cette potentialisation des synapses à ruban âgées pourrait en partie expliquer l’hyperacousie paradoxalement observée dans la presbyacousie

Synaptopathies of auditory hair cells due to Ca2+-dependent exocytosis dysruption

Les signaux acoustiques sont encodés en impulsions électriques nerveuses au niveau des synapses à ruban des cellules ciliées internes (CCI) de la cochlée. L'exocytose des vésicules synaptiques glutamatergiques des CCIs est déclenchée par l'activation des canaux calciques voltage-dépendent CaV1.3 et requiert un senseur calcique particulier : l'otoferline. Ces canaux calciques CaV1.3 s’organisent de façon spatialement resserrée autour des rubans synaptiques en formant un complexe avec l’harmonine et la clarin-1 associées à l’actine-F. Dans la première partie de notre étude, nous avons étudié le rôle possible du nouvellement identifié paralogue de la clarine-1, la clarine-2, dans les synapses à ruban des CCI. Nous démontrons que la clarine-2 est impliquée dans la régulation du potentiel d'activation des canaux calciques des CCIs et en conséquence de l’exocytose synaptique. Dans une deuxième partie de notre travail nous avons étudié le rôle de la SNAP-25 dans l’exocytose des CCI. La SNAP-25 est une protéine essentielle du complexe SNARE qui permet l'amarrage des vésicules synaptiques à la membrane et leur fusion dans les synapses neuronales centrales mais dont le rôle au niveau des CCI est encore largement débattu. En utilisant des souris dont la SNAP-25 est sélectivement supprimée dans les cellules ciliées, nous montrons que la SNAP-25 est essentielle à la fonction auditive et vestibulaire, du fait d’une perte de l’exocytose rapide des cellules ciliées, suivi par une dégénérescence progressive des synapses et des cellules ciliées elles-mêmes. Dans une troisième partie, nous avons étudié la dégénérescence des synapses à ruban des CCI au cours du vieillissement. Nous observons chez les souris C57BL/6J âgées que les synapses à rubans des CCIs restantes, qui n’ont pas encore dégénérées, présentent des rubans agrandis, une augmentation des microdomaines calciques autour des rubans et une plus grande capacité à soutenir l’exocytose suggérant une potentialisation de ces synapses. Cette potentialisation des synapses à ruban âgées pourrait en partie expliquer l’hyperacousie paradoxalement observée dans la presbyacousie.Acoustic signals are encoded into electrical nerve impulses at the ribbon synapses of the cochlear inner hair cells (IHC). Exocytosis of the synaptic glutamatergic vesicles at these ribbon synapses is triggered by the activation of the voltage-dependent calcium channels CaV1.3 and required a specific calcium sensor: otoferlin. In the first part of my thesis, we studied the role of clarin-2, a newly identified tetraspan protein paralog of clarin-1, in these synapses. We know from previous studies that clarin-1, by forming a complex associating CaV1.3 channels, harmonin-b and actin-F, is essential for a compact spatial organization of the Ca1.3 channels at the active zone of the IHC ribbons. We show that clarin-2 is essential for a normal voltage-dependent activation of the IHC calcium channels and is likely part of the clarin-1 complex at the ribbon synapses. In the second part of my thesis, we studied the role of SNAP-25, a protein of the SNARE complex implicated in the docking, priming and fusion of the synaptic vesicles at neuronal central synapses but whose implication at the ribbon hair cell synapses remain still under debate. We show that mutant mice specifically deleted for SNAP-25 in hair cells are profoundly deaf and have a severe vestibular deficit. Electrophysiological patch-clamp recordings in IHCs and utricular hair cells indicated a severe defect in fast exocytosis. This synaptic functional defect was associated with a progressive degeneration of the ribbon synapses and the hair cells themselves, suggesting that SNAP-25 is also essential for the release of important neurotrophic factors. In the third part of my thesis, we investigated the functional changes of the aging ribbon synapses in C57BL/6J mice. We found that the aging synaptic IHC ribbons, while progressively degenerating, become larger with enlarged Ca2+ microdomains to produce a more sustained exocytosis of synaptic vesicles. The expression and the voltage dependence of the fast BK channels, essential for fast IHC repolarization, were also severely affected with aging. All these results suggested that the IHC ribbon synapses become potentiated with aging, a process that may partly explain the paradoxical hyperacusis phenomenon in presbyacusis

Hyperacusis in the Adult Fmr1-KO Mouse Model of Fragile X Syndrome: The Therapeutic Relevance of Cochlear Alterations and BKCa Channels

International audienceHyperacusis, i.e., an increased sensitivity to sounds, is described in several neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). The mechanisms underlying hyperacusis in FXS are still largely unknown and effective therapies are lacking. Big conductance calcium-activated potassium (BKCa) channels were proposed as a therapeutic target to treat several behavioral disturbances in FXS preclinical models, but their role in mediating their auditory alterations was not specifically addressed. Furthermore, studies on the acoustic phenotypes of FXS animal models mostly focused on central rather than peripheral auditory pathways. Here, we provided an extensive characterization of the peripheral auditory phenotype of the Fmr1-knockout (KO) mouse model of FXS at adulthood. We also assessed whether the acute administration of Chlorzoxazone, a BKCa agonist, could rescue the auditory abnormalities of adult mutant mice. Fmr1-KO mice both at 3 and 6 months showed a hyperacusis-like startle phenotype with paradoxically reduced auditory brainstem responses associated with a loss of ribbon synapses in the inner hair cells (IHCs) compared to their wild-type (WT) littermates. BKCa expression was markedly reduced in the IHCs of KOs compared to WT mice, but only at 6 months, when Chlorzoxazone rescued mutant auditory dysfunction. Our findings highlight the age-dependent and progressive contribution of peripheral mechanisms and BKCa channels to adult hyperacusis in FXS, suggesting a novel therapeutic target to treat auditory dysfunction in NDDs

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses

International audienceAge-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca 2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis

Loss of Pex1 in Inner Ear Hair Cells Contributes to Cochlear Synaptopathy and Hearing Loss

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1Creor VGlut3Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function

Clarin-2 gene supplementation durably preserves hearing in a model of progressive hearing loss

http://dx.doi.org/10.13039/100019671 Fondation Pour l'Auditionhttp://dx.doi.org/10.13039/501100000262 Foundation Fighting Blindnesshttp://dx.doi.org/10.13039/501100000265 Medical Research Councilhttp://dx.doi.org/10.13039/501100000780 European Unionhttp://dx.doi.org/10.13039/501100001659 German Research Foundationhttp://dx.doi.org/10.13039/501100001665 French National Research Agencyhttp://dx.doi.org/10.13039/501100013121 Fondation Voir et Entendrehttp://dx.doi.org/10.13039/501100004431 Fondation de Franc

Clarin-2 gene supplementation durably preserves hearing in a model of progressive hearing loss

International audienceHearing loss is a major health concern affecting millions of people worldwide with currently limited treatment options. In clarin-2-deficient Clrn2/ mice, used here as a model of progressive hearing loss, we report synaptic auditory abnormalities in addition to the previously demonstrated defects of hair bundle structure and mechanoelectrical transduction. We sought an in-depth evaluation of viral-mediated gene deliveryas a therapy for these hearing-impaired mice. Supplementationwith either the murine Clrn2 or human CLRN2 genes preservednormal hearing in treated Clrn2/ mice. Conversely,mutated forms of CLRN2, identified in patients with postlingualmoderate to severe hearing loss, failed to prevent hearing loss. The ectopic expression of clarin-2 successfully prevented the loss of stereocilia, maintained normal mechanoelectrical transduction, preserved inner hair cell synaptic function, and ensured near-normal hearing thresholds over time. Maximal hearing preservation was observed when Clrn2 was delivered prior to the loss of transducing stereocilia. Our findings demonstrate that gene therapy is effective for the treatment of post-lingual hearing impairment and age-related deafness associated with CLRN2 patient mutations

The SNARE protein SNAP-25 is required for normal exocytosis at auditory hair cell ribbon synapses

International audienceHearing depends on fast and sustained calcium-dependent synaptic vesicle fusion at the ribbon synapses of cochlear inner hair cells (IHCs). The implication of the canonical neuronal SNARE complex in this exocytotic process has so far remained controversial. We investigated the role of SNAP-25, a key component of this complex, in hearing, by generating and analyzing a conditional knockout mouse model allowing a targeted postnatal deletion of Snap-25 in IHCs. Mice subjected to IHC Snap-25 inactivation after hearing onset developed severe to profound deafness because of defective IHC exocytosis followed by ribbon degeneration and IHC loss. Viral transfer of Snap-25 in these mutant mice rescued their hearing function by restoring IHC exocytosis and preventing synapses and hair cells from degeneration. These results demonstrate that SNAP-25 is essential for normal hearing function, most likely by ensuring IHC exocytosis and ribbon synapse maintenance