Tobias Moser

Otoferlin, Defective in a Human Deafness Form, Is Essential for Exocytosis at the Auditory Ribbon Synapse

The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.

Hair cell synaptic ribbons are essential for synchronous auditory signalling

Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.

Munc18-1 Promotes Large Dense-Core Vesicle Docking

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.

Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse

Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i above 8 microM caused a biphasic capacitance increase corresponding to the fusion of approximately 40,000 vesicles. The kinetics of exocytosis displayed a fifth-order Ca2+ dependence reaching maximal rates of >3 x 10(7) vesicle/s. Exocytosis was always followed by slow, compensatory endocytosis (tau congruent with 15 s). Higher [Ca2+]i increased the contribution of a faster mode of endocytosis with a Ca2+ independent time constant of approximately 300 ms. These properties provide for rapid and sustained transmitter release from this large presynaptic terminal.

Few CaV1.3 Channels Regulate the Exocytosis of a Synaptic Vesicle at the Hair Cell Ribbon Synapse

Hearing relies on faithful sound coding at hair cell ribbon synapses, which use Ca2+-triggered glutamate release to signal with submillisecond precision. Here, we investigated stimulus–secretion coupling at mammalian inner hair cell (IHC) synapses to explore the mechanisms underlying this high temporal fidelity. Using nonstationary fluctuation analysis on Ca2+ tail currents, we estimate that IHCs contain ∼1700 Ca2+ channels, mainly of CaV1.3 type. We show by immunohistochemistry that the CaV1.3 Ca2+ channels are localized preferentially at the ribbon-type active zones of IHCs. We argue that each active zone holds ∼80 Ca2+ channels, of which probably <10 open simultaneously during physiological stimulation. We then manipulated the Ca2+ current by primarily changing single-channel current or open-channel number. Effects on exocytosis of the readily releasable vesicle pool (RRP) were monitored by membrane capacitance recordings. Consistent with the high intrinsic Ca2+ cooperativity of exocytosis, RRP exocytosis changed nonlinearly with the Ca2+ current when varying the single-channel current. In contrast, the apparent Ca2+ cooperativity of RRP exocytosis was close to unity when primarily manipulating the number of open channels. Our findings suggest a Ca2+ channel–release site coupling in which few nearby CaV1.3 channels impose high nanodomain [Ca2+] on release sites in IHCs during physiological stimulation. We postulate that the IHC ribbon synapse uses this Ca2+ nanodomain control of exocytosis to signal with high temporal precision already at low sound intensities.

Cytosolic Ca2+ Acts by Two Separate Pathways to Modulate the Supply of Release-Competent Vesicles in Chromaffin Cells

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.

Tuning of synapse number, structure and function in the cochlea

Cochlear inner hair cells (IHCs) transmit acoustic information to spiral ganglion neurons through ribbon synapses. Here we have used morphological and physiological techniques to ask whether synaptic mechanisms differ along the tonotopic axis and within IHCs in the mouse cochlea. We show that the number of ribbon synapses per IHC peaks where the cochlea is most sensitive to sound. Exocytosis, measured as membrane capacitance changes, scaled with synapse number when comparing apical and midcochlear IHCs. Synapses were distributed in the subnuclear portion of IHCs. High-resolution imaging of IHC synapses provided insights into presynaptic Ca2+ channel clusters and Ca2+ signals, synaptic ribbons and postsynaptic glutamate receptor clusters and revealed subtle differences in their average properties along the tonotopic axis. However, we observed substantial variability for presynaptic Ca2+ signals, even within individual IHCs, providing a candidate presynaptic mechanism for the divergent dynamics of spiral ganglion neuron spiking.

Mice with altered KCNQ4 K + channels implicate sensory outer hair cells in human progressive deafness

KCNQ4 is an M‐type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4−/− mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the IK,n current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near‐normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.

Bassoon and the synaptic ribbon organize Ca2+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca²+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²+ channels and vesicles, and (2) promote vesicle replenishment.

Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca2+-dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca2+-uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca2+-dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca2+-sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.