Jakob Neef

Ca2+-binding proteins tune Ca2+-feedback to Cav1.3 channels in mouse auditory hair cells

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Cav1.3 voltage‐gated Ca2+ channels. Central to their role in this regard, Cav1.3 channels in inner hair cells show little Ca2+‐dependent inactivation, a fast negative feedback regulation by incoming Ca2+ ions, which depends on calmodulin association with the Ca2+ channel α1 subunit. Ca2+‐dependent inactivation characterizes nearly all voltage‐gated Ca2+ channels including Cav1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Cav1.3 in inner hair cells remains a mystery. Previously, we established calmodulin‐like Ca2+‐binding proteins in the brain and retina (CaBPs) as essential modulators of voltage‐gated Ca2+ channels. Here, we demonstrate that CaBPs differentially modify Ca2+ feedback to Cav1.3 channels in transfected cells and explore their significance for Cav1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca2+‐dependent inactivation of Cav1.3. CaBP1 and CaBP4 both interact with calmodulin‐binding sequences in Cav1.3, but CaBP4 more weakly inhibits Ca2+‐dependent inactivation than CaBP1. Ca2+‐dependent inactivation is marginally greater in inner hair cells from CaBP4−/− than from wild‐type mice, yet CaBP4−/− mice are not hearing‐impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild‐type and CaBP4−/− mice and therefore is positioned to modulate native Cav1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca2+ channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Cav1.3 Ca2+ currents required for auditory transmission.

Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins

SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and synaptobrevin-1 or synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs.

GaN-based micro-LED arrays on flexible substrates for optical cochlear implants

Currently available cochlear implants are based on electrical stimulation of the spiral ganglion neurons. Optical stimulation with arrays of micro-sized light-emitting diodes (µLEDs) promises to increase the number of distinguishable frequencies. Here, the development of a flexible GaN-based micro-LED array as an optical cochlear implant is reported for application in a mouse model. The fabrication of 15 µm thin and highly flexible devices is enabled by a laser-based layer transfer process of the GaN-LEDs from sapphire to a polyimide-on-silicon carrier wafer. The fabricated 50 × 50 µm2 LEDs are contacted via conducting paths on both p- and n-sides of the LEDs. Up to three separate channels could be addressed. The probes, composed of a linear array of the said µLEDs bonded to the flexible polyimide substrate, are peeled off the carrier wafer and attached to flexible printed circuit boards. Probes with four µLEDs and a width of 230 µm are successfully implanted in the mouse cochlea both in vitro and in vivo. The LEDs emit 60 µW at 1 mA after peel-off, corresponding to a radiant emittance of 6 mW mm−2.

Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis

Cochlear inner hair cells (IHCs) use Ca2+-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C2 domain protein essential for IHC exocytosis and hearing, may serve as a Ca2+ sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca2+ sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca2+ sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof −/− mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca2+ influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof −/− hippocampal neurons and in Syt1−/− IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C2 domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.

The Ca2+ channel subunit β2 regulates Ca2+ channel abundance and function in inner hair cells and is required for hearing.

Hearing relies on Ca2+ influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca2+ channel subunit CaVβ2 in hearing. Of the CaVβ1–4 mRNAs, IHCs predominantly contained CaVβ2. Hearing was severely impaired in mice lacking CaVβ2 in extracardiac tissues (CaVβ2−/−). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in CaVβ2−/− mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. CaVβ2−/− IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing CaV1.3 channels. Confocal Ca2+ imaging revealed presynaptic Ca2+ microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer CaV1.3 channels. The coupling of the remaining Ca2+ influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the CaVβ2−/− mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in CaVβ2−/− mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic CaV1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca2+ activated potassium channels in IHCs were impaired in the absence of CaVβ2. Moreover, we found the developmental efferent innervation to persist in CaVβ2-deficient IHCs. In summary, CaVβ2 has an essential role in regulating the abundance and properties of CaV1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.

Uniquantal release through a dynamic fusion pore is a candidate mechanism of hair cell exocytosis

The mechanisms underlying the large amplitudes and heterogeneity of excitatory postsynaptic currents (EPSCs) at inner hair cell (IHC) ribbon synapses are unknown. Based on electrophysiology, electron and superresolution light microscopy, and modeling, we propose that uniquantal exocytosis shaped by a dynamic fusion pore is a candidate neurotransmitter release mechanism in IHCs. Modeling indicated that the extended postsynaptic AMPA receptor clusters enable large uniquantal EPSCs. Recorded multiphasic EPSCs were triggered by similar glutamate amounts as monophasic ones and were consistent with progressive vesicle emptying during pore flickering. The fraction of multiphasic EPSCs decreased in absence of Ca(2+) influx and upon application of the Ca(2+) channel modulator BayK8644. Our experiments and modeling did not support the two most popular multiquantal release interpretations of EPSC heterogeneity: (1) Ca(2+)-synchronized exocytosis of multiple vesicles and (2) compound exocytosis fueled by vesicle-to-vesicle fusion. We propose that IHC synapses efficiently use uniquantal glutamate release for achieving high information transmission rates.

Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells

Hearing requires an optimal afferent innervation of sensory hair cells by spiral ganglion neurons in the cochlea. Here we report that complementary expression of ephrin-A5 in hair cells and EphA4 receptor among spiral ganglion neuron populations controls the targeting of type I and type II afferent fibres to inner and outer hair cells, respectively. In the absence of ephrin-A5 or EphA4 forward signalling, a subset of type I projections aberrantly overshoot the inner hair cell layer and invade the outer hair cell area. Lack of type I afferent synapses impairs neurotransmission from inner hair cells to the auditory nerve. By contrast, radial shift of type I projections coincides with a gain of presynaptic ribbons that could enhance the afferent signalling from outer hair cells. Ephexin-1, cofilin and myosin light chain kinase act downstream of EphA4 to induce type I spiral ganglion neuron growth cone collapse. Our findings constitute the first identification of an Eph/ephrin-mediated mutual repulsion mechanism responsible for specific sorting of auditory projections in the cochlea.

Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP‐2μ) is required for release site replenishment and hearing. We show that hair cell‐specific disruption of AP‐2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane‐proximal vesicles and intact endocytic membrane retrieval. Sound‐driven postsynaptic spiking was reduced in a use‐dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome‐like vacuoles, fewer clathrin‐coated endocytic intermediates, and vesicle depletion of the membrane‐distal synaptic ribbon in AP‐2μ‐deficient IHCs, indicating a further role of AP‐2μ in clathrin‐dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP‐2 sorts its IHC‐cargo otoferlin. We propose that binding of AP‐2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP‐2 in synaptic vesicle reformation.

Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells.

ABSTRACT Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca2+-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.

The Mechanosensory Structure of the Hair Cell Requires Clarin-1, a Protein Encoded by Usher Syndrome III Causative Gene

Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1−/−) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca2+ currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1−/− mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1−/− mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1N48K) supports our in vitro and Clrn1−/− mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1N48K mouse suggests that it is a valuable model for ear disease in CLRN1N48K, the most prevalent Usher syndrome III mutation in North America.