Cabp2-Gene Therapy Restores Inner Hair Cell Calcium Currents and Improves Hearing in a DFNB93 Mouse Model.

Clinical management of auditory synaptopathies like other genetic hearing disorders is currently limited to the use of hearing aids or cochlear implants. However, future gene therapy promises restoration of hearing in selected forms of monogenic hearing impairment, in which cochlear morphology is preserved over a time window that enables intervention. This includes non-syndromic autosomal recessive hearing impairment DFNB93, caused by defects in the CABP2 gene. Calcium-binding protein 2 (CaBP2) is a potent modulator of inner hair cell (IHC) voltage-gated calcium channels CaV1.3. Based on disease modeling in Cabp2-/- mice, DFNB93 hearing impairment has been ascribed to enhanced steady-state inactivation of IHC CaV1.3 channels, effectively limiting their availability to trigger synaptic transmission. This, however, does not seem to interfere with cochlear development and does not cause early degeneration of hair cells or their synapses. Here, we studied the potential of a gene therapeutic approach for the treatment of DFNB93. We used AAV2/1 and AAV-PHP.eB viral vectors to deliver the Cabp2 coding sequence into IHCs of early postnatal Cabp2-/- mice and assessed the level of restoration of hair cell function and hearing. Combining in vitro and in vivo approaches, we observed high transduction efficiency, and restoration of IHC CaV1.3 function resulting in improved hearing of Cabp2-/- mice. These preclinical results prove the feasibility of DFNB93 gene therapy.

The synaptic ribbon is critical for sound encoding at high rates and with temporal precision.

We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation.

Ca2+-binding protein 2 inhibits Ca2+-channel inactivation in mouse inner hair cells.

Ca2+-binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca2+ channels of type 1.3 (CaV1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2LacZ/LacZ ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs). Cabp2LacZ/LacZ mice displayed intact cochlear amplification but impaired auditory brainstem responses. Patch-clamp recordings from Cabp2LacZ/LacZ IHCs revealed enhanced Ca2+-channel inactivation. The voltage dependence of activation and the number of Ca2+ channels appeared normal in Cabp2LacZ/LacZ mice, as were ribbon synapse counts. Recordings from single SGNs showed reduced spontaneous and sound-evoked firing rates. We propose that CaBP2 inhibits CaV1.3 Ca2+-channel inactivation, and thus sustains the availability of CaV1.3 Ca2+ channels for synaptic sound encoding. Therefore, we conclude that human deafness DFNB93 is an auditory synaptopathy.

Hair cells use active zones with different voltage dependence of Ca2+ influx to decompose sounds into complementary neural codes.

For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca(2+) influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca(2+) influx ranged over ∼20 mV. Ca(2+) influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca(2+) channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca(2+) influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca(2+) influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca(2+) influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca(2+) channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.

Rab3-interacting molecules 2α and 2ß promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones.

Ca(2+) influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and ß (RIM2α and RIM2ß) in clustering voltage-gated CaV1.3 Ca(2+) channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2ß and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca(2+) imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca(2+) channels, resulting in reduced synaptic Ca(2+) influx. Using superresolution microscopy, we found that Ca(2+) channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca(2+) current, whereas the apparent Ca(2+) dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca(2+) influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2ß promote a large complement of synaptic Ca(2+) channels at IHC AZs and are required for normal hearing.

A mutation in CABP2, expressed in cochlear hair cells, causes autosomal-recessive hearing impairment.

CaBPs are a family of Ca(2+)-binding proteins related to calmodulin and are localized in the brain and sensory organs, including the retina and cochlea. Although their physiological roles are not yet fully elucidated, CaBPs modulate Ca(2+) signaling through effectors such as voltage-gated Ca(v) Ca(2+) channels. In this study, we identified a splice-site mutation (c.637+1G>T) in Ca(2+)-binding protein 2 (CABP2) in three consanguineous Iranian families affected by moderate-to-severe hearing loss. This mutation, most likely a founder mutation, probably leads to skipping of exon 6 and premature truncation of the protein (p.Phe164Serfs(∗)4). Compared with wild-type CaBP2, the truncated CaBP2 showed altered Ca(2+) binding in isothermal titration calorimetry and less potent regulation of Ca(v)1.3 Ca(2+) channels. We show that genetic defects in CABP2 cause moderate-to-severe sensorineural hearing impairment. The mutation might cause a hypofunctional CaBP2 defective in Ca(2+) sensing and effector regulation in the inner ear.