Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membraneanchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size. synaptogenesis | presynaptic development | ribbon synapse maturation | synaptic heterogeneity I n mammals, synaptic sound encoding occurs at the first auditory synapse between cochlear inner hair cells (IHCs) and postsynaptic neurites of afferent spiral ganglion neurons (SGNs). The highly specialized IHC presynaptic active zones (AZs) are characterized by the presence of proteinaceous electron-dense bodies, called "synaptic ribbons," which are primarily composed of the structural cytomatrix protein RIBEYE (1, 2). Ribbons provide presynaptic scaffolding, cluster and functionally regulate presynaptic Ca 2+ channels at the AZ membrane (3-5), and tether a halo of synaptic vesicles (SVs) (6). This latter feature is thought to enable rapid and indefatigable vesicular replenishment to the release site-even during periods of persistent stimulation (3,5,7,8).In mice, hearing onset occurs around postnatal day (P)12 (9) before which, IHC presynaptic AZs undergo a range of structural and functional refinements. For example, extrasynaptically localized Ca 2+ channels are progressively eliminated from the nonsynaptic basolateral plasma membrane and form-in concert with the corresponding postsynaptic glutamate receptor patchestightly confined clusters at mature presynaptic AZs (3, 10). Moreover, otoferlin-a large Ca 2+ -binding multi-C 2 domain protein (11, 12)-likely replaces synaptotagmins as the putative Ca 2+ sensor of IHC release during the first postnatal week (13). This finding reflects a key landmark of functional maturation of this unconventional high-throughput release machinery and is essentially required to faithfully orchestrate vesicular...

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.