Pietro Scimemi

The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30T5M/T5M mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca2+ signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

The novel PMCA2 pump mutation Tommy impairs cytosolic calcium clearance in hair cells and links to deafness in mice.

The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle.

Abnormal cochlear potentials from deaf patients with mutations in the otoferlin gene.

Otoferlin is involved in neurotransmitter release at the synapse between inner hair cells (IHCs) and auditory nerve fibres, and mutations in the OTOF gene result in severe to profound hearing loss. Abnormal sound-evoked cochlear potentials were recorded with transtympanic electrocochleography from four children with otoferlin (OTOF) mutations to evaluate physiological effects in humans of abnormal neurotransmitter release from IHCs. The subjects were profoundly deaf with absent auditory brainstem responses and preserved otoacoustic emissions consistent with auditory neuropathy. Two children were compound heterozygotes for mutations c.2732_2735dupAGCT and p.Ala964Glu; one subject was homozygous for mutation p.Phe1795Cys, and one was compound heterozygote for two novel mutations c.1609delG in exon 16 and c.1966delC in exon 18. Cochlear potentials evoked by clicks from 60 to 120 dB peak equivalent sound pressure level were compared to recordings obtained from 16 normally hearing children. Cochlear microphonic (CM) was recorded with normal amplitudes from all but one ear. After cancelling CM, cochlear potentials were of negative polarity with reduced amplitude and prolonged duration compared to controls. These cochlear potentials were recorded as low as 50–90 dB below behavioural thresholds in contrast to the close correlation in controls between cochlear potentials and behavioural threshold. Summating potential was identified in five out of eight ears with normal latency whilst auditory nerve compound action potentials were either absent or of low amplitude. Stimulation at high rates reduced amplitude and duration of the prolonged potentials, consistent with neural generation. This study suggests that mechano-electrical transduction and cochlear amplification are normal in patients with OTOF mutations. The low-amplitude prolonged negative potentials are consistent with decreased neurotransmitter release resulting in abnormal dendritic activation and impairment of auditory nerve firing.

BAAV Mediated GJB2 Gene Transfer Restores Gap Junction Coupling in Cochlear Organotypic Cultures from Deaf Cx26Sox10Cre Mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26Sox10Cre mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10–Cre line. Cx26Sox10Cre mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

Reduced phosphatidylinositol 4,5-bisphosphate synthesis impairs inner ear Ca2+ signaling and high-frequency hearing acquisition.

Phosphatidylinositol phosphate kinase type 1γ (PIPKIγ) is a key enzyme in the generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is expressed at high levels in the nervous system. Homozygous knockout mice lacking this enzyme die postnatally within 24 h, whereas PIPKIγ+/− siblings breed normally and have no reported phenotype. Here we show that adult PIPKIγ+/− mice have dramatically elevated hearing thresholds for high-frequency sounds. During the first postnatal week we observed a reduction of ATP-dependent Ca2+ signaling activity in cochlear nonsensory cells. Because Ca2+ signaling under these conditions depends on inositol-1,4,5-trisphosphate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P2, we conclude that (i) PIPKIγ is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P2 pool in the cell syncytia that supports auditory hair cells; (ii) spatially graded impairment of this signaling pathway in cochlear nonsensory cells causes a selective alteration in the acquisition of hearing in PIPKIγ+/− mice. This mouse model also suggests that PIPKIγ may determine the level of gap junction contribution to cochlear development.

OPA1-related auditory neuropathy: site of lesion and outcome of cochlear implantation

Santarelli et al. reveal that hearing impairments in patients carrying OPA1 missense mutations are the result of disordered synchrony in auditory nerve fibre activity owing to degeneration of terminal dendrites. Cochlear implantation improves speech perception and synchronous activation of auditory pathways in these patients by bypassing the lesion site.

Audiological and electrocochleography findings in hearing-impaired children with connexin 26 mutations and otoacoustic emissions

We recorded cochlear potentials by transtympanic electrocochleography (ECochG) in three hearing-impaired children with GJB2 mutation who showed otoacoustic emissions. Pure tone thresholds, distortion product otoacoustic emissions (DPOAEs) and, auditory brainstem responses (ABRs) were also obtained. Subjects 1 (35delG/35delG) and 3 (M34T/wt) had profound hearing loss and showed the picture of auditory neuropathy (AN) as DPOAEs were detected with absent ABRs in both ears. The hearing impairment found in subject 2 (35delG/35delG) was profound in the right ear and moderate in the left ear. Both DPOAEs and ABRs with normal latencies and morphology were recorded only from the left ear. On the ECochG recording the cochlear microphonic was obtained from all children. No compound action potential (CAP) was detected in subject 1. A neural response was recorded only from the left ear in subject 2 with a threshold corresponding to the audiometric threshold while no CAP was detected on the right side. The ECochG obtained from subject 3 showed a low-amplitude broad negative deflection which was identifiable down to low stimulus levels. This response decreased in amplitude and duration when utilizing a high-rate stimulation paradigm. The amount of amplitude reduction was close to that calculated for normal ears, thus revealing the presence of an adapting neural component. These findings indicate that patients with GJB2 mutations and preserved outer hair cells function could present with the picture of AN. The hearing impairment is underlain by a selective inner hair cell loss or a lesion involving the synapses and/or the auditory nerve terminals. We suggest that neonatal hyperbilirubinemia may play a role in protecting outer hair cells against the damage induced by GJB2 mutations.

Audibility, speech perception and processing of temporal cues in ribbon synaptic disorders due to OTOF mutations

Mutations in the OTOF gene encoding otoferlin result in a disrupted function of the ribbon synapses with impairment of the multivesicular glutamate release. Most affected subjects present with congenital hearing loss and abnormal auditory brainstem potentials associated with preserved cochlear hair cell activities (otoacoustic emissions, cochlear microphonics [CMs]). Transtympanic electrocochleography (ECochG) has recently been proposed for defining the details of potentials arising in both the cochlea and auditory nerve in this disorder, and with a view to shedding light on the pathophysiological mechanisms underlying auditory dysfunction. We review the audiological and electrophysiological findings in children with congenital profound deafness carrying two mutant alleles of the OTOF gene. We show that cochlear microphonic (CM) amplitude and summating potential (SP) amplitude and latency are normal, consistently with a preserved outer and inner hair cell function. In the majority of OTOF children, the SP component is followed by a markedly prolonged low-amplitude negative potential replacing the compound action potential (CAP) recorded in normally-hearing children. This potential is identified at intensities as low as 90 dB below the behavioral threshold. In some ears, a synchronized CAP is superimposed on the prolonged responses at high intensity. Stimulation at high rates reduces the amplitude and duration of the prolonged potentials, consistently with their neural generation. In some children, however, the ECochG response only consists of the SP, with no prolonged potential. Cochlear implants restore hearing sensitivity, speech perception and neural CAP by electrically stimulating the auditory nerve fibers. These findings indicate that an impaired multivesicular glutamate release in OTOF-related disorders leads to abnormal auditory nerve fiber activation and a consequent impairment of spike generation. The magnitude of these effects seems to vary, ranging from no auditory nerve fiber activation to an abnormal generation of EPSPs that occasionally trigger a synchronized electrical activity, resulting in high-threshold CAPs.

Presynaptic and postsynaptic mechanisms underlying auditory neuropathy in patients with mutations in the OTOF or OPA1 gene

Abstract Objective: Our objective was to compare acoustically- and electrically-evoked potentials of the auditory nerve in patients with postsynaptic or presynaptic auditory neuropathy with underlying mutations in the OPA1 or OTOF gene. Study design: Transtympanic electrocochleography (ECochG) was recorded from two adult patients carrying the R445H OPA1 mutation, and from five children with mutations in the OTOF gene. Cochlear potentials to clicks or tone-bursts were compared to recordings obtained from 16 normally hearing subjects. Electrically-evoked neural responses recorded through the cochlear implant were also obtained. Results: The cochlear microphonic (CM) was recorded from all subjects, with normal amplitudes. After cancelling the CM, cochlear potentials were of negative polarity with reduced amplitude and prolonged duration compared to controls in both groups of patients. Prolonged negative responses were recorded as low as 50–90dB below behavioural threshold in subjects with OTOF mutations whereas in the OPA1 disorder the prolonged potentials were correlated with hearing threshold. A compound action potential (CAP) was superimposed on the prolonged activity at high stimulation intensity in two children with mutations in the OTOF gene while CAPs were absent in the OPA1 disorder. Electrically-evoked compound action potentials (e-CAPs) were only recorded from subjects with OTOF mutations following cochlear implantation. Conclusions: The findings are consistent with abnormal function of distal portions of auditory nerve fibres in patients carrying the OPA1 mutation whereas the low-threshold prolonged potentials recorded from children with mutations in the OTOF gene are consistent with abnormal neurotransmitter release resulting in reduced dendritic activation and impairment of spike initiation.

Abnormal Cochlear Potentials in Friedreich's Ataxia Point to Disordered Synchrony of Auditory Nerve Fiber Activity.

Background: Friedreichs ataxia (FRDA) is a degenerative disorder caused by mutations of the FXN gene. Sensorineural hearing loss is one of the clinical features of FRDA, and the majority of hearing-impaired patients have shown evidence of auditory neuropathy. Objective: This study characterizes the cochlear receptor and auditory nerve potentials in a patient with FRDA who had the clinical profile of auditory neuropathy. The aim was to investigate the site of the lesion and the pathophysiological mechanisms behind the hearing dysfunction. Methods: Using transtympanic electrocochleography, both receptor (cochlear microphonic, CM, and summating potential, SP) and auditory nerve potentials were recorded in response to trains of clicks with stimulation intensities from 60 to 120 dB SPL. The results were compared with recordings obtained from two groups of subjects, i.e. 20 normally hearing controls and 19 subjects with cochlear hearing loss. Results: The results showed that the synchronized neural response seen in both normally hearing and hearing-impaired subjects was lacking in our patient, replaced by a prolonged, low-amplitude negative potential that decreased in both amplitude and duration for rapid stimulation rates, consistent with adaptation of neural sources. CMs were recorded with a normal amplitude, consistent with preserved outer hair cell function. SP peak latency was within normal limits, whereas SP amplitude was comparable with that of subjects with cochlear hearing loss, consistent with inner hair cell loss. Conclusion: These findings suggest that underlying auditory neuropathy in FRDA is a disordered synchrony in auditory nerve fiber discharge, possibly resulting from auditory nerve fiber degeneration and inner hair cell loss.