Pannexin1 channels dominate ATP release in the cochlea ensuring endocochlear potential and auditory receptor potential generation and hearing.
ABSTRACT: Pannexin1 (Panx1) is a gap junction gene in vertebrates whose proteins mainly function as non-junctional channels on the cell surface. Panx1 channels can release ATP under physiological conditions and play critical roles in many physiological and pathological processes. Here, we report that Panx1 deficiency can reduce ATP release and endocochlear potential (EP) generation in the cochlea inducing hearing loss. Panx1 extensively expresses in the cochlea, including the cochlear lateral wall. We found that deletion of Panx1 in the cochlear lateral wall almost abolished ATP release under physiological conditions. Positive EP is a driving force for current through hair cells to produce auditory receptor potential. EP generation requires ATP. In the Panx1 deficient mice, EP and auditory receptor potential as measured by cochlear microphonics (CM) were significantly reduced. However, no apparent hair cell loss was detected. Moreover, defect of connexin hemichannels by deletion of connexin26 (Cx26) and Cx30, which are predominant connexin isoforms in the cochlea, did not reduce ATP release under physiological conditions. These data demonstrate that Panx1 channels dominate ATP release in the cochlea ensuring EP and auditory receptor potential generation and hearing. Panx1 deficiency can reduce ATP release and EP generation causing hearing loss.
Project description:Gap junctions play a critical role in hearing. Connexin gap junction gene mutations can induce a high incidence of hearing loss. Pannexin (Panx) gene also encodes gap junction proteins in vertebrates. Panx1 is a predominant pannexin isoform and has extensive expression in the cochlea. Here, we report that deletion of Panx1 in the cochlea could produce a progressive hearing loss. The auditory brainstem response (ABR) recording showed that hearing loss was moderate to severe and severe at high-frequencies. Distortion product otoacoustic emission (DPOAE), which reflects the activity of active cochlear mechanics that can amply acoustic stimulation to enhance hearing sensitivity and frequency selectivity, was also reduced. We further found that Panx1 deficiency could activate Caspase-3 cell apoptotic pathway in the cochlea to cause hair cells and other types of cells degeneration. These data indicate that like connexins Panx1 deficiency can also induce hearing loss. These data also suggest that pannexins play important rather than redundant roles in the cochlea and hearing.
Project description:Connexin gap junctions play an important role in hearing function, but the mechanism by which this contribution occurs is unknown. Connexins in the cochlea are expressed only in supporting cells; no connexin expression occurs in auditory sensory hair cells. A gap junctional channel is formed by two hemichannels. Here, we show that connexin hemichannels in the cochlea can release ATP at levels that account for the submicromolar concentrations measured in the cochlear fluids in vivo. The release could be increased 3- to 5-fold by a reduction of extracellular Ca2+ or an increase in membrane stress, and blocked by gap junctional blockers. We also demonstrated that extracellular ATP at submicromolar levels apparently affected outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining cochlear sensitivity to sound stimulation in mammals. ATP reduced OHC electromotility and the slope factor of the voltage dependence and shifted the operating point to reduce the active amplifier gain. ATP also reduced the generation of distortion products. Immunofluorescent staining showed that purinergic receptors P2x2 and P2x7 were distributed on the OHC surface. Blockage of P2 receptors eliminated the effect of ATP on the OHC electromotility. The data revealed that there is a hemichannel-mediated, purinergic intercellular signaling pathway between supporting cells and hair cells in the cochlea to control hearing sensitivity. The data also demonstrated a potential source of ATP in the cochlea.
Project description:The sense of hearing is remarkable for its auditory dynamic range, which spans more than 10(12) in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2-null mice. At higher sound levels (?95 dB), additional processes dominated TTS, and P2RX2-null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2-null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.
Project description:Mutations of gap junction connexin genes induce a high incidence of nonsyndromic hearing loss. Pannexin genes also encode gap junctional proteins in vertebrates. Recent studies demonstrated that Pannexin-1 (Panx1) deficiency in mice and mutation in humans are also associated with hearing loss. So far, several Panx1 knockout (KO) mouse lines were established. In general, these Panx1 KO mouse lines demonstrate consistent phenotypes in most aspects, including hearing loss. However, a recent study reported that a Panx1 KO mouse line, which was created by Genentech Inc., had no hearing loss as measured by the auditory brainstem response (ABR) threshold at low-frequency range (<24 kHz). Here, we used multiple auditory function tests and re-examined hearing function in the Genentech Panx1 (Gen-Panx1) KO mouse. We found that ABR thresholds in the Gen-Panx1 KO mouse were significantly increased, in particular, in the high-frequency region. Moreover, consistent with the increase in ABR threshold, distortion product otoacoustic emission (DPOAE) and cochlear microphonics (CM), which reflect active cochlear amplification and auditory receptor current, respectively, were significantly reduced. These data demonstrated that the Gen-Panx1 KO mouse has hearing loss and further confirmed that Panx1 deficiency can cause deafness.
Project description:Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.
Project description:An estimated 466 million people suffer from hearing loss worldwide. Sensorineural hearing loss is characterized by degeneration of key structures of the sensory pathway in the cochlea such as the sensory hair cells, the primary auditory neurons and their synaptic connection to the hair cells - the ribbon synapse. Various strategies to protect or regenerate these sensory cells and structures are the subject of intensive research. Yet despite recent advances in our understandings of the capacity of the cochlea for repair and regeneration there are currently no pharmacological or biological interventions for hearing loss. Current research focusses on localized cochlear drug, gene and cell-based therapies. One of the more promising drug-based therapies is based on neurotrophic factors for the repair of the ribbon synapse after noise exposure, as well as preventing loss of primary auditory neurons and regrowth of the auditory neuron fibers after severe hearing loss. Drug therapy delivery technologies are being employed to address the specific needs of neurotrophin and other therapies for hearing loss that include the need for high doses, long-term delivery, localised or cell-specific targeting and techniques for their safe and efficacious delivery to the cochlea. Novel biomaterials are enabling high payloads of drugs to be administered to the cochlea with subsequent slow-release properties that are proving to be beneficial for treating hearing loss. In parallel, new gene therapy technologies are addressing the need for cell specificity and high efficacy for the treatment of both genetic and acquired hearing loss with promising reports of hearing recovery. Some biomaterials and cell therapies are being used in conjunction with the cochlear implant ensuring therapeutic benefit to the primary neurons during electrical stimulation. This review will introduce the auditory system, hearing loss and the potential for repair and regeneration in the cochlea. Drug delivery to the cochlea will then be reviewed, with a focus on new biomaterials, gene therapy technologies, cell therapy and the use of the cochlear implant as a vehicle for drug delivery. With the current pre-clinical research effort into therapies for hearing loss, including clinical trials for gene therapy, the future for the treatment for hearing loss is looking bright.
Project description:Connexins (Cxs) are ubiquitous membrane proteins that are found throughout vertebrate organs, acting as building blocks of the gap junctions (GJs) known to play vital roles in the normal function of many organs. Mutations in Cx genes (particularly GJB2, which encodes Cx26) cause approximately half of all cases of congenital hearing loss in newborns. Great progress has been made in understanding GJ function and the molecular mechanisms for the role of Cxs in the cochlea. Data reveal that multiple types of Cxs work together to ensure normal development and function of the cochlea. These findings include many aspects not proposed in the classic K+ recycling theory, such as the formation of normal cochlear morphology (e.g., the opening of the tunnel of Corti), the fine-tuning of the innervation of nerve fibers to the hair cells (HCs), the maturation of the ribbon synapses, and the initiation of the endocochlear potential (EP). New data, especially those collected from targeted modification of major Cx genes in the mouse cochlea, have demonstrated that Cx26 plays an essential role in the postnatal maturation of the cochlea. Studies also show that Cx26 and Cx30 assume very different roles in the EP generation, given that only Cx26 is required for normal hearing. This article will review our current understanding of the molecular structure, cellular distribution, and major functions of cochlear GJs. Potential implications of the knowledge of cochlear GJs on the design and implementation of translational studies of cochlear gene therapies for Cx mutations are also discussed.
Project description:Neurons in the developing auditory system fire bursts of action potentials before the onset of hearing. This spontaneous activity promotes the survival and maturation of auditory neurons and the refinement of synaptic connections in auditory nuclei; however, the mechanisms responsible for initiating this activity remain uncertain. Previous studies indicate that inner supporting cells (ISCs) in the developing cochlea periodically release ATP, which depolarizes inner hair cells (IHCs), leading to bursts of action potentials in postsynaptic spiral ganglion neurons (SGNs). To determine when purinergic signaling appears in the developing cochlea and whether it is responsible for initiating auditory neuron activity throughout the prehearing period, we examined spontaneous activity from ISCs, IHCs, and SGNs in cochleae acutely isolated from rats during the first three postnatal weeks. We found that ATP was released from ISCs within the cochlea from birth until the onset of hearing, which led to periodic inward currents, Ca(2+) transients, and morphological changes in these supporting cells. This spontaneous release of ATP also depolarized IHCs and triggered bursts of action potentials in SGNs for most of the postnatal prehearing period, beginning a few days after birth as IHCs became responsive to ATP, until the onset of hearing when ATP was no longer released from ISCs. When IHCs were not subject to purinergic excitation, SGNs exhibited little or no activity. These results suggest that supporting cells in the cochlea provide the primary excitatory stimulus responsible for initiating bursts of action potentials in auditory nerve fibers before the onset of hearing.
Project description:Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(12), during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y(2) and P2Y(4) protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca(2+)-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.