Mitochondrial dynamics regulate cell morphology in the developing cochlea.

In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.

Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma.

Schwannoma tumours typically arise on the eighth cranial nerve and are mostly caused by loss of the tumour suppressor Merlin (NF2). There are no approved chemotherapies for these tumours and the surgical removal of the tumour carries a high risk of damage to the eighth or other close cranial nerve tissue. New treatments for schwannoma and other NF2-null tumours such as meningioma are urgently required. Using a combination of human primary tumour cells and mouse models of schwannoma, we have examined the role of the Hippo signalling pathway in driving tumour cell growth. Using both genetic ablation of the Hippo effectors YAP and TAZ as well as novel TEAD palmitoylation inhibitors, we show that Hippo signalling may be successfully targeted in vitro and in vivo to both block and, remarkably, regress schwannoma tumour growth. In particular, successful use of TEAD palmitoylation inhibitors in a preclinical mouse model of schwannoma points to their potential future clinical use. We also identify the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) as a Hippo signalling target, driven by the TAZ protein in human and mouse NF2-null schwannoma cells, as well as in NF2-null meningioma cells, and examine the potential future role of this new target in halting schwannoma and meningioma tumour growth.

Age-related changes in P2Y receptor signalling in mouse cochlear supporting cells.

Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non-sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age-related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age-related changes in the expression and function of metabotropic purinergic receptors (P2Y1 , P2Y2 and P2Y4 ) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic-mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month-old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y2 and P2Y4 receptors and display purinergic-induced Ca2+ responses that mimic those observed during pre-hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. KEY POINTS: Age-related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech. We investigated age-related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non-sensory supporting cells of mice displaying early-onset (C57BL/6N) and late-onset (C3H/HeJ) hearing loss. The expression of P2Y1 , P2Y2 and P2Y4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y2 and P2Y4 was upregulated in the aged cochlea. P2Y2 and P2Y4 receptors were primarily responsible for the ATP-induced Ca2+ responses in the supporting cells. The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains. We propose that the upregulation of purinergic-mediated signalling in the aged cochlea is subsequent to age-related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium.

Functional P2X7 Receptors in the Auditory Nerve of Hearing Rodents Localize Exclusively to Peripheral Glia.

P2X7 receptors (P2X7Rs) are associated with numerous pathophysiological mechanisms, and this promotes them as therapeutic targets for certain neurodegenerative conditions. However, the identity of P2X7R-expressing cells in the nervous system remains contentious. Here, we examined P2X7R functionality in auditory nerve cells from rodents of either sex, and determined their functional and anatomic expression pattern. In whole-cell recordings from rat spiral ganglion cultures, the purinergic agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) activated desensitizing currents in spiral ganglion neurons (SGNs) but non-desensitizing currents in glia that were blocked by P2X7R-specific antagonists. In imaging experiments, BzATP gated sustained Ca2+ entry into glial cells. BzATP-gated uptake of the fluorescent dye YO-PRO-1 was reduced and slowed by P2X7R-specific antagonists. In rats, P2X7Rs were immuno-localized predominantly within satellite glial cells (SGCs) and Schwann cells (SCs). P2X7R expression was not detected in the portion of the auditory nerve within the central nervous system. Mouse models allowed further exploration of the distribution of cochlear P2X7Rs. In GENSAT reporter mice, EGFP expression driven via the P2rx7 promoter was evident in SGCs and SCs but was undetectable in SGNs. A second transgenic model showed a comparable cellular distribution of EGFP-tagged P2X7Rs. In wild-type mice the discrete glial expression was confirmed using a P2X7-specific nanobody construct. Our study shows that P2X7Rs are expressed by peripheral glial cells, rather than by afferent neurons. Description of functional signatures and cellular distributions of these enigmatic proteins in the peripheral nervous system (PNS) will help our understanding of ATP-dependent effects contributing to hearing loss and other sensory neuropathies.SIGNIFICANCE STATEMENT P2X7 receptors (P2X7Rs) have been the subject of much scrutiny in recent years. They have been promoted as therapeutic targets in a number of diseases of the nervous system, yet the specific cellular location of these receptors remains the subject of intense debate. In the auditory nerve, connecting the inner ear to the brainstem, we show these multimodal ATP-gated channels localize exclusively to peripheral glial cells rather than the sensory neurons, and are not evident in central glia. Physiologic responses in the peripheral glia display classical hallmarks of P2X7R activation, including the formation of ion-permeable and also macromolecule-permeable pores. These qualities suggest these proteins could contribute to glial-mediated inflammatory processes in the auditory periphery under pathologic disease states.

A rare genomic duplication in 2p14 underlies autosomal dominant hearing loss DFNA58.

Here we define a ~200 Kb genomic duplication in 2p14 as the genetic signature that segregates with postlingual progressive sensorineural autosomal dominant hearing loss (HL) in 20 affected individuals from the DFNA58 family, first reported in 2009. The duplication includes two entire genes, PLEK and CNRIP1, and the first exon of PPP3R1 (protein coding), in addition to four uncharacterized long non-coding (lnc) RNA genes and part of a novel protein-coding gene. Quantitative analysis of mRNA expression in blood samples revealed selective overexpression of CNRIP1 and of two lncRNA genes (LOC107985892 and LOC102724389) in all affected members tested, but not in unaffected ones. Qualitative analysis of mRNA expression identified also fusion transcripts involving parts of PPP3R1, CNRIP1 and an intergenic region between PLEK and CNRIP1, in the blood of all carriers of the duplication, but were heterogeneous in nature. By in situ hybridization and immunofluorescence, we showed that Cnrip1, Plek and Ppp3r1 genes are all expressed in the adult mouse cochlea including the spiral ganglion neurons, suggesting changes in expression levels of these genes in the hearing organ could underlie the DFNA58 form of deafness. Our study highlights the value of studying rare genomic events leading to HL, such as copy number variations. Further studies will be required to determine which of these genes, either coding proteins or non-coding RNAs, is or are responsible for DFNA58 HL.

Divergent membrane properties of mouse cochlear glial cells around hearing onset.

Spiral ganglion neurons (SGNs) are the primary afferent neurons of the auditory system, and together with their attendant glia, form the auditory nerve. Within the cochlea, satellite glial cells (SGCs) encapsulate the cell body of SGNs, whereas Schwann cells (SCs) wrap their peripherally- and centrally-directed neurites. Despite their likely importance in auditory nerve function and homeostasis, the physiological properties of auditory glial cells have evaded description. Here, we characterized the voltage-activated membrane currents of glial cells from the mouse cochlea. We identified a prominent weak inwardly rectifying current in SGCs within cochlear slice preparations (postnatal day P5-P6), which was also present in presumptive SGCs within dissociated cultures prepared from the cochleae of hearing mice (P14-P15). Pharmacological block by Ba2+ and desipramine suggested that channels belonging to the Kir4 family mediated the weak inwardly rectifying current, and post hoc immunofluorescence implicated the involvement of Kir4.1 subunits. Additional electrophysiological profiles were identified for glial cells within dissociated cultures, suggesting that glial subtypes may have specific membrane properties to support distinct physiological roles. Immunofluorescence using fixed cochlear sections revealed that although Kir4.1 is restricted to SGCs after the onset of hearing, these channels are more widely distributed within the glial population earlier in postnatal development (i.e., within both SGCs and SCs). The decrease in Kir4.1 immunofluorescence during SC maturation was coincident with a reduction of Sox2 expression and advancing neurite myelination. The data suggest a diversification of glial properties occurs in preparation for sound-driven activity in the auditory nerve.

Olfactory ensheathing cells from the nasal mucosa and olfactory bulb have distinct membrane properties.

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for the regeneration of damaged neurons. While they maintain tissue homeostasis in the olfactory mucosa (OM) and olfactory bulb (OB), their regenerative properties also support the normal sense of smell by enabling continual turnover and axonal regrowth of olfactory sensory neurons (OSNs). However, the molecular physiology of OECs is not fully understood, especially that of OECs from the mucosa. Here, we carried out whole-cell patch-clamp recordings from individual OECs cultured from the OM and OB of the adult rat, and from the human OM. A subset of OECs from the rat OM cultured 1-3 days in vitro had large weakly rectifying K+ currents, which were sensitive to Ba2+ and desipramine, blockers of Kir4-family channels. Kir4.1 immunofluorescence was detectable in cultured OM cells colabeled for the OEC marker S100, and in S100-labeled cells found adjacent to OSN axons in mucosal sections. OECs cultured from rat OB had distinct properties though, displaying strongly rectifying inward currents at hyperpolarized membrane potentials and strongly rectifying outward currents at depolarized potentials. Kir4.1 immunofluorescence was not evident in OECs adjacent to axons of OSNs in the OB. A subset of human OECs cultured from the OM of adults had membrane properties comparable to those of the rat OM that is dominated by Ba2+ -sensitive weak inwardly rectifying currents. The membrane properties of peripheral OECs are different to those of central OECs, suggesting they may play distinct roles during olfaction.

Disruption of SorCS2 reveals differences in the regulation of stereociliary bundle formation between hair cell types in the inner ear.

Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC) in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein) locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed by SorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia) of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC) lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle.

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions.

In the present study we investigated the life cycle, trafficking, assembly and cell surface dynamics of a poorly characterized connexin family member, connexin 30 (Cx30; also known as GJB6), which plays a critical role in skin health and hearing. Unexpectedly, Cx30 localization at the cell surface and gap junctional intercellular communication was not affected by prolonged treatments with the endoplasmic reticulum (ER)-Golgi transport inhibitor brefeldin A or the protein synthesis inhibitor cycloheximide, whereas Cx43 (also known as GJA1) was rapidly cleared. Fluorescent recovery after photobleaching revealed that Cx30 plaques were rebuilt from the outer edges in keeping with older channels residing in the inner core of the plaque. Expression of a dominant-negative form of Sar1 GTPase led to the accumulation of Cx30 within the ER, in contrast to a report that Cx30 traffics via a Golgi-independent pathway. Co-expression of Cx30 with Cx43 revealed that these connexins segregate into distinct domains within common gap junction plaques, suggesting that their assembly is governed by different mechanisms. In summary, Cx30 was found to be an unusually stable, long-lived connexin (half-life >12 h), which may underlie its specific role in the epidermis and cochlea.

Spinster homolog 2 (spns2) deficiency causes early onset progressive hearing loss.

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

Phosphoinositide Modulation of Heteromeric Kv1 Channels Adjusts Output of Spiral Ganglion Neurons from Hearing Mice.

Spiral ganglion neurons (SGNs) relay acoustic code from cochlear hair cells to the brainstem, and their stimulation enables electrical hearing via cochlear implants. Rapid adaptation, a mechanism that preserves temporal precision, and a prominent feature of auditory neurons, is regulated via dendrotoxin-sensitive low-threshold voltage-activated (LVA) K(+) channels. Here, we investigated the molecular physiology of LVA currents in SGNs cultured from mice following the onset of hearing (postnatal days 12-21). Kv1.1- and Kv1.2-specific toxins blocked the LVA currents in a comparable manner, suggesting that both subunits contribute to functional heteromeric channels. Confocal immunofluorescence in fixed cochlear sections localized both Kv1.1 and Kv1.2 subunits to specific neuronal microdomains, including the somatic membrane, juxtaparanodes, and the first heminode, which forms the spike initiation site of the auditory nerve. The spatial distribution of Kv1 immunofluorescence appeared mutually exclusive to that of Kv3.1b subunits, which mediate high-threshold voltage-activated currents. As Kv1.2-containing channels are positively modulated by membrane phosphoinositides, we investigated the influence of phosphatidylinositol-4,5-bisphosphate (PIP2) availability on SGN electrophysiology. Reducing PIP2 production using wortmannin, or sequestration of PIP2 using a palmitoylated peptide (PIP2-PP), slowed adaptation rate in SGN populations. PIP2-PP specifically inhibited the LVA current in SGNs, an effect reduced by intracellular dialysis of a nonhydrolysable analog of PIP2. PIP2-PP also inhibited heterologously expressed Kv1.1/Kv1.2 channels, recapitulating its effect in SGNs. Collectively, the data identify Kv1.1/Kv1.2 heteromeric channels as key regulators of action potential initiation and propagation in the auditory nerve, and suggest that modulation of these channels by endogenous phosphoinositides provides local control of membrane excitability. SIGNIFICANCE STATEMENT: Rapid spike adaptation is an important feature of auditory neurons that preserves temporal precision. In spiral ganglion neurons, the primary afferents in the cochlea, adaptation is regulated by heteromeric ion channels composed of Kv1.1 and Kv1.2 subunits. These subunits colocalize to common functional microdomains, such as juxtaparanodes and the somatic membrane. Activity of the heteromeric channels is controlled by cellular availability of PIP2, a membrane phospholipid. This mechanism provides an intrinsic regulation of output from the auditory nerve, which could be targeted for therapeutic adjustment of hearing sensitivity.

Gap junctional coupling is essential for epithelial repair in the avian cochlea.

The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration.

Connexin30-mediated intercellular communication plays an essential role in epithelial repair in the cochlea.

A role for connexin (Cx)30 in epithelial repair following injury was examined in the organ of Corti, the sensory epithelium of the cochlea. In this tissue, lesions caused by loss of the sensory hair cells are closed by the supporting cells that surround each one. Gap junctions in which Cx30 is the predominant connexin are large and numerous between supporting cells. In mice carrying a deletion in the gene (Gjb6) that encodes Cx30, the size and number of gap junction plaques, and the extent of dye transfer, between supporting cells was greatly reduced compared with normal animals. This corresponded with unique peculiarities of the lesion closure events during the progressive hair cell loss that occurs in these animals in comparison with other models of hair cell loss, whether acquired or as a result of a mutation. Only one, rather than all, of the supporting cells that contacted an individual dying hair closed the lesion, indicating disturbance of the co-ordination of cellular responses. The cell shape changes that the supporting cells normally undergo during repair of the organ of Corti did not occur. Also, there was disruption of the migratory activities that normally lead to the replacement of a columnar epithelium with a squamous-like one. These observations demonstrate a role for Cx30 and intercellular communication in regulating repair responses in an epithelial tissue.

Connexins and gap junctions in the inner ear--it's not just about K⁺ recycling.

Normal development, function and repair of the sensory epithelia in the inner ear are all dependent on gap junctional intercellular communication. Mutations in the connexin genes GJB2 and GJB6 (encoding CX26 and CX30) result in syndromic and non-syndromic deafness via various mechanisms. Clinical vestibular defects, however, are harder to connect with connexin dysfunction. Cx26 and Cx30 proteins are widely expressed in the epithelial and connective tissues of the cochlea, where they may form homomeric or heteromeric gap junction channels in a cell-specific and spatiotemporally complex fashion. Despite the study of mutant channels and animal models for both recessive and dominant autosomal deafness, it is still unclear why gap junctions are essential for auditory function, and why Cx26 and Cx30 do not compensate for each other in vivo. Cx26 appears to be essential for normal development of the auditory sensory epithelium, but may be dispensable during normal hearing. Cx30 appears to be essential for normal repair following sensory cell loss. The specific modes of intercellular signalling mediated by inner ear gap junction channels remain undetermined, but they are hypothesised to play essential roles in the maintenance of ionic and metabolic homeostasis in the inner ear. Recent studies have highlighted involvement of gap junctions in the transfer of essential second messengers between the non-sensory cells, and have proposed roles for hemichannels in normal hearing. Here, we summarise the current knowledge about the molecular and functional properties of inner ear gap junctions, and about tissue pathologies associated with connexin mutations.

Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates.

The evolutionarily conserved planar cell polarity (PCP) pathway (or noncanonical Wnt pathway) drives several important cellular processes, including epithelial cell polarization, cell migration and mitotic spindle orientation. In vertebrates, PCP genes have a vital role in polarized convergent extension movements during gastrulation and neurulation. Here we show that mice with mutations in genes involved in Bardet-Biedl syndrome (BBS), a disorder associated with ciliary dysfunction, share phenotypes with PCP mutants including open eyelids, neural tube defects and disrupted cochlear stereociliary bundles. Furthermore, we identify genetic interactions between BBS genes and a PCP gene in both mouse (Ltap, also called Vangl2) and zebrafish (vangl2). In zebrafish, the augmented phenotype results from enhanced defective convergent extension movements. We also show that Vangl2 localizes to the basal body and axoneme of ciliated cells, a pattern reminiscent of that of the BBS proteins. These data suggest that cilia are intrinsically involved in PCP processes.

Developmental shaping of node of Ranvier geometry contributes to spike timing maturation in primary auditory afferents.

Sound is encoded by action potentials in spiral ganglion neurons (SGNs), the auditory afferents from the cochlea. Rapid action potential transmission along SGNs is crucial for quick reactions to sounds, and binaural differences in action potential arrival time at the SGN output synapses enable sound localization based on interaural time or phase differences. SGN myelination increases conduction speed but other cellular changes may contribute. We show that nodes of Ranvier along peripherally and centrally directed SGN neurites form around hearing onset, but peri-somatic nodes mature later. There follows an adjustment of nodal geometry, notably a decrease in length and increase in diameter. Computational modeling predicts this increases conduction speed by >4%, and that four additional myelin wraps would be required on internodes to achieve the same conduction speed increase. We propose that nodal geometry changes optimize signal conduction for mature sound coding and decrease the energy needed for myelination.

Alström Syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity.

Alström Syndrome is a life-threatening disease characterized primarily by numerous metabolic abnormalities, retinal degeneration, cardiomyopathy, kidney and liver disease, and sensorineural hearing loss. The cellular localization of the affected protein, ALMS1, has suggested roles in ciliary function and/or ciliogenesis. We have investigated the role of ALMS1 in the cochlea and the pathogenesis of hearing loss in Alström Syndrome. In neonatal rat organ of Corti, ALMS1 was localized to the basal bodies of hair cells and supporting cells. ALMS1 was also evident at the basal bodies of differentiating fibrocytes and marginal cells in the lateral wall. Centriolar ALMS1 expression was retained into maturity. In Alms1-disrupted mice, which recapitulate the neurosensory deficits of human Alström Syndrome, cochleae displayed several cyto-architectural defects including abnormalities in the shape and orientation of hair cell stereociliary bundles. Developing hair cells were ciliated, suggesting that ciliogenesis was largely normal. In adult mice, in addition to bundle abnormalities, there was an accelerated loss of outer hair cells and the progressive appearance of large lesions in stria vascularis. Although the mice progressively lost distortion product otoacoustic emissions, suggesting defects in outer hair cell amplification, their endocochlear potentials were normal, indicating the strial atrophy did not affect its function. These results identify previously unrecognized cochlear histopathologies associated with this ciliopathy that (i) implicate ALMS1 in planar cell polarity signaling and (ii) suggest that the loss of outer hair cells causes the majority of the hearing loss in Alström Syndrome.

Structural changes in the human stria vascularis induced by aminoglycosides and loop diuretics.

The human stria vascularis has been examined both by scanning and transmission electron microscopy in normal controls and from individuals who had received loop diuretics, aminoglycoside antibiotics or some combination of the two prior to their deaths. The tissues were preserved by perilymphatic perfusion of fixative within an hour of death and preservation was adequate. The normal ultrastructure is described and does not differ significantly from that found in experimental animals. The loop diuretics are associated with structural changes that cannot be distinguished from those found in animals treated with large doses of the same drugs. The aminoglycosides caused some changes, but the patients had been in renal failure and this probably contributed to the structural alterations. The combination of a loop diuretic and aminoglycoside was associated with a range of alterations from mild to severe. Overall, the three treatment groups had a series of ultrastructural changes resembling those found in animal models thereby justifying the use of experimental animals to predict human ototoxicity.

The timing of auditory sensory deficits in Norrie disease has implications for therapeutic intervention.

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.

Compartmentalized and signal-selective gap junctional coupling in the hearing cochlea.

Gap junctional intercellular communication (GJIC) plays a major role in cochlear function. Recent evidence suggests that connexin 26 (Cx26) and Cx30 are the major constituent proteins of cochlear gap junction channels, possibly in a unique heteromeric configuration. We investigated the functional and structural properties of native cochlear gap junctions in rats, from birth to the onset of hearing [postnatal day 12 (P12)]. Confocal immunofluorescence revealed increasing Cx26 and Cx30 expression from P0 to P12. Functional GJIC was assessed by coinjection of Lucifer yellow (LY) and Neurobiotin (NBN) during whole-cell recordings in cochlear slices. At P0, there was restricted dye transfer between supporting cells around outer hair cells. Transfer was more extensive between supporting cells around inner hair cells. At P8, there was extensive transfer of both dyes between all supporting cell types. By P12, LY no longer transferred between the supporting cells immediately adjacent to hair cells but still transferred between more peripheral cells. NBN transferred freely, but it did not transfer between inner and outer pillar cells. Freeze fracture further demonstrated decreasing GJIC between inner and outer pillar cells around the onset of hearing. These data are supportive of the appearance of signal-selective gap junctions around the onset of hearing, with specific properties required to support auditory function. Furthermore, they suggest that separate medial and lateral buffering compartments exist in the hearing cochlea, which are individually dedicated to the homeostasis of inner hair cells and outer hair cells.