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.

A comparative study of gland cells implicated in the nerve dependence of salamander limb regeneration.

Limb regeneration in salamanders proceeds by formation of the blastema, a mound of proliferating mesenchymal cells surrounded by a wound epithelium. Regeneration by the blastema depends on the presence of regenerating nerves and in earlier work it was shown that axons upregulate the expression of newt anterior gradient (nAG) protein first in Schwann cells of the nerve sheath and second in dermal glands underlying the wound epidermis. The expression of nAG protein after plasmid electroporation was shown to rescue a denervated newt blastema and allow regeneration to the digit stage. We have examined the dermal glands by scanning and transmission electron microscopy combined with immunogold labelling of the nAG protein. It is expressed in secretory granules of ductless glands, which apparently discharge by a holocrine mechanism. No external ducts were observed in the wound epithelium of the newt and axolotl. The larval skin of the axolotl has dermal glands but these are absent under the wound epithelium. The nerve sheath was stained post-amputation in innervated but not denervated blastemas with an antibody to axolotl anterior gradient protein. This antibody reacted with axolotl Leydig cells in the wound epithelium and normal epidermis. Staining was markedly decreased in the wound epithelium after denervation but not in the epidermis. Therefore, in both newt and axolotl the regenerating axons induce nAG protein in the nerve sheath and subsequently the protein is expressed by gland cells, under (newt) or within (axolotl) the wound epithelium, which discharge by a holocrine mechanism. These findings serve to unify the nerve dependence of limb regeneration.

Gap junctions in the inner ear: comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals.

The distribution and size of gap junctions (GJ) in the sensory epithelia of the inner ear have been examined in a reptile (gecko), birds (chicken and owl), and mammals (mouse, guinea pig, gerbil, and bat), and the connexin composition of GJs in the mammalian inner ear has been assessed. Freeze fracture revealed a common pattern of GJ distribution in auditory and vestibular sensory epithelia in the different vertebrate classes. In all these tissues, GJs are numerous, often occupying more than 25% of the plasma membrane area of supporting cells and sometimes composed of more than 100,000 channels. Screening for 12 members of the connexin family in the mammalian inner ear by RT-PCR, Western blotting, and immunohistochemistry revealed four connexin isotypes, cx26, cx30, cx31, and cx43, in the cochlea and three, cx26, cx30, and cx43, in the vestibular organs. With antibodies characterised for their specificity, cx26 and cx30 colocalised in supporting cells of the organ of Corti, in the basal cell region of the stria vascularis, and in type 1 fibrocytes of the spiral ligament. No other connexin was detected in these regions. Cx31 was localised among type 2 fibrocytes below the spiral prominence, a region where cx30 was not expressed and cx26 expression appeared to be low. Cx43 was detected only in the region of "tension fibrocytes" lining the inner aspect of the otic capsule. This suggests separate functional compartments in the cochlea. In addition to cx26 and cx30, cx43 was detected in supporting cells of the vestibular sensory epithelia. Where cx26 and cx30 were colocalised, double immunogold labelling of thin sections showed both cx26 and cx30 evenly distributed in individual GJ plaques, a pattern consistent with the presence of heteromeric connexons. Coimmunoprecipitation of cochlear membrane proteins solubilised with a procedure that preserves the oligomeric structure of connexons confirmed the presence of heteromeric cx26/cx30 connexons. Heteromeric cx26/cx30 connexons may be unique to the inner ear, which could be one factor underlying the non-syndromic character of the deafness caused by mutations in cx26.

The Membrane Properties of Cochlear Root Cells are Consistent with Roles in Potassium Recirculation and Spatial Buffering.

Auditory transduction, amplification, and hair cell survival depend on the regulation of extracellular [K(+)] in the cochlea. K(+) is removed from the vicinity of sensory hair cells by epithelial cells, and may be distributed through the epithelial cell syncytium, reminiscent of "spatial buffering" in glia. Hypothetically, K(+) is then transferred from the epithelial syncytium into the connective tissue syncytium within the cochlear lateral wall, enabling recirculation of K(+) back into endolymph. This may involve secretion of K(+) from epithelial root cells, and its re-uptake via transporters into spiral ligament fibrocytes. The molecular basis of this secretion is not known. Using a combination of approaches we demonstrated that the resting conductance in guinea pig root cells was dominated by K(+) channels, most likely composed of the Kir4.1 subunit. Dye injections revealed extensive intercellular gap junctional coupling, and delineated the root cell processes that penetrated the spiral ligament. Following uncoupling using 1-octanol, individual cells had Ba(2+)-sensitive weakly rectifying currents. In the basal (high-frequency encoding) cochlear region K(+) loads are predicted to be the highest, and root cells in this region had the largest surface area and the highest current density, consistent with their role in K(+) secretion. Kir4.1 was localized within root cells by immunofluorescence, and specifically to root cell process membranes by immunogold labeling. These results support a role for root cells in cochlear K(+) regulation, and suggest that channels composed of Kir4.1 subunits may mediate K(+) secretion from the epithelial gap junction network.

Rapid hair cell loss: a mouse model for cochlear lesions.

In comparison to other mammals, mice have proved extremely resistant to aminoglycoside-induced hair cell ablation in vivo. In this paper we examine the pattern and extent of cochlear lesions rapidly induced with a combination of a single dose of aminoglycoside (kanamycin) followed by a loop diuretic (bumetanide). With this protocol, the vestibular system was unaffected, but in the cochlea, there was extensive loss of outer hair cells (OHC) that commenced in the basal coil and progressed apically so that, by 48 h, OHC loss was almost complete. TUNEL-positive nuclei and activated caspase-3 labeling demonstrated that most OHC died via a classical apoptotic pathway. However, scattered debris within the OHC region suggested that many apoptotic cells ruptured prior to completion of apoptosis. Following lesion repair, supporting cells retained characteristics of differentiated cells but positional shift occurred. In comparison to OHC loss, inner hair cell (IHC) death was delayed and only observed in 50% of all cochleae examined even after extensive reorganization of the tissue. The coadmininstration of diuretic with FM1-43, used as a tracer for aminoglycoside uptake, indicated entry into IHC as readily as OHC, suggesting that the differential response to aminoglycoside was not due to differential uptake. Where IHC death was ongoing, there were indications of different modes of cell death: cells with morphological features of autophagy, necrosis, and apoptosis were apparent. In addition to damage to the organ of Corti, there was a significant and progressive decrease in strial thickness beginning as early as 7 days posttreatment. This was due predominantly to degeneration of marginal cells. The strial pathology resembled that reported after noise damage and with aging. This in vivo protocol provides a robust model in which to obtain extensive OHC loss in the mature cochleae of mice and is a means with which to examine different aspects of cochlear pathology in transgenic or mutant strains.