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.

Characterizing human vestibular sensory epithelia for experimental studies: new hair bundles on old tissue and implications for therapeutic interventions in ageing.

Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is loss of sensory cells from the vestibular sensory epithelia of the inner ear. However, inaccessibility of inner ear tissue in humans severely restricts possibilities for experimental manipulation to develop therapies to ameliorate this loss. We provide a structural and functional analysis of human vestibular sensory epithelia harvested at trans-labyrinthine surgery. We demonstrate the viability of the tissue and labeling with specific markers of hair cell function and of ion homeostasis in the epithelium. Samples obtained from the oldest patients revealed a significant loss of hair cells across the tissue surface, but we found immature hair bundles present in epithelia harvested from patients >60 years of age. These results suggest that the environment of the human vestibular sensory epithelium could be responsive to stimulation of developmental pathways to enhance hair cell regeneration, as has been demonstrated successfully in the vestibular organs of adult mice.

In vitro preparation of newt inner ear sensory epithelia as a model for repair and regeneration.

The sensory "hair" cells of the inner ear transform sound energy into electrical signals, but are readily lost through aging, excessive noise, and ototoxic agents. The newt provides an excellent model in which to explore regeneration and whilst loss of hair cells from inner ear epithelia does not require whole organ regeneration, new hair cells are generated from differentiated supporting cells that transdifferentiate without an intervening mitotic event. Here we describe the methods for maintaining the sensory epithelia in long term culture; for the use of the aminoglycoside, gentamicin, to kill the hair cells; and for the examination of the tissue by electron microscopy or fluorescence microscopy. Demembranation of the epithelium reveals the underlying ultrastructure of the tissue for examination by scanning electron microscopy (SEM) and is a technique that can be utilized with immunogold labelling.

Hearing loss in a mouse model of 22q11.2 Deletion Syndrome.

22q11.2 Deletion Syndrome (22q11DS) arises from an interstitial chromosomal microdeletion encompassing at least 30 genes. This disorder is one of the most significant known cytogenetic risk factors for schizophrenia, and can also cause heart abnormalities, cognitive deficits, hearing difficulties, and a variety of other medical problems. The Df1/+ hemizygous knockout mouse, a model for human 22q11DS, recapitulates many of the deficits observed in the human syndrome including heart defects, impaired memory, and abnormal auditory sensorimotor gating. Here we show that Df1/+ mice, like human 22q11DS patients, have substantial rates of hearing loss arising from chronic middle ear infection. Auditory brainstem response (ABR) measurements revealed significant elevation of click-response thresholds in 48% of Df1/+ mice, often in only one ear. Anatomical and histological analysis of the middle ear demonstrated no gross structural abnormalities, but frequent signs of otitis media (OM, chronic inflammation of the middle ear), including excessive effusion and thickened mucosa. In mice for which both in vivo ABR thresholds and post mortem middle-ear histology were obtained, the severity of signs of OM correlated directly with the level of hearing impairment. These results suggest that abnormal auditory sensorimotor gating previously reported in mouse models of 22q11DS could arise from abnormalities in auditory processing. Furthermore, the findings indicate that Df1/+ mice are an excellent model for increased risk of OM in human 22q11DS patients. Given the frequently monaural nature of OM in Df1/+ mice, these animals could also be a powerful tool for investigating the interplay between genetic and environmental causes of OM.

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.

Defining the cellular environment in the organ of Corti following extensive hair cell loss: a basis for future sensory cell replacement in the Cochlea.

BACKGROUND: Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss. METHODOLOGY/PRINCIPAL FINDINGS: Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca's, suggesting genetic background influences the rate of re-organisation. CONCLUSIONS/SIGNIFICANCE: The lack of dedifferentiation amongst supporting cells and their replacement by cells from the outer side of the organ of Corti are factors that may need to be considered in any attempt to promote endogenous hair cell regeneration. The variability of the cellular environment along an individual cochlea arising from patch-like generation of flat epithelium, and the possible variability between individuals resulting from genetic influences on the rate at which remodelling occurs may pose challenges to devising the appropriate regenerative therapy for a deaf patient.

A congenital activating mutant of WASp causes altered plasma membrane topography and adhesion under flow in lymphocytes.

Leukocytes rely on dynamic actin-dependent changes in cell shape to pass through blood vessels, which is fundamental to immune surveillance. Wiskott-Aldrich Syndrome protein (WASp) is a hematopoietic cell-restricted cytoskeletal regulator important for modulating cell shape through Arp2/3-mediated actin polymerization. A recently identified WASp(I294T) mutation was shown to render WASp constitutively active in vivo, causing increased filamentous (F)-actin polymerization, high podosome turnover in macrophages, and myelodysplasia. The aim of this study was to determine the effect of WASp(I294T) expression in lymphocytes. Here, we report that lymphocytes isolated from a patient with WASp(I294T), and in a cellular model of WASp(I294T), displayed abnormal microvillar architecture, associated with an increase in total cellular F-actin. Microvillus function was additionally altered as lymphocytes bearing the WASp(I294T) mutation failed to roll normally on L-selectin ligand under flow. This was not because of defects in L-selectin expression, shedding, cytoskeletal anchorage, or membranal positioning; however, under static conditions of adhesion, WASp(I294T)-expressing lymphocytes exhibited altered dynamic interaction with L-selectin ligand, with a significantly reduced rate of adhesion turnover. Together, our results demonstrate that WASp(I294T) significantly affects lymphocyte membrane topography and L-selectin-dependent adhesion, which may be linked to defective hematopoiesis and leukocyte function in affected patients.

Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear.

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.

MicroRNAs and regeneration: Let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt.

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.

Hair cell regeneration in sensory epithelia from the inner ear of a urodele amphibian.

The capacity of urodele amphibians to regenerate a variety of body parts is providing insight into mechanisms of tissue regeneration in vertebrates. In this study the ability of the newt, Notophthalmus viridescens, to regenerate inner ear hair cells in vitro was examined. Intact otic capsules were maintained in organotypic culture. Incubation in 2 mM gentamicin for 48 hours resulted in ablation of all hair cells from the saccular maculae. Thus, any hair cell recovery was not due to repair of damaged hair cells. Immature hair cells were subsequently observed at approximately 12 days posttreatment. Their number increased over the following 7-14 days to reach approximately 30% of the normal number. Following incubation of damaged tissue with bromodeoxyuridine (BrdU), labeled nuclei were confined strictly within regions of hair cell loss, indicating that supporting cells entered S-phase. Double labeling of tissue with two different hair cell markers and three different antibodies to BrdU in various combinations, however, all showed that the nuclei of cells that labeled with hair cell markers did not label for BrdU. This suggested that the new hair cells were not derived from those cells that had undergone mitosis. When mitosis was blocked with aphidicolin, new hair cells were still generated. The results suggest that direct phenotypic conversion of supporting cells into hair cells without an intervening mitotic event is a major mechanism of hair cell regeneration in the newt. A similar mechanism has been proposed for the hair cell recovery phenomenon observed in the vestibular organs of mammals.

Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate.

How do organisms respond adaptively to environmental stress? Although some gene-specific responses have been explored, others remain to be identified, and there is a very poor understanding of the system-wide integration of response, particularly in complex, multitissue animals. Here, we adopt a transcript screening approach to explore the mechanisms underpinning a major, whole-body phenotypic transition in a vertebrate animal that naturally experiences extreme environmental stress. Carp were exposed to increasing levels of cold, and responses across seven tissues were assessed by using a microarray composed of 13,440 cDNA probes. A large set of unique cDNAs (approximately 3,400) were affected by cold. These cDNAs included an expression signature common to all tissues of 252 up-regulated genes involved in RNA processing, translation initiation, mitochondrial metabolism, proteasomal function, and modification of higher-order structures of lipid membranes and chromosomes. Also identified were large numbers of transcripts with highly tissue-specific patterns of regulation. By unbiased profiling of gene ontologies, we have identified the distinctive functional features of each tissue's response and integrate them into a comprehensive view of the whole-body transition from one strongly adaptive phenotype to another. This approach revealed an expression signature suggestive of atrophy in cooled skeletal muscle. This environmental genomics approach by using a well studied but nongenomic species has identified a range of candidate genes endowing thermotolerance and reveals a previously unrecognized scale and complexity of responses that impacts at the level of cellular and tissue function.