Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

[The study of AAV9 expression in cochleae of mice at different ages].

Objective: To determine the extent of transfection and expression of adeno-associated virus (AAV) serotype 9 (AAV9) in the cochleae of mice at different ages. Methods: AAV9-green fluorescent protein (GFP) was inoculated into the cochlea of mice via the round window membrane (RWM) or through cochleostomy at different ages. Four groups were divided according to ages and injection sites: P1SM group, AAV9-GFP was delivered to the scala media by cochleostomy at postnatal day 1; P1RW group, AAV9-GFP was delivered to the scala tympani via RWM at postnatal day 1; P9RW group: AAV9-GFP was injected through RWM at postnatal day 9; and P30RW group, adult mice (P30) were injected through RWM. GFP expression in cochlear whole mount was analyzed and auditory brainstem response (ABR) tests were conducted one month after virus injection (for each animal, only left cochlea was injected and the right side was used as a control). GraphPad Prism 5 statistical software was used for data analysis. Results: All of inner hair cells (IHCs) and most of outer hair cells (OHCs) were transfected via two approaches at P1 injection. There was no significant difference in ABR threshold between injected ears and untreated ears (P>0.05). All of the IHCs and parts of OHCs (69% in apical turn) were transfected via RWM at P9. The strongest GFP expression was observed near the apical turn. Cochlear inoculation via RWM at P30 led to transgene expression in only IHCs. The ABR threshold of injected ears in P9RW group and P30RW group was significantly higher than that of contralateral ears (P<0.01). Conclusions: AAV9 can be highly expressed in the inner and outer hair cells of the cochlea and hearing sensitivity can be preserved if virus injections are performed in neonatal mice. After AAV9 is transfected into the inner ear of adult mice, it is only expressed in the inner hair cells, which leads to the increase of the ABR response threshold of mice. Transfection efficiency is significant higher in neonate mice than in P9 and adult mice.

Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection.

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1). The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells. Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium. Supporting cells are also important mediators of hair cell survival and death. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Prox1 interacts with Atoh1 and Gfi1, and regulates cellular differentiation in the inner ear sensory epithelia.

Inner ear hair cells and supporting cells arise from common precursors and, in mammals, do not show phenotypic conversion. Here, we studied the role of the homeodomain transcription factor Prox1 in the inner ear sensory epithelia. Adenoviral-mediated Prox1 transduction into hair cells in explant cultures led to strong repression of Atoh1 and Gfi1, two transcription factors critical for hair cell differentiation and survival. Luciferase assays showed that Prox1 can repress transcriptional activity of Gfi1 independently of Atoh1. Prox1 transduction into cochlear outer hair cells resulted in degeneration of these cells, consistent with the known phenotype of Gfi1-deficient mice. These results together with the widespread expression of endogenous Prox1 within the population of inner ear supporting cells point to the role for Prox1 in antagonizing the hair cell phenotype in these non-sensory cells. Further, in vivo analyses of hair cells from Gfi1-deficient mice suggest that the cyclin-dependent kinase inhibitor p57(Kip2) mediates the differentiation- and survival-promoting functions of Gfi1. These data reveal novel gene interactions and show that these interactions regulate cellular differentiation within the inner ear sensory epithelia. The data point to the tight regulation of phenotypic characteristics of hair cells and supporting cells.

An in vitro model system to study gene therapy in the human inner ear.

The confined fluid-filled labyrinth of the human inner ear presents an opportunity for introduction of gene therapy reagents designed to treat hearing and balance dysfunction. Here we present a novel model system derived from the sensory epithelia of human vestibular organs and show that the tissue can survive up to 5 days in vitro. We generated organotypic cultures from 26 human sensory epithelia excised at the time of labyrinthectomy for intractable Meniere's disease or vestibular schwannoma. We applied multiply deleted adenoviral vectors at titers between 10(5) and 10(8) viral particles/ml directly to the cultures for 4-24 h and examined the tissue 12-96 h post-transfection. We noted robust expression of the exogenous transgene, green fluorescent protein (GFP), in hair cells and supporting cells suggesting both were targets of adenoviral transfection. We also transfected cultures with a vector that carried the genes for GFP and KCNQ4, a potassium channel subunit that causes dominant-progressive hearing loss when mutated. We noted a positive correlation between GFP fluorescence and KCNQ4 immunolocalization. We conclude that our in vitro model system presents a novel and effective experimental paradigm for evaluation of gene therapy reagents designed to restore cellular function in patients who suffer from inner ear disorders.

A glucocorticoid reduces adverse effects of adenovirus vectors in the cochlea.

Gene transfer using a recombinant adenovirus is a powerful tool for research and clinical applications, but its cytotoxicity and immune response limit its use, especially when repeated application of the vector is necessary. This study investigated the effects of dexamethasone (DEX)-induced immunosuppression on the outcome of adenovirus gene transfer in guinea pig inner ears. Animals received DEX for 29 days. Their inner ear was inoculated with 5 micro l of adenovirus vector twice, on days 5 and 26. Auditory brainstem response was measured on days 1, 8 and 29. The animals were sacrificed on day 29, and reporter gene expression was evaluated. In control animals that received no DEX, postinoculation threshold shifts and lesions in the organ of Corti were observed and reporter gene expression was absent. In contrast, DEX-treated ears were largely protected, and transduction of inner ear cells was readily apparent. These data demonstrate that immunosuppressive treatment can reduce the negative consequences of repeated adenovirus-mediated gene therapy.

Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer.

Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.

To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.