Biomedical subjects

Yehoash Raphael

Publications and source records attributed to Yehoash Raphael.

35 records · Page 2Linked to original sources

PubMed2003

Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo.

Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells express Math1, and nonsensory cells do not. We set out to determine the outcome of overexpression of Math1 in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate that in vivo inoculation of adenovirus with the Math1 gene insert into the endolymph of the mature guinea pig cochlea results in Math1 overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells. Math1 overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo and that Math1 is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.

Adenoviridae↗

PubMed2003

Hearing and hair cells are protected by adenoviral gene therapy with TGF-beta1 and GDNF.

Glial cell line-derived neurotrophic factor (GDNF) overexpression in the inner ear can protect hair cells against degeneration induced by aminoglycoside ototoxicity. The protective efficiency of GDNF increases when it is combined with co-factors such as transforming growth factor beta1 (TGF-beta1), a ubiquitous cytokine. The aim of this study was to determine whether TGF-beta1 receptors are expressed in the inner ear and whether a cocktail of GDNF and TGF-beta1 transgenes provides enhanced protection of the inner ear against ototoxic trauma. Using RT-PCR analysis, we determined that both TGF-beta1 receptors, type 1 and 2 are present in rat cochlea. We co-inoculated two adenoviral vectors, one encoding human TGF-beta1 gene (Ad.TGF-beta1) and the other encoding human GDNF gene (Ad.GDNF) into guinea pig cochleae 4 days prior to injecting an ototoxic dose of aminoglycosides. Inoculated ears had better hearing and fewer missing inner hair cells after exposure to the aminoglycoside ototoxicity, as compared with controls and ears treated only with Ad.GDNF. Cochleae with TGF-beta1 overexpression exhibited fibrosis in the scala tympani regardless of the presence of GDNF. Our results suggest that the adenovirus-mediated overexpression of GDNF and TGF-beta1 can be used in combination to protect cochlear hair cells and hearing from ototoxic trauma.

Adenoviridae↗

PubMed2003

Prospects for gene therapy in hearing loss.

Deafness is the most common form of sensory impairment in humans. Depending on the age of onset, hearing impairment can affect oral language acquisition, cognitive development and psychosocial development. Here, we cover the latest advances in gene therapy for alleviating or preventing hearing loss. This review is not meant to be comprehensive, but to highlight some of the most recent developments in the field. Several recent reviews have described potential therapeutic approaches.

Cell Death↗

PubMed2003

Gene-based diagnostic and treatment methods for tinnitus.

The etiology of tinnitus combines hereditary and environmental factors. To help develop optimal therapies for tinnitus, it is necessary to characterize the genetic contributors to the pathophysiology and to design treatments at the level of the gene. Inner ear gene therapy involves delivery of genes into the vestibular or auditory portions of the inner ear for preventive or reparative therapies at the level of the sensory epithelium or the eighth nerve neurons. BDNF and GDNF are among the neurotrophic factors shown to be overexpressed with gene therapy and to protect the inner ear against trauma. Combined treatment with Ad.GDNF and electrical stimulation provided enhanced preservation of denervated spiral ganglion neurons. The use of viral vectors for gene therapy may involve side effects, including immune response to the viral proteins. Treatment with immunosuppressive medications can reduce the negative consequences of adenovirus-mediated gene therapy.

Adenoviruses, Human↗

PubMed2002

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation.

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line-derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad-GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad-GDNF or ES or both. Ad-GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES-treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad-GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES-treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF- and ES-mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. .

Animals↗

PubMed2002

Mutation of the novel gene Tmie results in sensory cell defects in the inner ear of spinner, a mouse model of human hearing loss DFNB6.

The recessive mutation at the mouse spinner (sr) locus results in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified the mutant locus responsible for this pathology. The affected gene (Tmie) lies within a 40 kb deletion in the original sr allele. In a newly identified allele, Tmie contains a nonsense mutation expected to truncate the C-terminal end of its product. The 153 amino acid protein encoded by the gene shows no similarity to other known proteins, and is predicted to contain a signal peptide and at least one transmembrane domain. Tmie transcripts were identified in several tissues, including the cochlea. Loss of function of Tmie results in postnatal alterations of sensory hair cells in the cochlea, including defects in stereocilia, the apical projections of hair cells that are important in mechanotransduction of sound. These morphological defects are associated with a profound failure to develop normal auditory function. Consistent with a conserved role for this gene in the cochlea, the genetic mapping data presented here support human TMIE as the gene affected at DFNB6, a non-syndromic hearing loss locus. The spinner mutant is thus a valuable model for insight into mechanisms of human deafness and development of sensory cell function.

Amino Acid Sequence↗

PubMed2002

WDR1 colocalizes with ADF and actin in the normal and noise-damaged chick cochlea.

Auditory hair cells of birds, unlike hair cells in the mammalian organ of Corti, can regenerate following sound-induced loss. We have identified several genes that are upregulated following such an insult. One gene, WDR1, encodes the vertebrate homologue of actin-interacting protein 1, which interacts with actin depolymerization factor (ADF) to enhance the rate of actin filament cleavage. We examined WDR1 expression in the developing, mature, and noise-damaged chick cochlea by in situ hybridization and immunocytochemistry. In the mature cochlea, WDR1 mRNA was detected in hair cells, homogene cells, and cuboidal cells, all of which contain high levels of F-actin. In the developing inner ear, WDR1 mRNA was detected in homogene cells and cuboidal cells by embryonic day 7, in the undifferentiated sensory epithelium by day 9, and in hair cells at embryonic day 16. We also demonstrated colocalization of WDR1, ADF, and F-actin in all three cell types in the normal and noise-damaged cochlea. Immediately after acoustic overstimulation, WDR1 mRNA was seen in supporting cells. These cells contribute to the structural integrity of the basilar papilla, the maintenance of the ionic barrier at the reticular lamina, and the generation of new hair cells. These results indicate that one of the immediate responses of the supporting cell after noise exposure is to induce WDR1 gene expression and thus to increase the rate of actin filament turnover. These results suggest that WDR1 may play a role either in restoring cytoskeletal integrity in supporting cells or in a cell signaling pathway required for regeneration.

Actin Depolymerizing Factors↗

PubMed2002

Transgene expression in neonatal mouse inner ear explants mediated by first and advanced generation adenovirus vectors.

The mouse serves as a valuable model for treatment leading to the prevention and therapy of inner ear disease. Transgenic correction of genetic inner ear disease in mice may help develop treatment for human genetic inner ear disease. In mutations involving hair cells (HCs) or supporting cells (SCs), it is necessary to insert the wild-type transgenes directly into these cells. We used inner ear explants to characterize the transgenic expression using adenovirus-mediated reporter genes (bacterial lacZ). The variable parameters were the age of the explants (P1-P5), the type of vector (first and advanced generation adenovirus) and the genotype of the mouse (wild-type versus shaker-2 mutant). Transduction of cochlear HCs was detected at P1 and in some of the P3 cochleae. Low efficiency transduction of SCs was observed in P1 explants, but the efficiency increased with age and reached high levels at P5. The pattern of transduction was similar regardless of the genotype and the type of vector used. The data demonstrate that differentiating HCs and SCs in mouse explants can be transduced by adenovirus vectors, suggesting that cultures of mouse ears are a valuable model for developing inner ear gene therapy protocols.

Adenoviruses, Human↗

PubMed2002

Gene transfer into supporting cells of the organ of Corti.

To utilize the rapidly accumulating genetic information for developing new therapeutic technologies for inner ear disease, it is necessary to design technologies for expressing transgenes in the inner ear, especially in the organ of Corti. We examined the outcome of an adenovirus gene transfer into the organ of Corti via the scala media in guinea pigs. The transgene insert is the bacterial lacZ gene driven by a cytomegalovirus promoter. We demonstrate that the inoculation is detrimental to the hair cells that surround the site of inoculation, but the supporting cells in the organ of Corti survive and retain the ability to express the reporter transgene beta-gal. The ability to deliver transgenes that are expressed in the supporting cells is an important step in the development of clinically applicable treatments that involve hair cell regeneration.

Adenoviridae↗

PubMed2002

Cochlear pathology, sensory cell death and regeneration.

Loss of cochlear hair cells leads to permanent hearing loss. Hair cells may degenerate due to hereditary or environmental causes, or a combination of the two. Cochlear supporting cells actively participate in the process of hair cell elimination and scar formation by rapidly expanding and sealing the reticular lamina, the barrier between endolymph and perilymph. This scarring process helps preserve the remaining hair cells and hearing. Anti-apoptotic agents, anti-oxidants and several growth factors have been shown to protect hair cells and hearing against environmental insults. Characterization of the genes that regulate the development of the inner ear and its response to trauma has been helpful in designing strategies for enhancing protection of the inner ear and for inducing hair cell regeneration. This chapter discusses the potential for some of these approaches.

Aging↗

PubMed2001

Differential Gene Expression Following Noise Trauma in Birds and Mammals.

Acoustic overstimulation has very different outcomes in birds and mammals. When noise exposure kills hair cells in birds, these cells can regenerate and hearing will recover. In mammals, however, the hair cell loss, and resulting hearing loss, is permanent. Changes in gene expression form the basis for important biological processes, including repair, regeneration, and plasticity. We are therefore using a battery of molecular approaches to identify and compare changes in gene expression following noise trauma in birds and mammals. Both differential display and subtractive hybridisation were used to identify genes whose expression increased in the chick basilar papilla immediately following exposure to an octave band noise (118 dB, centre frequency 1.5 kHz) for 4-6 hr. Among those upregulated genes were two involved in actin signalling: the CDC42 gene encoding a Rho GTPase, and WDR1, which encodes a protein involved in actin dynamics. A third gene, UBE3B, encodes an E3 ubiquitin ligase involved in protein turnover. A fourth gene encodes a cystein-rich secreted protein that may interact with calcium channels. To examine the mammalian response, gene microarrays on nylon membranes (Clontech Atlas Gene Arrays) were used to examine global changes in gene expression 30 minutes after TTS (110 dB broadband noise 50% duty cycle) or PTS (125 dB, 100% duty cycle) noise overstimulation (each for 90 minutes) in the rat cochlea. Several genes, including classic immediate early response genes such as c-fos, EGR1/NGFI-A, and NGFI-B, were upregulated at this early time point following the PTS exposure, but were not upregulated following the TTS exposure.

Journal Article↗

PubMed2001

Gene-based therapy for inner ear disease.

Environmental inner ear insults often lead to hair cell injury and loss. Therapeutic measures for the prevention of hair cell loss are currently limited. Several reports have demonstrated the applicability of growth factors for hair cell protection. The goal of the experiments presented here was to assess the protective capability of the human GDNF transgene against noise trauma in the guinea pig cochlea. The left ears of guinea pigs were inoculated with a recombinant adenovirus with a human GDNF insert (Ad.GDNF). Four days later, animals were exposed to noise trauma. One week later, animals were sacrificed and hair cells counted in the left (inoculated) and right (non-inoculated) ears. Auditory brainstem thresholds were measured before the inoculation and just prior to sacrifice. Control groups included inoculation with a reporter gene vector (Ad.lacZ) and Ad.GDNF in normal ears with no noise exposure. The results show that intracochlear inoculation with adenovirus into normal ears does not compromise hair cell counts and ABR thresholds. Both Ad.GDNF and Ad.lacZ vectors can protect the cochlear hair cells and hearing from the noise insult. The difference between the protection afforded by Ad.GDNF and that of the Ad.lacZ vector is not statistically significant. The mechanism of Ad.lacZ protection needs to be elucidated. The data demonstrate the general feasibility of gene therapy for over-expression of neurotrophic factors against noise trauma, and emphasize the complexity of the technique and the problems of variability between subjects.

Journal Article↗

PubMed2001

Absence of hair cell protection by exogenous FGF-1 and FGF-2 delivered to guinea pig cochlea in vivo.

Recent findings that glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and transforming growth factor a can protect the auditory hair cells from acoustic trauma or aminoglycoside ototoxicity in vivo raise the question of whether other neurotrophic factors can also protect the hair cells in vivo. Fibroblast growth factor-2 (FGF-2) can protect hair cells from neomycin ototoxicity in vitro, and in vivo study has shown upregulation of FGF receptor-3 in the cochlea following noise exposure, suggesting that some FGF family members might play a role in protection or repair of the cochlea from damage. We therefore examined if FGF-1 and FGF-2 chronically delivered to the cochlea prior to noise overstimulation can attenuate noise-induced hair cell damage in vivo under conditions in which GDNF and NT-3 were effective. Pigmented female guinea pigs underwent left scala tympani implantation of a microcannula attached to an osmotic pump filled with artificial perilymph only or containing FGFs (10 or 1 mg/ml FGF-1 or 10 mg/ml FGF-2). They were exposed to noise (4 kHz octave band, 115 dB SPL, 5 hr) 4 days after surgery. Threshold shifts 10 days postexposure were essentially equivalent at all frequencies tested across different treatment groups. No significant difference in threshold shifts was observed between the treated and untreated ears in any of the groups. The extent of hair cell damage was also comparable among the different treatment groups. These findings indicate that exogenous FGF-1 or FGF-2 does not influence noise-induced hair cell damage under the experimental conditions used in this study, suggesting that these FGFs are not good candidates as auditory hair cell protectors in vivo.

Journal Article↗

PubMedDate not supplied

From gene identification to gene therapy.

Inner ear disease due to hair cell loss is common, and no restorative treatments for the balance and hearing impairment are currently available. To develop clinical means for enhancing protection and regeneration in the inner ear, it is necessary to understand the molecular basis for hereditary and acquired deafness and vestibular disorders. One approach is to identify and characterize genes that regulate protection or repair in other systems. For that purpose, we have used the differential display assay and compared gene expression between normal and acoustically traumatized inner ears of chicks. Several chick cDNAs that were identified are considered as candidates for roles in the reparative process that follows trauma in the basilar papilla. The mammalian vestibular epithelium has a limited regenerative capability. To identify genes that may participate in the regenerative response, we have used gene arrays profiling, comparing normal to drug-traumatized vestibular epithelia. We identified several genes that are differentially expressed in traumatized vestibular epithelium, including several insulin-like growth factor-I binding proteins. To use this molecular knowledge for enhancing protection and repair in the organ of Corti, it is necessary to overexpress the genes of choice in the inner ear. Using viral-mediated gene transfer, we overexpressed transgenic glial cell line-derived neurotrophic factor and demonstrated a robust protective effect against acoustic and ototoxic inner ear trauma. Future identification of the genes that are important for protection and regeneration, along with improved gene transfer technology, will allow the use of gene therapy for treating hereditary and environmental inner ear disease.

Animals↗

PubMedDate not supplied

The cytocaud: a hair cell pathology in the waltzing Guinea pig.

The waltzing guinea pig displays severe inner ear dysfunction that involves both an auditory and a vestibular manifestation. The aim of this study was to characterize a pathological tail-like extension of the vestibular hair cells, the cytocaud. Our data suggest that nearly all type I hair cells in the waltzing guinea pig have cytocauds, which appear as membrane-bound tails containing mitochondria and cytoplasm that proceed in a basal direction toward the basement membrane. The extensions either attach to the basement membrane or penetrate it, and further proceed into the extracellular matrix. A core made of a thick and long (30 microm) actin-rich structure supports the slender long process. The actin core has cross-links that are periodically placed along the length of the cytocaud. Our data suggest that the cytocauds in vestibular hair cells of the waltzing guinea pig are highly organized structures associated with a failure to detach from the basement membrane.

Actins↗

PubMedDate not supplied

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.

Adenoviridae Infections↗

PubMedDate not supplied

Adenovirus-mediated expression of brain-derived neurotrophic factor protects spiral ganglion neurons from ototoxic damage.

Hair cell loss, the most common cause of deafness, is often associated with auditory nerve degeneration. Our goal was to determine the influence of combined ciliary-derived neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) gene therapy on the survival of spiral ganglion neurons (SGNs) after elimination of inner hair cells in the mature guinea pig ear. Seven days after bilateral deafening, a 5-microl suspension of CNTF and/or BDNF adenovirus vectors was injected into the left scala tympani through the round window. Animals were sacrificed 28 days after deafening, and their inner ears were prepared for SGN counts. The SGN counts revealed that BDNF alone and the combined CNTF and BDNF treatment significantly enhanced SGN survival. CNTF did not significantly enhance the protective effect of BDNF. These data present possible strategies for enhancing SGN survival in cochlear implant procedures.

Adenoviridae↗