RESUMO
Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5' and the other the 3' portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea of Otof-/- mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5' and 3' cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.
Assuntos
Surdez/terapia , Dependovirus/genética , Terapia Genética , Proteínas de Membrana/genética , Animais , Surdez/genética , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Células Ependimogliais/metabolismo , Proteínas de Membrana/biossíntese , Retina/metabolismo , Síndromes de Usher/metabolismo , Animais , Glicosilação , Humanos , Hibridização In Situ , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , RNA Mensageiro/genética , Síndromes de Usher/patologiaRESUMO
Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.
Assuntos
Síndromes de Usher/genética , Síndromes de Usher/terapia , Animais , Animais Recém-Nascidos , DNA Complementar/administração & dosagem , DNA Complementar/genética , Dependovirus/genética , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Terapia Genética/métodos , Vetores Genéticos , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Síndromes de Usher/fisiopatologia , Vestíbulo do Labirinto/patologia , Vestíbulo do Labirinto/fisiopatologiaRESUMO
Saposin B (Sap B) is an essential activator protein for arylsulfatase A in the hydrolysis of sulfatide, a lipid component of myelin. To study Sap B's role in hearing and balance, a Sap B-deficient (B(-/-)) mouse was evaluated. At both light and electron microscopy (EM) levels, inclusion body accumulation was seen in satellite cells surrounding spiral ganglion (SG) neurons from postnatal month 1 onward, progressing into large vacuoles preceding satellite cell degeneration, and followed by SG degeneration. EM also revealed reduced or absent myelin sheaths in SG neurons from postnatal month 8 onwards. Hearing loss was initially seen at postnatal month 6 and progressed thereafter for frequency-specific stimuli, whereas click responses became abnormal from postnatal month 13 onward. The progressive hearing loss correlated with the accumulation of inclusion bodies in the satellite cells and their subsequent degeneration. Outer hair cell numbers and efferent function measures (distortion product otoacoustic emissions and contralateral suppression) were normal in the B(-/-) mice throughout this period. Alcian blue staining of SGs demonstrated that these inclusion bodies corresponded to sulfatide accumulation. In contrast, changes in the vestibular system were much milder, but caused severe physiologic deficits. These results demonstrate that loss of Sap B function leads to progressive sulfatide accumulation in satellite cells surrounding the SG neurons, leading to satellite cell degeneration and subsequent SG degeneration with a resultant loss of hearing. Relative sparing of the efferent auditory and vestibular neurons suggests that alternate glycosphingolipid metabolic pathways predominate in these other systems.
Assuntos
Transtornos da Audição/etiologia , Leucodistrofia Metacromática/complicações , Leucodistrofia Metacromática/genética , Degeneração Neural/etiologia , Saposinas/deficiência , Células Satélites Perineuronais/patologia , Gânglio Espiral da Cóclea/patologia , Estimulação Acústica , Animais , Morte Celular/genética , Cóclea/metabolismo , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Lateralidade Funcional , Testes Auditivos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Emissões Otoacústicas Espontâneas/genética , Saposinas/genética , Gânglio Espiral da Cóclea/ultraestrutura , Natação/psicologiaRESUMO
Achalasia is a motility disorder of the esophagus in which the lower esophageal sphincter fails to relax. Megaesophagus is a rare complication of achalasia characterized by severe dilatation of the esophagus, often indicative of end-stage achalasia. Typical presenting symptoms include dysphagia, nausea, vomiting, weight loss, and chest pain. The majority of patients with achalasia typically have excellent outcomes after surgical intervention with Heller myotomy. We discuss an interesting case of unsuccessful surgical intervention and hypothesize the reason for its failure in our patient.
RESUMO
The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Perda Auditiva Neurossensorial/genética , Convulsões/genética , Estimulação Acústica/métodos , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Eletroencefalografia/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Células Ciliadas Auditivas/metabolismo , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/patologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Neurônios/patologia , Neurônios/ultraestrutura , Quinoxalinas/farmacologia , Reflexo de Sobressalto/fisiologia , Convulsões/etiologia , Gânglio Espiral da Cóclea/patologiaRESUMO
PURPOSE OF REVIEW: This review highlights recent advances in cochlear gene therapy over the past several years. Cochlear gene therapy has undergone tremendous advances over the past decade. Beginning with some groundbreaking work in 2005 documenting hair cell regeneration using virally mediated delivery of the mouse atonal 1 gene, gene therapy is now being explored as a possible treatment for a variety of causes of hearing loss. RECENT FINDINGS: Recent advances in cochlear gene therapy include improved methods of gene delivery with a better delineation of viral vectors that are suitable for this purpose, additional improvements in hair cell regeneration, and directed research toward autoimmune hearing loss, ototoxicity, spiral ganglion survival, and genetic forms of hearing loss. SUMMARY: If successful, cochlear gene therapy will dramatically alter our ability to treat a variety of forms of acquired and genetic hearing loss.
Assuntos
Cóclea/fisiopatologia , Doenças Cocleares/genética , Doenças Cocleares/terapia , Terapia Genética/métodos , Perda Auditiva/genética , Perda Auditiva/terapia , Animais , Cóclea/patologia , Técnicas de Transferência de Genes , Células Ciliadas Auditivas/fisiologia , Perda Auditiva/etiologia , Humanos , Regeneração/fisiologiaRESUMO
Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-ß (TGFß)-responsive pathway that controls osteoblast differentiation. Deregulated TGFß or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/â» mice, inhibition of TGFß signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.
Assuntos
Condução Óssea , Matriz Óssea/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Desenvolvimento Ósseo/fisiologia , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/metabolismo , Modelos Animais de Doenças , Módulo de Elasticidade , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The goal of this study was to determine the role of an influx copper transporter, CTR1, in the ototoxicity induced by cisplatin, a potent anticancer platinum analog used in the treatment of a variety of solid tumors. As determined through reverse transcriptase-PCR (RT-PCR), quantitative RT-PCR, Western blot, and immunohistochemistry, mouse CTR1 (Ctr1) was found to be abundantly expressed and highly localized at the primary sites of cisplatin toxicity in the inner ear, mainly outer hair cells (OHCs), inner hair cells, stria vascularis, spiral ganglia, and surrounding nerves in the mouse cochlea. A CTR1 substrate, copper sulfate, decreased the uptake and cytotoxicity of cisplatin in HEI-OC1, a cell line that expresses many molecular markers reminiscent of OHCs. Small interfering RNA-mediated knockdown of Ctr1 in this cell line caused a corresponding decrease in cisplatin uptake. In mice, intratympanic administration of copper sulfate 30 min before intraperitoneal administration of cisplatin was found to prevent hearing loss at click stimulus and 8, 16, and 32 kHz frequencies. To date, the utility of cisplatin remains severely limited because of its ototoxic effects. The studies described in this report suggest that cisplatin-induced ototoxicity and cochlear uptake can be modulated by administration of a CTR1 inhibitor, copper sulfate. The possibility of local administration of CTR1 inhibitors during cisplatin therapy as a means of otoprotection is thereby raised.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cisplatino/farmacologia , Cóclea/metabolismo , Perda Auditiva/metabolismo , Estimulação Acústica , Animais , Western Blotting , Proteínas de Transporte de Cátions/genética , Contagem de Células , Linhagem Celular , Cóclea/efeitos dos fármacos , Transportador de Cobre 1 , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Perda Auditiva/induzido quimicamente , Humanos , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adeno-associated virus (AAV)-mediated gene therapy has evolved from the bench to the bedside, and is now considered the therapy of choice for certain inherited diseases. AAVs are attractive vectors for several reasons: they are nonpathogenic, result in long-term transgene expression, have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms allowing the targeting of specific cell types. However, one of the greatest limitations of AAVs is the limited genome-packaging capacity of â¼4.7 kb. Given that numerous diseases are caused by mutations in genes with coding sequences exceeding this capacity, packaging into a single AAV capsid is currently unfeasible for larger genes. Taking advantage of the AAV genome's ability to concatemerize, multiple strategies have been explored to overcome the size limit of AAV vectors. One strategy is to split large transgenes into two or three parts, generating dual or triple AAV vectors. Coinfection of a cell with these two or three AAVs will then, through a variety of mechanisms, result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector. This review: 1) documents AAV dual and triple vector strategies currently employed in a variety of tissues, and highlights the advantages and disadvantages of each method; 2) describes the first successful studies using the dual vector approach to restore hearing and prevent deafness in a mouse model of non-syndromic deafness due to absence of the otoferlin protein function, and the implications of these findings for the future of gene therapy in the human inner ear; and 3) highlights additional different deafness genes that could be potential future targets for gene therapy using the dual vector approach.
Assuntos
Dependovirus , Orelha Interna , Animais , Surdez , Dependovirus/genética , Vetores Genéticos , Proteínas de Membrana , TransgenesRESUMO
Hearing impairment is a major health and economic concern worldwide. Currently, the cochlear implant (CI) is the standard of care for remediation of severe to profound hearing loss, and in general, contemporary CIs are highly successful. But there is great variability in outcomes among individuals, especially in children, with many CI users deriving much less or even marginal benefit. Much of this variability is related to differences in auditory nerve survival, and there has been substantial interest in recent years in exploring potential therapies to improve survival of the cochlear spiral ganglion neurons (SGN) after deafness. Preclinical studies using osmotic pumps and other approaches in deafened animal models to deliver neurotrophic factors (NTs) directly to the cochlea have shown promising results, especially with Brain-Derived Neurotrophic Factor (BDNF). More recent studies have focused on the use of NT gene therapy to force expression of NTs by target cells within the cochlea. This could provide the means for a one-time treatment to promote long-term NT expression and improve neural survival after deafness. This review summarizes the evidence for the efficacy of exogenous NTs in preventing SGN degeneration after hearing loss and reviews the animal research to date suggesting that NT gene therapy can elicit long-term NT expression in the cochlea, resulting in significantly improved SGN and radial nerve fiber survival after deafness. In addition, we discuss NT gene therapy in other non-auditory applications and consider some of the remaining issues with regard to selecting optimal vectors, timing of treatment, and place/method of delivery, etc. that must be resolved prior to considering clinical application.
Assuntos
Surdez , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Surdez/genética , Surdez/terapia , Terapia Genética , Humanos , Neurônios , Neurotrofina 3/genética , Gânglio Espiral da CócleaRESUMO
The consequences of neonatal white matter injury are devastating and represent a major societal problem as currently there is no cure. Prematurity, low weight birth and maternal pre-natal infection are the most frequent causes of acquired myelin deficiency in the human neonate leading to cerebral palsy and cognitive impairment. In the developing brain, oligodendrocyte (OL) maturation occurs perinatally, and immature OLs are particularly vulnerable. Cell replacement therapy is often considered a viable option to replace progenitors that die due to glutamate excitotoxicity. We previously reported directed specification and mobilization of endogenous committed and uncommitted neural progenitors by the combination of transferrin and insulin growth factor 1 (TSC1). Here, considering cell replacement and integration as therapeutic goals, we examined if OL progenitors (OLPs) grafted into the brain parenchyma of mice that were subjected to an excitotoxic insult could rescue white matter injury. For that purpose, we used a well-established model of glutamate excitotoxic injury. Four-day-old mice received a single intraparenchymal injection of the glutamate receptor agonist N-methyl-D-aspartate alone or in conjunction with TSC1 in the presence or absence of OLPs grafted into the brain parenchyma. Energetics and expression of stress proteins and OL developmental specific markers were examined. A comparison of the proteomic profile per treatment was also ascertained. We found that OLPs did not survive in the excitotoxic environment when grafted alone. In contrast, when combined with TSC1, survival and integration of grafted OLPs was observed. Further, energy metabolism in OLPs was significantly increased by N-methyl-D-aspartate and modulated by TSC1. The proteomic profile after the various treatments showed elevated ubiquitination and stress/heat shock protein 90 in response to N-methyl-D-aspartate. These changes were reversed in the presence of TSC1 and ubiquitination was decreased. The results obtained in this pre-clinical study indicate that the use of a combinatorial intervention including both trophic support and healthy OLPs constitutes a promising approach for long-term survival and successful graft integration. We established optimal conditioning of the host brain environment to promote long-term survival and integration of grafted OLPs into an inflamed neonate host brain. Experimental procedures were performed under the United States Public Health Service Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee at (UCLA) (ARC #1992-034-61) on July 1, 2010.
RESUMO
Nothing is known about the regulation of nicotinic acetylcholine receptors (nAChRs) in hair cells of the inner ear. MuSK, rapsyn and RIC-3 are accessory molecules associated with muscle and brain nAChR function. We demonstrate that these accessory molecules are expressed in the inner ear raising the possibility of a muscle-like mechanism for clustering and assembly of nAChRs in hair cells. We focused our investigations on rapsyn and RIC-3. Rapsyn interacts with the cytoplasmic loop of nAChR alpha9 subunits but not nAChR alpha10 subunits. Although rapsyn and RIC-3 increase nAChR alpha9 expression, rapsyn plays a greater role in receptor clustering while RIC-3 is important for acetylcholine-induced calcium responses. Our data suggest that RIC-3 facilitates receptor function, while rapsyn enhances receptor clustering at the cell surface.
Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Musculares/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Nicotínicos/metabolismo , Animais , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Feminino , Células Ciliadas Auditivas Internas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/citologia , Células LLC-PK1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/genética , Suínos , Sinapses/metabolismo , TransfecçãoRESUMO
Cochlear gene therapy has made tremendous strides over the past 5 years. The first study documenting successful restoration of congenital hearing loss using AAV-mediated gene therapy occurred in a mouse model of deafness lacking vesicular glutamate transporter 3 (VGLUT 3). This study utilized a trans-bulla round window membrane (RWM) delivery approach. Since this study, these methodologies have been applied to a number of other mouse models of genetic deafness with varying degrees of success, lending promise for future clinical application of this burgeoning technology. Here we describe a method of virally mediated gene delivery into the cochlear scala tympani through the RWM. This method involves negligible damage to essential structures of the middle and inner ear while preserving hearing. The efficacy of this surgical technique will be demonstrated by the restoration of hearing to the VGLUT3 knockout mice (a mouse model of congenital deafness) after delivery of VGLUT3 gene to the inner ear using an adeno-associated virus as a vector.
Assuntos
Dependovirus/genética , Orelha Interna/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Animais , Tronco Encefálico/metabolismo , Imunofluorescência , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Camundongos , Camundongos Knockout , Transdução Genética , TransgenesRESUMO
Over 450 million people worldwide suffer from hearing loss, leading to an estimated economic burden of â¼$750 billion. The past decade has seen significant advances in the understanding of the molecular mechanisms that contribute to hearing, and the environmental and genetic factors that can go awry and lead to hearing loss. This in turn has sparked enormous interest in developing gene therapy approaches to treat this disorder. This review documents the most recent advances in cochlear gene therapy to restore hearing loss, and will cover viral vectors and construct designs, potential routes of delivery into the inner ear, and, lastly, the most promising genes of interest.
Assuntos
Cóclea/patologia , Terapia Genética/métodos , Terapia Genética/tendências , Perda Auditiva/terapia , Animais , Cóclea/anatomia & histologia , Cóclea/fisiologia , Vetores Genéticos , HumanosRESUMO
Contemporary cochlear implants (CI) are generally very effective for remediation of severe to profound sensorineural hearing loss, but outcomes are still highly variable. Auditory nerve survival is likely one of the major factors underlying this variability. Neurotrophin therapy therefore has been proposed for CI recipients, with the goal of improving outcomes by promoting improved survival of cochlear spiral ganglion neurons (SGN) and/or residual hair cells. Previous studies have shown that glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor, and neurotrophin-3 can rescue SGNs following insult. The current study was designed to determine whether adeno-associated virus vector serotype 5 (AAV-5) encoding either green fluorescent protein or GDNF can transduce cells in the mouse cochlea to express useful levels of neurotrophin and to approximate the optimum therapeutic dose(s) for transducing hair cells and SGN. The findings demonstrate that AAV-5 is a potentially useful gene therapy vector for the cochlea, resulting in extremely high levels of transgene expression in the cochlear inner hair cells and SGN. However, overexpression of human GDNF in newborn mice caused severe neurological symptoms and hearing loss, likely due to Purkinje cell loss and cochlear nucleus pathology. Thus, extremely high levels of transgene protein expression should be avoided, particularly for proteins that have neurological function in neonatal subjects.
Assuntos
Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Perda Auditiva/genética , Perda Auditiva/terapia , Neurônios/metabolismo , Animais , Biomarcadores , Cóclea/metabolismo , Cóclea/patologia , Modelos Animais de Doenças , Imunofluorescência , Técnicas de Transferência de Genes , Terapia Genética/métodos , Perda Auditiva/diagnóstico , Perda Auditiva/fisiopatologia , Humanos , Imuno-Histoquímica , Camundongos , FenótipoRESUMO
Outcomes with contemporary cochlear implants (CI) depend partly upon the survival and condition of the cochlear spiral ganglion (SG) neurons. Previous studies indicate that CI stimulation can ameliorate SG neural degeneration after deafness, and brain-derived neurotrophic factor (BDNF) delivered by an osmotic pump can further improve neural survival. However, direct infusion of BDNF elicits undesirable side effects, and osmotic pumps are impractical for clinical application. In this study, we explored the potential for two adeno-associated viral vectors (AAV) to elicit targeted neurotrophic factor expression in the cochlea and promote improved SG and radial nerve fiber survival. Juvenile cats were deafened prior to hearing onset by systemic aminoglycoside injections. Auditory brainstem responses showed profound hearing loss by 16-18 days postnatal. At ~ 4 weeks of age, AAV2-GFP (green fluorescent protein), AAV5-GFP, AAV2-hBDNF, or AAV5-hGDNF (glial-derived neurotrophic factor) was injected through the round window unilaterally. For GFP immunofluorescence, animals were studied ~ 4 weeks post-injection to assess cell types transfected and their distributions. AAV2-GFP immunofluorescence demonstrated strong expression of the GFP reporter gene in residual inner (IHCs), outer hair cells (OHCs), inner pillar cells, and in some SG neurons throughout the cochlea. AAV5-GFP elicited robust transduction of IHCs and some SG neurons, but few OHCs and supporting cells. After AAV-neurotrophic factor injections, animals were studied ~ 3 months post-injection to evaluate neural survival. AAV5-hGDNF elicited a modest neurotrophic effect, with 6 % higher SG density, but had no trophic effect on radial nerve fiber survival, and undesirable ectopic fiber sprouting occurred. AAV2-hBDNF elicited a similar 6 % increase in SG survival, but also resulted in greatly improved radial nerve fiber survival, with no ectopic fiber sprouting. A further study assessed whether AAV2-hBDNF neurotrophic effects would persist over longer post-injection periods. Animals examined 6 months after virus injection showed substantial neurotrophic effects, with 14 % higher SG density and greatly improved radial nerve fiber survival. Our results suggest that AAV-neurotrophin gene therapy can elicit expression of physiological concentrations of neurotrophins in the cochlea, supporting improved SG neuronal and radial nerve fiber survival while avoiding undesirable side effects. These studies also demonstrate the potential for application of cochlear gene therapy in a large mammalian cochlea comparable to the human cochlea and in an animal model of congenital/early acquired deafness.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Surdez/terapia , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Gânglio Espiral da Cóclea/citologia , Animais , Animais Recém-Nascidos , Gatos , Dependovirus , Nervo RadialRESUMO
AIM: To better elucidate the cellular dynamics by which perforations in the tympanic membrane (TM) are healed. BACKGROUND: Under normal conditions, epidermal cells are born and then migrate radially outward from the malleus in the TM. It is unknown what the relative contribution of newly proliferated cells from different lineages is in the healing of TM perforations. METHODS: Thirty-six female mice were used in this study. Ethynyl deoxyuridine, a thymidine analogue that labels newly proliferated cells, was injected intraperitoneally into each mouse and then subsequently supplied in the drinking water. Acute perforations were performed on the right TM and the left TM served as the control and remained intact. The animals were sacrificed at six time points between 2âhours and 6 days. We stained for proliferative, epithelial, mesenchymal markers, and ethynyl deoxyuridine and analyzed the distribution of cells. RESULTS: In control TMs, newly proliferated cells were detected around the malleus handle and then migrated radially outward. Perforated TMs had a significantly higher number of newly proliferated cells throughout the tympanic membrane with a marked proliferative response of epithelial, mesenchymal, and mucosal cells in the region of the malleus and perforation. The majority of cells in the healed perforation were newly proliferated. In the anterior TM opposite the perforation, an increased turnover of keratinocytes was noted, but not mesenchymal cells. CONCLUSIONS: Perforation of the TM alters the cellular dynamics throughout the entire TM, rather than simply adjacent to the perforation. This argues for long distance signaling occurring in the perforated TM.
Assuntos
Perfuração da Membrana Timpânica/fisiopatologia , Cicatrização/fisiologia , Animais , Proliferação de Células , Feminino , Camundongos , Membrana Timpânica/fisiopatologiaRESUMO
Hearing loss is a significant public health concern, affecting over 250 million people worldwide. Both genetic and environmental etiologies are linked to hearing loss, but in many cases the underlying cellular pathophysiology is not well understood, highlighting the importance of further discovery. We found that inactivation of the gene Tmtc4 (transmembrane and tetratricopeptide repeat 4), which was broadly expressed in the mouse cochlea, caused acquired hearing loss in mice. Our data showed Tmtc4 enriched in the endoplasmic reticulum, and that it functioned by regulating Ca2+ dynamics and the unfolded protein response (UPR). Given this genetic linkage of the UPR to hearing loss, we demonstrated a direct link between the more common noise-induced hearing loss (NIHL) and the UPR. These experiments suggested a novel approach to treatment. We demonstrated that the small-molecule UPR and stress response modulator ISRIB (integrated stress response inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout of Tmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing loss disorders.
Assuntos
Deleção de Genes , Perda Auditiva/metabolismo , Proteínas de Membrana/deficiência , Resposta a Proteínas não Dobradas , Acetamidas/farmacologia , Animais , Cicloexilaminas/farmacologia , Modelos Animais de Doenças , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Perda Auditiva/tratamento farmacológico , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Camundongos , Camundongos Knockout , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1ex4-/-) and postnatal hair cell-specific conditional (Clrn1ex4fl/fl Myo15-Cre+/-) knockout mice. Clrn1ex4-/- mice were profoundly deaf, whereas Clrn1ex4fl/fl Myo15-Cre+/- mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and CaV1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic CaV1.3 Ca2+ channel complex via the Cavß2 auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1ex4fl/fl Myo15-Cre+/- mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.