RESUMO
Mutations in the Pejvakin (PJVK) gene are thought to cause auditory neuropathy and hearing loss of cochlear origin by affecting noise-induced peroxisome proliferation in auditory hair cells and neurons. Here we demonstrate that loss of pejvakin in hair cells, but not in neurons, causes profound hearing loss and outer hair cell degeneration in mice. Pejvakin binds to and colocalizes with the rootlet component TRIOBP at the base of stereocilia in injectoporated hair cells, a pattern that is disrupted by deafness-associated PJVK mutations. Hair cells of pejvakin-deficient mice develop normal rootlets, but hair bundle morphology and mechanotransduction are affected before the onset of hearing. Some mechanotransducing shorter row stereocilia are missing, whereas the remaining ones exhibit overextended tips and a greater variability in height and width. Unlike previous studies of Pjvk alleles with neuronal dysfunction, our findings reveal a cell-autonomous role of pejvakin in maintaining stereocilia architecture that is critical for hair cell function.SIGNIFICANCE STATEMENT Two missense mutations in the Pejvakin (PJVK or DFNB59) gene were first identified in patients with audiological hallmarks of auditory neuropathy spectrum disorder, whereas all other PJVK alleles cause hearing loss of cochlear origin. These findings suggest that complex pathogenetic mechanisms underlie human deafness DFNB59. In contrast to recent studies, we demonstrate that pejvakin in auditory neurons is not essential for normal hearing in mice. Moreover, pejvakin localizes to stereociliary rootlets in hair cells and is required for stereocilia maintenance and mechanosensory function of the hair bundle. Delineating the site of the lesion and the mechanisms underlying DFNB59 will allow clinicians to predict the efficacy of different therapeutic approaches, such as determining compatibility for cochlear implants.
Assuntos
Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Mecanotransdução Celular , Proteínas/metabolismo , Animais , Linhagem Celular , Audição , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Proteínas/genética , Estereocílios/metabolismo , Estereocílios/patologiaRESUMO
Mutations in the Pejvakin (Pjvk) gene cause autosomal recessive hearing loss DFNB59 with audiological features of auditory neuropathy spectrum disorder (ANSD) or cochlear dysfunction. The precise mechanisms underlying the variable clinical phenotypes of DFNB59 remain unclear. Here, we demonstrate that mice with conditional ablation of the Pjvk gene in all sensory hair cells or only in outer hair cells (OHCs) show similar auditory phenotypes with early-onset profound hearing loss. By contrast, loss of Pjvk in adult OHCs causes a slowly progressive hearing loss associated with OHC degeneration and delayed loss of inner hair cells (IHCs), indicating a primary role for pejvakin in regulating OHC function and survival. Consistent with this model, synaptic transmission at the IHC ribbon synapse is largely unaffected in sirtaki mice that carry a C-terminal deletion mutation in Pjvk. Using the C-terminal domain of pejvakin as bait, we identified in a cochlear cDNA library ROCK2, an effector for the small GTPase Rho, and the scaffold protein IQGAP1, involved in modulating actin dynamics. Both ROCK2 and IQGAP1 associate via their coiled-coil domains with pejvakin. We conclude that pejvakin is required to sustain OHC activity and survival in a cell-autonomous manner likely involving regulation of Rho signaling.
Assuntos
Células Ciliadas Auditivas Externas/metabolismo , Perda Auditiva/metabolismo , Proteínas/metabolismo , Animais , Sobrevivência Celular/fisiologia , Progressão da Doença , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/patologia , Células HeLa , Perda Auditiva/patologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Proteínas/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Sinapses/metabolismo , Técnicas de Cultura de Tecidos , Proteínas Ativadoras de ras GTPase/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Disordered protein ubiquitination has been linked to neurodegenerative disease, yet its role in inner ear homeostasis and hearing loss is essentially unknown. Here we show that progressive hearing loss in the ethylnitrosourea-generated mambo mouse line is caused by a mutation in Usp53, a member of the deubiquitinating enzyme family. USP53 contains a catalytically inactive ubiquitin-specific protease domain and is expressed in cochlear hair cells and a subset of supporting cells. Although hair cell differentiation is unaffected in mambo mice, outer hair cells degenerate rapidly after the first postnatal week. USP53 colocalizes and interacts with the tight junction scaffolding proteins TJP1 and TJP2 in polarized epithelial cells, suggesting that USP53 is part of the tight junction complex. The barrier properties of tight junctions of the stria vascularis appeared intact in a biotin tracer assay, but the endocochlear potential is reduced in adult mambo mice. Hair cell degeneration in mambo mice precedes endocochlear potential decline and is rescued in cochlear organotypic cultures in low potassium milieu, indicating that hair cell loss is triggered by extracellular factors. Remarkably, heterozygous mambo mice show increased susceptibility to noise injury at high frequencies. We conclude that USP53 is a novel tight junction-associated protein that is essential for the survival of auditory hair cells and normal hearing in mice, possibly by modulating the barrier properties and mechanical stability of tight junctions. SIGNIFICANCE STATEMENT: Hereditary hearing loss is extremely prevalent in the human population, but many genes linked to hearing loss remain to be discovered. Forward genetics screens in mice have facilitated the identification of genes involved in sensory perception and provided valuable animal models for hearing loss in humans. This involves introducing random mutations in mice, screening the mice for hearing defects, and mapping the causative mutation. Here, we have identified a mutation in the Usp53 gene that causes progressive hearing loss in the mambo mouse line. We demonstrate that USP53 is a catalytically inactive deubiquitinating enzyme and a novel component of tight junctions that is necessary for sensory hair cell survival and inner ear homeostasis.
Assuntos
Progressão da Doença , Perda Auditiva/genética , Perda Auditiva/patologia , Heterozigoto , Mutação/genética , Proteases Específicas de Ubiquitina/genética , Sequência de Aminoácidos , Animais , Cóclea/patologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by a prominent loss of nigrostriatal dopamine (DA) neurons with an accompanying neuroinflammation. The peptide angiotensin II (AngII) plays a role in oxidative-stress induced disorders and is thought to mediate its detrimental actions via activation of AngII AT1 receptors. The brain renin-angiotensin system is implicated in neurodegenerative disorders including PD. Blockade of the angiotensin converting enzyme or AT1 receptors provides protection in acute animal models of parkinsonism. We demonstrate here that treatment of mice with the angiotensin converting enzyme inhibitor captopril protects the striatum from acutely administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP), and that chronic captopril protects the nigral DA cell bodies from degeneration in a progressive rat model of parkinsonism created by the chronic intracerebral infusion of 1-methyl-4-phenylpyridinium (MPP+). The accompanying activation of microglia in the substantia nigra of MPP+-treated rats was reduced by the chronic captopril treatment. These findings indicate that captopril is neuroprotective for nigrostriatal DA neurons in both acute and chronic rodent PD models. Targeting the brain AngII pathway may be a feasible approach to slowing neurodegeneration in PD.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Encéfalo/efeitos dos fármacos , Captopril/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Intoxicação por MPTP/tratamento farmacológico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Imuno-Histoquímica , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Masculino , Camundongos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/farmacologia , RatosRESUMO
Parkinson's disease (PD) is characterized by a prominent degeneration of nigrostriatal dopamine (DA) neurons with an accompanying neuroinflammation. Despite clinical and preclinical studies of neuroprotective strategies for PD, there is no effective treatment for preventing or slowing the progression of neurodegeneration. The inverse correlation between caffeine consumption and risk of PD suggests that caffeine may exert neuroprotection. Whether caffeine is neuroprotective in a chronic progressive model of PD has not been evaluated nor is it known if delayed caffeine treatment can stop DA neuronal loss. We show that a chronic unilateral intra-cerebroventricular infusion of 1-methyl-4-phenylpyridinium in the rat brain for 28 days produces a progressive loss of DA and tyrosine hydroxylase in the ipsilateral striatum and a loss of DA cell bodies and microglial activation in the ipsilateral substantia nigra. Chronic caffeine consumption prevented the degeneration of DA cell bodies in the substantia nigra. Importantly, neuroprotection was still apparent when caffeine was introduced after the onset of the neurodegenerative process. These results add to the clinical relevance for adenosine receptors as a disease-modifying drug target for PD.
Assuntos
Cafeína/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , Substância Negra/efeitos dos fármacos , Animais , Cafeína/uso terapêutico , Contagem de Células , Modelos Animais de Doenças , Progressão da Doença , Neurônios Dopaminérgicos/patologia , Masculino , Degeneração Neural/patologia , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Substância Negra/patologiaRESUMO
An electrospun nonwoven matrix of polyamide nanofibers was employed as a new model for the capillary basement membrane at the blood-brain barrier (BBB). The basement membrane separates astrocytes from endothelial cells and is associated with growth factors, such as fibroblast growth factor-2 (FGF-2). FGF-2 is produced by astrocytes and induces specialized functions in endothelial cells, but also has actions on astrocytes. To investigate potential autocrine actions of FGF-2 at the BBB, astrocytes were cultured on unmodified nanofibers or nanofibers covalently modified with FGF-2. The former assumed an in vivo-like stellate morphology that was enhanced in the presence of cross-linked FGF-2. Furthermore, astrocyte monolayers established on unmodified nanofibers were more permissive for neurite outgrowth when cultured with an overlay of neurons than similar monolayers established on standard tissue culture surfaces, while astrocytes cultured on FGF-2-modifed nanofibers were yet more permissive. The observed differences were due in part to progressively increasing amounts of FGF-2 secreted by the astrocytes into the medium; hence FGF-2 increases its own expression in astrocytes to modulate astrocyte-neuron interactions. Soluble FGF-2 was unable to replicate the effects of cross-linked FGF-2. Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, these results indicate that FGF-2-modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of lost tissue following central nervous system (CNS) injury or disease.