RESUMEN
Noise-induced hearing loss (NIHL) is a prevalent health risk. Inbred mouse strains 129S6/SvEvTac (129S6) and MOLF/EiJ (MOLF) show strong NIHL resistance (NR) relative to CBA/CaJ (CBACa). In this study, we developed quantitative trait locus (QTL) maps for NR. We generated F1 animals by intercrossing (129S6 × CBACa) and (MOLF × CBACa). In each intercross, NR was recessive. N2 animals were produced by backcrossing F1s to their respective parental strain. The 232 N2-129S6 and 225 N2-MOLF progenies were evaluated for NR using auditory brainstem response. In 129S6, five QTL were identified on chromosomes (Chr) 17, 18, 14, 11, and 4, referred to as loci nr1, nr2, nr3, nr4, and nr5, respectively. In MOLF, four QTL were found on Chr 4, 17, 6, and 12, referred to as nr7, nr8, nr9, and nr10, respectively. Given that NR QTL were discovered on Chr 4 and 17 in both the N2-129S6 and N2-MOLF cross, we generated two consomic strains by separately transferring 129S6-derived Chr 4 and 17 into an otherwise CBACa background and a double-consomic strain by crossing the two strains. Phenotypic analysis of the consomic strains indicated that whole 129S6 Chr 4 contributes strongly to mid-frequency NR, while whole 129S6 Chr 17 contributes markedly to high-frequency NR. Therefore, we anticipated that the double-consomic strain containing Chr 4 and 17 would demonstrate NR across the mid- and high-frequency range. However, whole 129S6 Chr 17 masks the expression of mid-frequency NR from whole 129S6 Chr 4. To further dissect NR on 129S6 Chr 4 and 17, CBACa.129S6 congenic strains were generated for each chromosome. Phenotypic analysis of the Chr 17 CBACa.129S6 congenic strains further defined the NR region on proximal Chr 17, uncovered another NR locus (nr6) on distal Chr 17, and revealed an epistatic interaction between proximal and distal 129S6 Chr 17.
Asunto(s)
Pérdida Auditiva Provocada por Ruido/genética , Sitios de Carácter Cuantitativo , Animales , Mapeo Cromosómico , Ratones , Ratones Endogámicos CBARESUMEN
Voltage-gated K(+) channels (Kv) represent the largest family of genes in the K(+) channel family. The Kv1 subfamily plays an essential role in the initiation and shaping of action potentials, influencing action potential firing patterns and controlling neuronal excitability. Overlapping patterns with differential expression and precise localization of Kv1.1 and Kv1.2 channels targeted to specialized subcellular compartments contribute to distinctive patterns of neuronal excitability. Dynamic regulation of the components in these subcellular domains help to finely tune the cellular and regional networks. Disruption of the expression, distribution, and density of these channels through deletion or mutation of the genes encoding these channels, Kcna1 and Kcna2, is associated with neurologic pathologies including epilepsy and ataxia in humans and in rodent models. Kv1.1 and Kv1.2 knockout mice both have seizures beginning early in development; however, each express a different seizure type (pathway), although the channels are from the same subfamily and are abundantly coexpressed. Voltage-gated ion channels clustered in specific locations may present a novel therapeutic target for influencing excitability in neurologic disorders associated with some channelopathies.
Asunto(s)
Canal de Potasio Kv.1.1/genética , Canal de Potasio Kv.1.2/genética , Convulsiones/genética , Animales , Axones/metabolismo , Encéfalo/crecimiento & desarrollo , Química Encefálica/genética , Química Encefálica/fisiología , Epilepsia/genética , Epilepsia/fisiopatología , Humanos , Ratones , Ratones Noqueados , Mutación/genética , Mutación/fisiología , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Nódulos de Ranvier/metabolismo , Convulsiones/fisiopatologíaRESUMEN
Mutations within MYO7A can lead to recessive and dominant forms of inherited hearing loss. We previously identified a large pedigree (referred to as the HL2 family) with hearing loss that first impacts the low and mid frequencies segregating a dominant MYO7A mutation in exon 17 at DNA residue G2164C. The MYO7A(G2164C) mutation predicts a nonconservative glycine-to-arginine (G722R) amino acid substitution at a highly conserved glycine residue. The degree of low and mid frequency hearing loss varies markedly in the family, suggesting the presence of a genetic modifier that either rescues or exacerbates the primary MYO7A(G2164C) mutation. Here we describe a single nucleotide polymorphism (SNP) T/C at position -4128 in the wild-type MYO7A promoter allele that sorts with the degree of hearing loss severity in the pedigree. Electrophoretic mobility shift assay analysis indicates that the SNP differentially regulates the binding of the YY1 transcription factor with the T(-4128) allele creating an YY1 binding site. Immunocytochemistry demonstrates that Yy1 is expressed in hair cell nuclei within the cochlea. Given that Myo7a is also expressed in cochlear hair cells, Yy1 shows the appropriate localization to regulate Myo7a transcription within the inner ear. YY1 appears to be acting as a transcriptional repressor as the MYO7A promoter allele containing the T(-4128) SNP drives 41 and 46% less reporter gene expression compared with the C(-4128) SNP in the ARPE-19 and HeLa cell lines, respectively. The T(-4128) SNP may be contributing to the severe hearing loss phenotype in the HL2 pedigree by reducing expression of the wild-type MYO7A allele.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Pérdida Auditiva/genética , Miosinas/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Factor de Transcripción YY1/metabolismo , Adolescente , Adulto , Sitios de Unión , Línea Celular , Cóclea/química , Familia , Femenino , Células Ciliadas Auditivas Internas/química , Humanos , Masculino , Miosina VIIa , Factor de Transcripción YY1/análisis , Factor de Transcripción YY1/genéticaRESUMEN
Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.
Asunto(s)
Vías Auditivas/fisiología , Canal de Potasio Kv.1.1/metabolismo , Neuronas/fisiología , Núcleo Olivar/fisiología , Localización de Sonidos/fisiología , Estimulación Acústica , Potenciales de Acción/fisiología , Animales , Electrofisiología , Inmunohistoquímica , Canal de Potasio Kv.1.1/genética , Ratones , Ratones Noqueados , Modelos NeurológicosRESUMEN
Usher syndrome is the leading cause of combined deaf-blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction, and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in Exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock-in mice. Absent auditory-evoked brainstem responses indicated that the mutant mice are deaf at 1 month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock-in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Sordera/fisiopatología , Modelos Animales de Enfermedad , Degeneración Retiniana/fisiopatología , Síndromes de Usher/fisiopatología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Envejecimiento , Animales , Proteínas de Ciclo Celular , Cóclea/patología , Cóclea/fisiopatología , Cóclea/ultraestructura , Proteínas del Citoesqueleto , Sordera/patología , Electrorretinografía , Potenciales Evocados Auditivos del Tronco Encefálico , Exones , Técnicas de Sustitución del Gen , Células Ciliadas Auditivas/patología , Células Ciliadas Auditivas/fisiología , Louisiana , Ratones , Ratones Transgénicos , Mutación Missense , Sitios de Empalme de ARN , ARN Mensajero/metabolismo , Retina/patología , Retina/fisiopatología , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/patología , Células Fotorreceptoras Retinianas Bastones/fisiología , Síndromes de Usher/patologíaRESUMEN
Genes Kcna1 and Kcna2 code for the voltage-dependent potassium channel subunits Kv1.1 and Kv1.2, which are coexpressed in large axons and commonly present within the same tetramers. Both contribute to the low-voltage-activated potassium current I Kv1, which powerfully limits excitability and facilitates temporally precise transmission of information, e.g., in auditory neurons of the medial nucleus of the trapezoid body (MNTB). Kcna1-null mice lacking Kv1.1 exhibited seizure susceptibility and hyperexcitability in axons and MNTB neurons, which also had reduced I Kv1. To explore whether a lack of Kv1.2 would cause a similar phenotype, we created and characterized Kcna2-null mice (-/-). The -/- mice exhibited increased seizure susceptibility compared with their +/+ and +/- littermates, as early as P14. The mRNA for Kv1.1 and Kv1.2 increased strongly in +/+ brain stems between P7 and P14, suggesting the increasing importance of these subunits for limiting excitability. Surprisingly, MNTB neurons in brain stem slices from -/- and +/- mice were hypoexcitable despite their Kcna2 deficit, and voltage-clamped -/- MNTB neurons had enlarged I Kv1. This contrasts strikingly with the Kcna1-null MNTB phenotype. Toxin block experiments on MNTB neurons suggested Kv1.2 was present in every +/+ Kv1 channel, about 60% of +/- Kv1 channels, and no -/- Kv1 channels. Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, which may explain why MNTB neurons with larger proportions of such channels had larger I Kv1. If channel voltage dependence is determined by how many Kv1.2 subunits each contains, neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density.
Asunto(s)
Canal de Potasio Kv.1.2/deficiencia , Canal de Potasio Kv.1.2/fisiología , Convulsiones/genética , Canales de Potasio de la Superfamilia Shaker/fisiología , Envejecimiento , Animales , Tronco Encefálico/fisiología , Tronco Encefálico/fisiopatología , Vectores Genéticos , Genoma , Genotipo , Esperanza de Vida , Ratones , Ratones Noqueados , Neuronas/fisiología , Sistemas de Lectura Abierta , Mapeo RestrictivoRESUMEN
Voltage-gated sodium channels (Na(V)) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a-/- mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/- mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of Na(V)1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/- and Scn1a-/- mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of Na(V)1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/- heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.
Asunto(s)
Potenciales de Acción/fisiología , Epilepsias Mioclónicas/fisiopatología , Interneuronas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Canales de Sodio/fisiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsias Mioclónicas/genética , Genotipo , Humanos , Immunoblotting , Lactante , Interneuronas/citología , Interneuronas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.1 , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Fenotipo , Convulsiones/genética , Convulsiones/mortalidad , Convulsiones/fisiopatología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Tasa de Supervivencia , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Although there is increasing awareness of adverse effects associated with use of the high-fat ketogenic diet, very little is known regarding its long-term clinical consequences, especially in relation to cardiovascular health. Recent reports have highlighted rare but significant cardiac problems in patients treated with the ketogenic diet. Given the inherent limitations in conducting detailed pathologic assessments in patients, we asked whether histologic changes might develop in the brain and skeletal muscle of mice fed a high-fat diet for 2 to 3 months. We found no evidence of gross morphologic or histochemical alterations in muscle or brain after administration of the ketogenic diet. Further, there was no abnormal lipid storage or mitochondrial enzymatic staining. Our data suggest that patients chronically treated with a ketogenic diet are not likely to develop a lipid myopathy or neuronal inclusions.
Asunto(s)
Encéfalo/patología , Dietoterapia/efectos adversos , Grasas de la Dieta/efectos adversos , Músculo Esquelético/patología , Animales , Epilepsia/dietoterapia , Femenino , Cetonas , Masculino , Ratones , Ratones Endogámicos C3HRESUMEN
Tuberous sclerosis (TSC) is an autosomal dominant disorder, caused by mutations of either the TSC1 or TSC2 gene. Characteristic brain pathologies (including cortical tubers and subependymal hamartomas/giant astrocytomas) are thought to cause epilepsy, as well as other neurological dysfunction. The Eker rat, which carries a spontaneous germline mutation of the TSC2 gene (TSC2+/-), provides a unique animal model in which to study the relationship between TSC cortical pathologies and epilepsy. In the present study, we have analyzed the seizure propensity and histopathological features of a modified Eker rat preparation, in which early postnatal irradiation was employed as a "second hit" stimulus in an attempt to exacerbate cortical malformations and increase seizure propensity. Irradiated Eker rats had a tendency toward lower seizure thresholds (latencies to flurothyl-induced seizures) than seen in non-irradiated Eker rats (significant difference) or irradiated wild-type rats (non-significant difference). The majority of irradiated Eker rats exhibited dysplastic cytomegalic neurons and giant astrocyte-like cells, similar to cytopathologies observed in TSC lesions of patients. The most prominent features in these brains were hamartoma-like lesions involving large eosinophilic cells, similar to giant tuber cells in human TSC. In some cells from these hamartomas, immunocytochemistry revealed features of both neuronal and glial phenotypes, suggesting an undifferentiated or immature cell population. Both normal-appearing and dysmorphic neurons, as well as cells in the hamartomas, exhibited immunopositivity for tuberin, the protein product of the TSC2 gene.