RESUMO
Adding rapamycin or acarbose to diet at 9-10 months of age has been shown to significantly increase life span in both male and female UM-HET3 mice. The current study examined cochleae of male and female UM-HET3 mice at 22 months of age to determine if either treatment also influenced age-related loss of cochlear hair cells. A large loss of cochlear outer hair cells was observed at 22 months of age in untreated mice in both apical and basal halves of the cochlear spiral. Addition of acarbose to diet had no significant effect on the amount of outer hair cell loss at 22 months of age or in its pattern, with large loss in both apical and basal halves. The addition of rapamycin to diet, however, significantly reduced outer hair cell loss in the basal half of the cochlea at 22 months of age when compared to untreated mice. There was no significant difference between male and female mice in any of the conditions. Age-related outer hair cell loss in the apical cochlea precedes outer hair cell loss in the base in many mouse strains. The results of the present study suggest that rapamycin but not acarbose treatment can delay age-related loss of outer hair cells at doses at which each drug increases life span.
Assuntos
Acarbose/farmacologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Audição/efeitos dos fármacos , Presbiacusia/prevenção & controle , Sirolimo/farmacologia , Fatores Etários , Animais , Morte Celular/efeitos dos fármacos , Citoproteção , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Células Ciliadas Auditivas Externas/patologia , Audição/genética , Masculino , Camundongos Transgênicos , Fenótipo , Presbiacusia/genética , Presbiacusia/patologia , Presbiacusia/fisiopatologiaRESUMO
This study compared the timing of appearance of three components of age-related hearing loss that determine the pattern and severity of presbycusis: the functional and structural pathologies of sensory cells and neurons and changes in gap detection (GD), the latter as an indicator of auditory temporal processing. Using UM-HET4 mice, genetically heterogeneous mice derived from four inbred strains, we studied the integrity of inner and outer hair cells by position along the cochlear spiral, inner hair cell-auditory nerve connections, spiral ganglion neurons (SGN), and determined auditory thresholds, as well as pre-pulse and gap inhibition of the acoustic startle reflex (ASR). Comparisons were made between mice of 5-7, 22-24 and 27-29 months of age. There was individual variability among mice in the onset and extent of age-related auditory pathology. At 22-24 months of age a moderate to large loss of outer hair cells was restricted to the apical third of the cochlea and threshold shifts in the auditory brain stem response were minimal. There was also a large and significant loss of inner hair cell-auditory nerve connections and a significant reduction in GD. The expression of Ntf3 in the cochlea was significantly reduced. At 27-29 months of age there was no further change in the mean number of synaptic connections per inner hair cell or in GD, but a moderate to large loss of outer hair cells was found across all cochlear turns as well as significantly increased ABR threshold shifts at 4, 12, 24 and 48 kHz. A statistical analysis of correlations on an individual animal basis revealed that neither the hair cell loss nor the ABR threshold shifts correlated with loss of GD or with the loss of connections, consistent with independent pathological mechanisms.
Assuntos
Envelhecimento/fisiologia , Percepção Auditiva/fisiologia , Nervo Coclear/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Envelhecimento/patologia , Animais , Vias Auditivas/patologia , Vias Auditivas/fisiologia , Limiar Auditivo/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Contagem de Células , Nervo Coclear/patologia , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/fisiologia , Camundongos , Distribuição Aleatória , Reflexo de Sobressalto/fisiologia , Gânglio Espiral da Cóclea/patologia , Gânglio Espiral da Cóclea/fisiologiaRESUMO
Hearing loss is the most common sensory deficit in humans and can result from genetic, environmental or combined etiologies that prevent normal function of the cochlea, the peripheral sensory organ. Recent advances in understanding the genetic pathways that are critical for the development and maintenance of cochlear function, as well as the molecular mechanisms that underlie cell trauma and death, have provided exciting opportunities for modulating these pathways to correct genetic mutations, to enhance the endogenous protective pathways for hearing preservation and to regenerate lost sensory cells with the possibility of ameliorating hearing loss. A number of recent animal studies have used gene-based therapies in innovative ways toward realizing these goals. With further refinement, some of the protective and regenerative approaches reviewed here may become clinically applicable.
Assuntos
Cóclea/fisiopatologia , Surdez/genética , Surdez/terapia , Terapia Genética/métodos , Animais , Terapia Combinada , Surdez/fisiopatologia , Auxiliares de Audição , Humanos , MutaçãoRESUMO
The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions "cytocauds." The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.
Assuntos
Surdez/genética , Surdez/patologia , Células Ciliadas Auditivas/anormalidades , Células Ciliadas Auditivas/ultraestrutura , Camundongos Mutantes Neurológicos/anormalidades , Doenças Vestibulares/genética , Doenças Vestibulares/patologia , Citoesqueleto de Actina/patologia , Citoesqueleto de Actina/ultraestrutura , Animais , Animais Recém-Nascidos/anormalidades , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/fisiologia , Cílios/patologia , Cílios/ultraestrutura , Surdez/fisiopatologia , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos/genética , Camundongos Mutantes Neurológicos/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Órgão Espiral/anormalidades , Órgão Espiral/patologia , Órgão Espiral/ultraestrutura , Faloidina , Doenças Vestibulares/fisiopatologia , Vestíbulo do Labirinto/anormalidades , Vestíbulo do Labirinto/patologia , Vestíbulo do Labirinto/ultraestruturaRESUMO
Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.
Assuntos
Cinesinas/genética , Proteínas do Tecido Nervoso/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Galinhas , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Feminino , Expressão Gênica , Variação Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Muridae , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The voltage-gated sodium channel SCN8A is associated with inherited neurological disorders in the mouse that include ataxia, dystonia, severe muscle weakness, and paralysis. We report the complete coding sequence and exon organization of the human SCN8A gene. The predicted 1980 amino acid residues are distributed among 28 exons, including two pairs of alternatively spliced exons. The SCN8A protein is evolutionarily conserved, with 98.5% amino acid sequence identity between human and mouse. Consensus sites for phosphorylation of serine/threonine and tyrosine residues are present in cyoplasmic loop domains. The polymorphic (CA)n microsatellite marker D12S2211, with PIC = 0.68, was isolated from intron 10C of SCN8A. Single nucleotide polymorphisms in intron 19 and exon 22 were also identified. We localized SCN8A to chromosome band 12q13.1 by physical mapping on a YAC contig. The cDNA clone CSC-1 was reported by others to be a cardiac-specific sodium channel, but sequence comparison demonstrates that it is derived from exon 24 of human SCN8A. The genetic information described here will be useful in evaluating SCN8A as a candidate gene for human neurological disease.
Assuntos
Canais de Sódio/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Clonagem Molecular , Sequência Conservada/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/genética , Mapeamento Físico do Cromossomo , Análise de Sequência de DNARESUMO
The voltage-gated sodium channel Scn8a is broadly distributed in brain and spinal cord. We have identified a missense mutation in Scn8a that is associated with cerebellar ataxia in the jolting mutant, a mild allele of the "motor endplate disease" locus. The jolting mutation results in substitution of Thr for an evolutionarily conserved Ala residue in the cytoplasmic S4-S5 linker of domain III. Introduction of the corresponding mutation into the rat brain IIA sodium channel shifted the voltage dependence of activation by 14 mV in the depolarizing direction, without affecting the kinetics of fast inactivation or recovery from inactivation. A shift in the threshold of the Scn8a channel could account for the reduced spontaneous activity of Purkinje cells, reduced inhibitory output from the cerebellum, and loss of motor control observed in jolting mice.
Assuntos
Ataxia Cerebelar/genética , Mutação , Canais de Sódio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Encéfalo/metabolismo , Sequência Conservada , Eletrofisiologia , Cinética , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Canais de Sódio/fisiologia , Transcrição GênicaRESUMO
Analysis of a transgene-induced mutation at the mouse med locus led to the identification of the novel voltage-gated sodium channel gene Scn8a (Burgess, D. L., Kohrman, D. C., Galt, J., Plummer, N. W., Jones, J. M., Spear, B., and Meisler, M. H.(1995) Nat. Genet. 10, 461-465). We now report the identification of splicing defects in two spontaneous mutations of Scn8a. The original med mutation was caused by insertion of a truncated LINE element into exon 2 of Scn8a. The med transcript is spliced from exon 1 to a cryptic acceptor site in intron 2. A 4-base pair deletion within the 5' donor site of exon 3 in the medJ allele results in splicing from exon 1 to exon 4. Both mutant transcripts have altered reading frames with premature stop codons close to the N terminus of the protein. Loss of Scn8a expression results in progressive paralysis and early death. Intron 2 of Scn8a is flanked by minor class AT-AC splice sites. The observed splicing patterns of the med and medJ mutant transcripts provide the first evidence for preferential in vivo splicing between donor and acceptor sites of the same class. The apparent functional incompatibility may be a consequence of the different composition of spliceosomes bound to major and minor splice sites.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso , Mutação Puntual , Canais de Sódio/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Éxons , Genes Recessivos , Humanos , Íntrons , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Doença dos Neurônios Motores/genética , Canal de Sódio Disparado por Voltagem NAV1.6 , Homologia de Sequência do Ácido Nucleico , Canais de Sódio/biossíntese , Transcrição GênicaRESUMO
The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locus ax. To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci.
Assuntos
Evolução Biológica , Cromossomos Humanos Par 18 , Fenda Labial/genética , Fissura Palatina/genética , Face/anormalidades , Crânio/anormalidades , Alelos , Animais , Sequência de Bases , Mapeamento Cromossômico , Sequência Conservada , Primers do DNA , DNA Satélite , Frequência do Gene , Biblioteca Gênica , Ligação Genética , Marcadores Genéticos , Humanos , Células Híbridas , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da PolimeraseRESUMO
The location of three mutations on proximal Chromosome (Chr) 18 was determined by analysis of the offspring of several backcrosses. The results demonstrate that ataxia and the insertional mutation TgN9257Mm are separated by less than 1 cM and are located approximately 3 cM from the centromere, while the balding locus is 7 cM more distal. Previous data demonstrated that the twirler locus also maps within 1 cM of ataxia. The corrected locations will contribute to identification of appropriate candidate genes for these mutations. Two polymorphic microsatellite markers for proximal Chr 18 are described, D18Umi1 and D18Umi2. The Lama3 locus encoding the alpha 3 subunit of nicein was mapped distal to ataxia and did not recombine with Tg9257.
Assuntos
Ataxia/genética , Mapeamento Cromossômico , Mutação , Animais , Sequência de Bases , Ligação Genética/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Transgenes/genéticaRESUMO
The mouse neurological mutant 'motor endplate disease' (med) is characterized by early onset progressive paralysis of the hind limbs, severe muscle atrophy, degeneration of Purkinje cells and juvenile lethality. We have isolated a voltage-gated sodium channel gene, Scn8a, from the flanking region of a transgene-induced allele of med. Scn8a is expressed in brain and spinal cord but not in skeletal muscle or heart, and encodes a predicted protein of 1,732 amino acids. An intragenic deletion at the transgene insertion site results in loss of expression. Scn8a is closely related to other sodium channel alpha subunits, with greatest similarity to a brain transcript from the pufferfish Fugu rubripes. The human homologue, SCN8A, maps to chromosome 12q13 and is a candidate gene for inherited neurodegenerative disease.
Assuntos
Placa Motora , Proteínas do Tecido Nervoso , Doenças do Sistema Nervoso/genética , Deleção de Sequência , Canais de Sódio/genética , Sequência de Aminoácidos , Animais , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.6 , Ratos , TransfecçãoRESUMO
Homozygous transgenic mice from line A4 have an early-onset progressive neuromuscular disorder characterized by paralysis of the rear limbs, muscle atrophy, and lethality by 4 weeks of age. The transgene insertion site was mapped to distal chromosome 15 close to the locus motor endplate disease (med). The sequence of mouse DNA flanking the insertion site junctions was determined. A small (< 20 kb) deletion was detected at the insertion site, with no evidence of additional rearrangement of the chromosomal DNA. Noncomplementation of the transgene-induced mutation and med was demonstrated in a cross with medJ/+mice. The new allele is designated medTgNA4Bs (medtg). The homologous human locus MED was assigned to chromosome 12. Synaptotagmin 1 and contactin 1 were eliminated as candidate genes for the med mutation. The transgene-induced allele provides molecular access to the med gene, whose function is required for synaptic transmission at the neuromuscular junction and long-term survival of cerebellar Purkinje cells.
Assuntos
Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais , Glicoproteínas de Membrana/genética , Placa Motora/patologia , Proteínas do Tecido Nervoso/genética , Doenças Neuromusculares/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Contactina 1 , Contactinas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Mutagênese Insercional , Células de Purkinje/patologia , Deleção de Sequência , Sinaptotagmina I , SinaptotagminasRESUMO
We have isolated an SV40 transformation revertant cell line, CL1L, by selection for normal cells whose growth is inhibited under low serum conditions. This line expresses a single, wild-type copy of large T antigen, yet is not transformed. It is not retransformable by transfection of SV40 DNA or infection with a recombinant retrovirus encoding large T antigen. Resistance to transformation therefore appears to be due to a cellular mutation. Fusion of CL1L cells to normal 3T3 cells or SV40-transformed cells results in somatic cell hybrids that are transformed, indicating that resistance is recessive. In addition, fusion of CL1L cells to another SV40 transformation-resistant line, A27, results in transformed hybrids, indicating the existence of discrete complementation groups with respect to SV40 transformation.
Assuntos
Vírus 40 dos Símios/genética , Células 3T3 , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Divisão Celular , Fusão Celular , Transformação Celular Viral , DNA Viral/genética , Genes Recessivos , Teste de Complementação Genética , Células Híbridas/citologia , Camundongos , Oncogenes , Ligação ProteicaRESUMO
Stable interactions between simian virus 40 large T antigen and host proteins are believed to play a major role in the ability of the viral protein to transform cells in culture and induce tumors in vivo. Two of these host proteins, the retinoblastoma susceptibility protein (pRB) and p53, are products of tumor suppressor genes, suggesting that T antigen exerts at least a portion of its transforming activity by complexing with and inactivating the function of these proteins. While analyzing T antigen-host protein complexes in mouse cells, we noted a protein of 185 kDa (p185) which specifically coimmunoprecipitates with T antigen. Coimmunoprecipitation results from the formation of stable complexes between T antigen and p185. Complex formation is independent of the interactions of T antigen with pRB, p120, and p53. Furthermore, analysis of T-antigen mutants suggests that T antigen-p185 complex formation may be important in transformation by simian virus 40.