RESUMEN
The calcium channel CACNA1A gene encodes the pore-forming, voltage-sensitive subunit of the voltage-dependent calcium Ca(v)2.1 type channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine, episodic ataxia 2 and spinocerebellar ataxia type 6. The mouse homologue, Cacna1a, is associated with the tottering, Cacna1a(tg), mutant series. Here we describe two new missense mutant alleles, Cacna1a(tg-4J) and Cacna1a(Tg-5J). The Cacna1a(tg-4J) mutation is a valine to alanine mutation at amino acid 581, in segment S5 of domain II. The recessive Cacna1a(tg-4J) mutant exhibited the ataxia, paroxysmal dyskinesia and absence seizures reminiscent of the original tottering mouse. The Cacna1a(tg-4J) mutant also showed altered activation and inactivation kinetics of the Ca(v)2.1 channel, not previously reported for other tottering alleles. The semi-dominant Cacna1a(Tg-5J) mutation changed a conserved arginine residue to glutamine at amino acid 1252 within segment S4 of domain III. The heterozygous mouse was ataxic and homozygotes rarely survived. The Cacna1a(Tg-5J) mutation caused a shift in both voltage activation and inactivation to lower voltages, showing that this arginine residue is critical for sensing Ca(v)2.1 voltage changes. These two tottering mouse models illustrate how novel allelic variants can contribute to functional studies of the Ca(v)2.1 calcium channel.
Asunto(s)
Canales de Calcio Tipo N/genética , Mutación , Enfermedades del Sistema Nervioso , Alanina/genética , Animales , Animales Recién Nacidos , Calbindinas , Células Cultivadas , Cisteína/genética , Modelos Animales de Enfermedad , Glicina/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Activación del Canal Iónico/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Moleculares , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/fisiopatología , Técnicas de Placa-Clamp , Células de Purkinje/patología , Células de Purkinje/fisiología , Células de Purkinje/ultraestructura , Proteína G de Unión al Calcio S100/metabolismo , Tinción con Nitrato de Plata/métodos , Treonina/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Rocker (gene symbol rkr), a new neurological mutant phenotype, was found in descendents of a chemically mutagenized male mouse. Mutant mice display an ataxic, unstable gait accompanied by an intention tremor, typical of cerebellar dysfunction. These mice are fertile and appear to have a normal life span. Segregation analysis reveals rocker to be an autosomal recessive trait. The overall cytoarchitecture of the young adult brain appears normal, including its gross cerebellar morphology. Golgi-Cox staining, however, reveals dendritic abnormalities in the mature cerebellar cortex characterized by a reduction of branching in the Purkinje cell dendritic arbor and a "weeping willow" appearance of the secondary branches. Using simple sequence length polymorphism markers, the rocker locus was mapped to mouse chromosome 8 within 2 centimorgans of the calcium channel alpha1a subunit (Cacna1a, formerly known as tottering) locus. Complementation tests with the leaner mutant allele (Cacna1a(la)) produced mutant animals, thus identifying rocker as a new allele of Cacna1a (Cacna1a(rkr)). Sequence analysis of the cDNA revealed rocker to be a point mutation resulting in an amino acid exchange: T1310K between transmembrane regions 5 and 6 in the third homologous domain. Important distinctions between rocker and the previously characterized alleles of this locus include the absence of aberrant tyrosine hydroxylase expression in Purkinje cells and the separation of the absence seizures (spike/wave type discharges) from the paroxysmal dyskinesia phenotype. Overall these findings point to an important dissociation between the seizure phenotypes and the abnormalities in catecholamine metabolism, and they emphasize the value of allelic series in the study of gene function.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Enfermedades Cerebelosas/genética , Alelos , Animales , Ataxia/etiología , Canales de Calcio Tipo N , Canales de Calcio Tipo P , Canales de Calcio Tipo Q , Enfermedades Cerebelosas/patología , Enfermedades Cerebelosas/fisiopatología , Cerebelo/patología , Cerebelo/fisiopatología , Mapeo Cromosómico , Cruzamientos Genéticos , Análisis Mutacional de ADN , Femenino , Genes Recesivos , Prueba de Complementación Genética , Ligamiento Genético/fisiología , Marcadores Genéticos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/genética , Mutación Puntual , Células de Purkinje/patología , Temblor/etiologíaRESUMEN
We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.
Asunto(s)
Cerebelo/metabolismo , Lisosomas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Recuento de Células , Proteínas de Ciclo Celular , Cerebelo/química , Cerebelo/ultraestructura , Citoplasma/química , Proteínas de Unión al ADN , Femenino , Ganglios Espinales/química , Ganglios Espinales/citología , Inmunohistoquímica , Lisosomas/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Mutantes , Microscopía Electrónica , Neuronas/química , Proteínas Serina-Treonina Quinasas/análisis , Células de Purkinje/química , Células de Purkinje/citología , Células de Purkinje/ultraestructura , Proteínas Supresoras de TumorRESUMEN
mGluR1 mutant mice are viable but show characteristic cerebellar symptoms such as ataxic gait and intention tremor. The anatomy of the cerebellum is not overtly disturbed. Excitatory synaptic transmission from parallel fibers (PFs) to Purkinje cells and that from climbing fibers (CFs) to Purkinje cells appear to be functional, and voltage-gated Ca2+ channels of Purkinje cells are normal. Both PF and CF synapses display normal short-term synaptic plasticity to paired stimuli. By marked contrast, long-term depression (LTD) is clearly deficient and conditioned eyeblink response is impaired. We conclude that mGluR1 is required for the induction of LTD and that the ataxic behavior and impaired eyeblink conditioning of the mGluR1 mutant mice are primarily due to deficient LTD.
Asunto(s)
Aprendizaje por Asociación/fisiología , Ataxia/fisiopatología , Cerebelo/fisiología , Memoria/fisiología , Células de Purkinje/fisiología , Receptores de Glutamato/fisiología , Animales , Parpadeo , Calcio/fisiología , Cerebelo/anatomía & histología , Condicionamiento Clásico , Activación del Canal Iónico , Ratones , Ratones Noqueados , Plasticidad Neuronal , Transmisión SinápticaRESUMEN
Fluorescence in situ hybridization using two or three probes was utilized to estimate the incidence of diploidy, the incidence of disomy for the sex chromosomes and chromosomes 16 and 18, and the proportion of Y- and X-chromosome bearing sperm, in a series of normal males. Our results demonstrate the importance of using an approach capable of distinguishing disomy from diploidy, as most donors had levels of diploidy higher than the disomy levels of individual chromosomes. Our analyses suggest the existence of chromosome-specific mechanisms of paternal non-disjunction, as sex chromosome disomy was approximately 1.5 times as common as disomy 16, and over two times as common as disomy 18. In studies of gametic sex ratio, we found little evidence for marked deviation from an expected 1:1 ratio.
Asunto(s)
Cromosomas Humanos Par 16 , Cromosomas Humanos Par 18 , No Disyunción Genética , Espermatozoides/citología , Cromosoma X , Cromosoma Y , Diploidia , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Meiosis , Ploidias , Embarazo , Aberraciones Cromosómicas Sexuales , Razón de Masculinidad , Espermatozoides/fisiología , TrisomíaRESUMEN
OBJECTIVE: To determine the cause of absent sexual development in a 17-year-old girl with end-stage renal disease. DESIGN: Case study. PARTICIPANT: Seventeen-year-old girl with end-stage renal failure. INTERVENTIONS: None. MEASUREMENTS/MAIN RESULTS: The patient had phenotypically normal external female genitalia, müllerian duct hypoplasia, and no ovaries. Her serum gonadotropin levels were in the castrate range at baseline and after gonadotropin-releasing hormone stimulation. Her karyotype, in lymphocytes and cultured fibroblasts, was 46,XX. Analysis of genomic DNA, following polymerase chain reaction-amplication with oligonucleotide primers corresponding to the Y-encoded zinc finger protein ZFY and the testis-determining SRY gene, showed Y chromosome material in a male control but none in the patient. CONCLUSIONS: The results suggest a diagnosis of Frasier syndrome, a disorder characterized by true gonadal dysgenesis and end-stage renal disease occurring in normal phenotypic girls. Although previously reported only in individuals with a 46,XX karyotype, our studies indicate that Frasier syndrome may also occur in 46,XX girls. Delayed puberty is not uncommon in renal failure. This case illustrates the importance of measuring gonadotropin levels in teenage girls with delayed puberty and renal failure, particularly if the origin of the renal disease is obscure.