Dataset Information

New ataxic tottering-6j mouse allele containing a Cacna1a gene mutation.

ABSTRACT: Voltage-gated Ca(2+) (Ca(v)) channels control neuronal functions including neurotransmitter release and gene expression. The Cacna1a gene encodes the ?1 subunit of the pore-forming Ca(v)2.1 channel. Mice with mutations in this gene form useful tools for defining channel functions. The recessive ataxic tottering-6j strain that was generated in the Neuroscience Mutagenesis Facility at The Jackson Laboratory has a mutation in the Cacna1a gene. However, the effect of this mutation has not been investigated in detail. In this study, mutation analysis shows a base substitution (C-to-A) in the consensus splice acceptor sequence linked to exon 5, which results in the skipping of exon 5 and the splicing of exon 4 directly to exon 6. The effect of this mutation is expected to be severe as the expressed ?1 subunit protein lacks a significant part of the S4-S5 linker, S5, and part of S5-S6 linker in domain I. Tottering-6j mice display motor dysfunctions in the footprint, rotating rod, and hind-limb extension tests. Although cytoarchitecture of the mutant brains appears normal, tyrosine hydroxylase was persistently expressed in cerebellar Purkinje cells in the adult mutant mice. These results indicate that tottering-6j is a useful model for functional studies of the Ca(v)2.1 channel.

SUBMITTER: Li W

PROVIDER: S-EPMC3432086 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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New ataxic tottering-6j mouse allele containing a Cacna1a gene mutation.

Li Weidong W Zhou Ying Y Tian Xiaoli X Kim Tae Yeon TY Ito Namiko N Watanabe Kaori K Tsuji Akiko A Niimi Kimie K Aoyama Yo Y Arai Takashi T Takahashi Eiki E

PloS one 20120831 8

Voltage-gated Ca(2+) (Ca(v)) channels control neuronal functions including neurotransmitter release and gene expression. The Cacna1a gene encodes the α1 subunit of the pore-forming Ca(v)2.1 channel. Mice with mutations in this gene form useful tools for defining channel functions. The recessive ataxic tottering-6j strain that was generated in the Neuroscience Mutagenesis Facility at The Jackson Laboratory has a mutation in the Cacna1a gene. However, the effect of this mutation has not been inves ...[more]

PMID: 22952933

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Project description:Spinocerebellar ataxias (SCAs) are diseases characterized by cerebellar atrophy and loss of Purkinje neurons caused by mutations in diverse genes. In SCA14, the disease is caused by point mutations or small deletions in protein kinase C γ (PKCγ), a crucial signaling protein in Purkinje cells. It is still unclear whether increased or decreased PKCγ activity may be involved in the SCA14 pathogenesis. In this study, we present a new knock-in mouse model related to SCA14 with a point mutation in the pseudosubstrate domain, PKCγ-A24E, known to induce a constitutive PKCγ activation. In this protein conformation, the kinase domain of PKCγ is activated, but at the same time the protein is subject to dephosphorylation and protein degradation. As a result, we find a dramatic reduction of PKCγ protein expression in PKCγ-A24E mice of either sex. Despite this reduction, there is clear evidence for an increased PKC activity in Purkinje cells from PKCγ-A24E mice. Purkinje cells derived from PKCγ-A24E have short thickened dendrites typical for PKC activation. These mice also develop a marked ataxia and signs of Purkinje cell dysfunction making them an interesting new mouse model related to SCA. Recently, a similar mutation in a human patient was discovered and found to be associated with overt SCA14. RNA profiling of PKCγ-A24E mice showed a dysregulation of related signaling pathways, such as mGluR1 or mTOR. Our results show that the induction of PKCγ activation in Purkinje cells results in the SCA-like phenotype indicating PKC activation as one pathogenetic avenue leading to a SCA.SIGNIFICANCE STATEMENT Spinocerebellar ataxias (SCAs) are hereditary diseases affecting cerebellar Purkinje cells and are a one of neurodegenerative diseases. While mutation in several genes have been identified as causing SCAs, it is unclear how these mutations cause the disease phenotype. Mutations in PKCγ cause one subtype of SCAs, SCA14. In this study, we have generated a knock-in mouse with a mutation in the pseudosubstrate domain of PKCγ, which keeps PKCγ in the constitutive active open conformation. We show that this mutation leading to a constant activation of PKCγ results in a SCA-like phenotype in these mice. Our findings establish the constant activation of PKC signaling as one pathogenetic avenue leading to an SCA phenotype and a mechanism causing a neurodegenerative disease.

Project description:Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T>C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs137 showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions.

Dataset Information

New ataxic tottering-6j mouse allele containing a Cacna1a gene mutation.

Publications

New ataxic tottering-6j mouse allele containing a Cacna1a gene mutation.

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