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Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells.

ABSTRACT: Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the ?1A subunit of the P/Q type voltage-gated Ca²⁺ channel Ca_V2.1 but also the ?1ACT protein, a 75?kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the ?1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding ?1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of Ca_V2.1 channel protein. The ?1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in Ca_V2.1 channel function were observed, they were found in the expression levels of the ?1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability.

SUBMITTER: Bavassano C

PROVIDER: S-EPMC5684673 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells.

Bavassano Carlo C Eigentler Andreas A Stanika Ruslan R Obermair Gerald J GJ Boesch Sylvia S Dechant Georg G Nat Roxana R

Stem cells and development 20171030 22

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca<sup>2+</sup> channel Ca<sub>V</sub>2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both pro ...[more]

PMID: 28946818

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Project description:Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3. The accumulation of the mutant ataxin-3 proteins carrying expanded polyglutamine (polyQ) leads to selective degeneration of neurons. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies have been identified, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3. Induced pluripotent stem cells (iPSCs) can be used as the ideal cell model for the molecular pathogenesis of polyQ diseases. Abnormal CAG expansions mediated by CRISPR/Cas9 genome engineering technologies have shown promising potential for the treatment of polyQ diseases, including SCA3. In this study, SCA3-iPSCs can be corrected by the replacement of the abnormal CAG expansions (74 CAG) with normal repeats (17 CAG) using CRISPR/Cas9-mediated homologous recombination (HR) strategy. Besides, corrected SCA3-iPSCs retained pluripotent and normal karyotype, which can be differentiated into a neural stem cell (NSCs) and neuronal cells, and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the neuronal differentiation from normal control iPSCs (Ctrl-iPSCs), SCA3-iPSCs, and isogenic control SCA3-iPSCs. Furthermore, this study proved that the phenotypic abnormalities in SCA3 neurons, including aggregated IC2-polyQ protein, decreased mitochondrial membrane potential (MMP) and glutathione expressions, increased reactive oxygen species (ROS), intracellular Ca2+ concentrations, and lipid peroxidase malondialdehyde (MDA) levels, all were rescued in the corrected SCA3-NCs. For the first time, this study demonstrated the feasibility of CRISPR/Cas9-mediated HR strategy to precisely repair SCA3-iPSCs, and reverse the corresponding abnormal disease phenotypes. In addition, the importance of genetic control using CRISPR/Cas9-mediated iPSCs for disease modeling. Our work may contribute to providing a potential ideal model for molecular mechanism research and autologous stem cell therapy of SCA3 or other polyQ diseases, and offer a good gene therapy strategy for future treatment.

Dataset Information

Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells.

Publications

Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells.

Similar Datasets

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