Article

The carboxy-terminal fragment of α1A calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells

Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan.
Acta Neuropathologica (Impact Factor: 9.78). 04/2010; 119(4):447-64. DOI: 10.1007/s00401-009-0630-0
Source: PubMed

ABSTRACT Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4-20Q; SCA6: 20-33Q) in the carboxyl(C)-terminal cytoplasmic domain of the alpha(1A) voltage-dependent calcium channel (Ca(v)2.1). Although a 75-85-kDa Ca(v)2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Ca(v)2.1 in human SCA6 brains. New antibodies against the Ca(v)2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Ca(v)2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus.

Download full-text

Full-text

Available from: Hidehiro Mizusawa, Jul 30, 2015
0 Followers
 · 
244 Views
  • Source
    • "However, given the level of Ca V 2.1 knockdown observed in human SK-N-SH cells following SIS-RNAi, it will be important to assess, in vivo, the extent to which SIS-RNAi targeting of polyQ-encoding Ca V 2.1 mRNAs affects overall Ca V 2.1 levels. Finally, several studies support a model of SCA6 pathogenesis that includes the accumulation and aberrant nuclear translocation of an expanded polyQ-containing Ca V 2.1 fragment (Ishiguro et al., 2009; Kordasiewicz et al., 2006; Kubodera et al., 2003; Marqueze-Pouey et al., 2008). Thus, SIS-RNAi-mediated suppression of the polyQ-encoding Ca V 2.1 splice variant should also reduce levels of putative pathogenic Ca V 2.1 fragments and be of therapeutic benefit in SCA6. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the Ca(V)2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a Ca(V)2.1 mini-gene reporter system, we found that pathogenic CAG expansions in Ca(V)2.1 enhance splicing activity at the 3'end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding Ca(V)2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding Ca(V)2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding Ca(V)2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases.
    Neurobiology of Disease 04/2011; 43(3):533-42. DOI:10.1016/j.nbd.2011.04.016 · 5.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: This paper describes an attempt to use a biodynamical measure to evaluate the performance of human-robot interfaces that should be comfortable for human operators. Since the issue of comfort is human related, we should adopt some biodynamical measures to evaluate the comforts of human operator. This paper proposes a quantitative biodynamical measure that depends on the heart rate, instantaneous lung volume and arterial blood pressure of the human operator. Experiments show that there exists a clear relation between the measure and the task stress of operators
    Robot and Human Communication, 1995. RO-MAN'95 TOKYO, Proceedings., 4th IEEE International Workshop on; 08/1995
  • [Show abstract] [Hide abstract]
    ABSTRACT: A number of neurodegenerative diseases, including Alzheimer's disease, tauopathies, Parkinson's disease, and synucleinopathies, polyglutamine diseases, including Huntington's disease, amyotrophic lateral sclerosis, and transmissible spongiform encephalopathy, are characterized by the existence of a protein or peptide prone to aggregation specific to the disease: amyloid-β, tau protein, α-synuclein, atrophin 1, androgen receptor, prion protein, copper-zinc superoxide dismutase, α 1A subunit of CaV2.1, TATA-box binding protein, huntingtin, and ataxins 1, 2, 3, and 7. Beside this common molecular feature, we have found three additional main properties related to the disease-connected protein or peptide, which are shared by all those neurological disorders: first, proneness to aggregation, which, in many cases, seems to be bound to the lack of a clearly defined secondary structure; second, reported presence of the disease-related protein inside the nucleus; and finally, an apparently unspecific interaction with DNA. These findings, together with the lack of clear details to explain the molecular origin of these neurodegenerative diseases, invite a hypothesis that, together with other plausible molecular explanations, may contribute to find the molecular basis of these diseases: I propose here the hypothesis that many neurological disorders may be the consequence, at least in part, of an aberrant interaction of the disease-related protein with nucleic acids, therefore affecting the normal DNA expression and giving place to a genetic stress which, in turn, alters the expression of proteins needed for the normal cellular function and regulation.
    Journal of Alzheimer's disease: JAD 01/2010; 22(2):375-91. DOI:10.3233/JAD-2010-100189 · 4.15 Impact Factor
Show more