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: 10.76). 04/2010; 119(4):447-64. DOI: 10.1007/s00401-009-0630-0
Source: PubMed


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.

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Available from: Hidehiro Mizusawa, Sep 29, 2015
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    • "Western blotting analysis was performed as described previously [25]. Transfer and detection were carried out according to the protocol provided with the ECL Detection System (Amersham Pharmacia Biotech, Piscataway, NJ, USA). "
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    ABSTRACT: p25¿/tubulin polymerization promoting protein (TPPP) is an oligodendroglial protein that plays crucial roles including myelination, and the stabilization of microtubules. In multiple system atrophy (MSA), TPPP is suggested to relocate from the myelin sheath to the oligodendroglial cell body, before the formation of glial cytoplasmic inclusions (GCIs), the pathologic hallmark of MSA. However, much is left unknown about the re-distribution of TPPP in MSA. We generated new antibodies against the N- and C-terminus of TPPP, and analyzed control and MSA brains, including the brain of a familial MSA patient carrying homozygous mutations in the coenzyme Q2 gene (COQ2). In control brain tissues, the TPPP was localized not only in the cytoplasmic component of the oligodendroglia including perinuclear cytoplasm and peripheral processes in the white matter, but also in the nucleus of a fraction (62.4%) of oligodendroglial cells. Immunoelectron microscopic analysis showed TPPP in the nucleus and mitochondrial membrane of normal oligodendroglia, while western blot also supported its nuclear and mitochondrial existence. In MSA, the prevalence of nuclear TPPP was 48.6% in the oligodendroglia lacking GCIs, whereas it was further decreased to 19.6% in the oligodendroglia with phosphorylated ¿-synuclein (p¿-syn)-positive GCIs, both showing a significant decrease compared to controls (62.4%). In contrast, TPPP accumulated in the perinuclear cytoplasm where mitochondrial membrane (TOM20 and cytochrome C) and fission (DRP1) proteins were often immunoreactive. We conclude that in MSA-oligodendroglia, the TPPP is reduced, not only in the peripheral cytoplasm, but also in the nucleus and relocated to the perinuclear cytoplasm.
    09/2014; 2(1):136. DOI:10.1186/PREACCEPT-3606739711332434
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    • "In the presence of the NLS tag, the rCTF-Q13-NLS dramatically translocated to the nucleus, whereas the NES tag made the rCTF-Q13-NES anchor in the cytoplasm (Figure 1B). This result was confirmed in human embryonic kidney (HEK) 293T cells (Figure S2), and was consistent with the location of native CTF in human brain [18]. The expanded version of the CTF, rCTF-Q28 was also expressed mainly in the cytoplasm with some obvious amounts in the nucleus (Figure 1C). "
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    ABSTRACT: The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.
    PLoS ONE 03/2013; 8(3):e50121. DOI:10.1371/journal.pone.0050121 · 3.23 Impact Factor
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    • "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. "
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    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 · 5.08 Impact Factor
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