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

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 α1A voltage-dependent calcium channel (Cav2.1). Although a 75–85-kDa Cav2.1 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 Cav2.1 in human SCA6 brains. New antibodies against the Cav2.1 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 Cav2.1 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.

Development of Purkinje cell degeneration in a knockin mouse model reveals lysosomal involvement in the pathogenesis of SCA6

“…Cerebellar extracts from the MPI 118Q/118Q mice gave an intense 1C2 IR signal, showing an apparent molecular mass of ∼250 kDa, suggesting that the polyQ-expanded Ca v 2.1 channel was expressed in the cerebellum of the MPI 118Q/118Q mice. Recently, it was reported that the expanded polyQ-containing carboxyl-terminal fragment, which showed molecular masses ranging from 75 to 110 kDa depending on the CAG repeat size, was mislocalized to the nuclear fraction in the SCA6 cerebellum (11). However, we could not detect such a fragment in the cerebellum of the MPI 118Q/118Q mice.…”

“…We analyzed the formation of NIs in the mutant PCs by immunohistochemistry using an A6RPT-polyQ Ab (10,11). This Ab reacts specifically with humanized Ca v 2.1 and revealed NI formation in the cytoplasm of the Sca6 84Q/84Q Purkinje neurons (10).…”

“…S7A). We also conducted a double-immunofluorescence analysis on the postmortem cerebellar tissues of an SCA6 patient who had 22 CAG repeats in her mutant allele (11) and found that many of the 1C2-positive inclusions showed co-IF with LIMP2 (38 ± 6.4%, n = 5; Fig. 3E).…”

Endogenous TRPV1 stimulation leads to the activation of the inositol phospholipid pathway necessary for sustained Ca2+ oscillations

“…Since Ca 2+ regulates the cell cycle at several stages, Ca 2+ signaling is importantly involved in cell-fate determination (quiescent state, proliferation or cell death). Mitogenic compounds such as platelet-derived growth factor, vasopressin, prostaglandin, bombesin or EGF evoke repetitive Ca 2+ transients and also induce inositol trisphosphate (InsP 3 ) production [1,2]. In Swiss 3T3 cells, increases in c cyt evoked by mitogenic compounds are essential, but not sufficient to induce DNA synthesis and proliferation [3].…”

Relocation of p25¿/tubulin polymerization promoting protein from the nucleus to the perinuclear cytoplasm in the oligodendroglia of sporadic and COQ2 mutant multiple system atrophy

“…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).…”