The carboxy-terminal fragment of alpha(1A) calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells.
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.
Ishiguro
Taro
T
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan.
Ishikawa
Kinya
K
Takahashi
Makoto
M
Obayashi
Masato
M
Amino
Takeshi
T
Sato
Nozomu
N
Sakamoto
Masaki
M
Fujigasaki
Hiroto
H
Tsuruta
Fuminori
F
Dolmetsch
Ricardo
R
Arai
Takao
T
Sasaki
Hidenao
H
Nagashima
Kazuro
K
Kato
Takeo
T
Yamada
Mitsunori
M
Takahashi
Hitoshi
H
Hashizume
Yoshio
Y
Mizusawa
Hidehiro
H
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001344:Autopsy; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism / Q000473:pathology*; D003600:Cytosol / Q000378:metabolism; D003712:Dendrites / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011689:Purkinje Cells / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020754:Spinocerebellar Ataxias / Q000378:metabolism* / Q000473:pathology
D020864:Calcium Channels, N-Type; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; C097188:polyglutamine