title: The carboxy-terminal fragment of alpha(1A) 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 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.
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Ishiguro
authors.forename: Kinya
authors.initials: K
authors.lastname: Ishikawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Takahashi
authors.forename: Masato
authors.initials: M
authors.lastname: Obayashi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Amino
authors.forename: Nozomu
authors.initials: N
authors.lastname: Sato
authors.forename: Masaki
authors.initials: M
authors.lastname: Sakamoto
authors.forename: Hiroto
authors.initials: H
authors.lastname: Fujigasaki
authors.forename: Fuminori
authors.initials: F
authors.lastname: Tsuruta
authors.forename: Ricardo
authors.initials: R
authors.lastname: Dolmetsch
authors.forename: Takao
authors.initials: T
authors.lastname: Arai
authors.forename: Hidenao
authors.initials: H
authors.lastname: Sasaki
authors.forename: Kazuro
authors.initials: K
authors.lastname: Nagashima
authors.forename: Takeo
authors.initials: T
authors.lastname: Kato
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Yamada
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hashizume
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Mizusawa
mesh_terms: 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
chemical_list: D020864:Calcium Channels, N-Type; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; C097188:polyglutamine