Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

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.

DnaJ-1 and karyopherin α3 suppress degeneration in a newDrosophilamodel of Spinocerebellar Ataxia Type 6

“…Restriction of nuclear localization of polyQ proteins leads to significantly milder toxicity in cultured cells and in mouse models of polyQ diseases, including SCA6, whereas nuclear localization can be strongly toxic (29,31,(35)(36)(37). It deserves mention that a polyQ-expanded C-terminal fragment of the α1A protein that localized to the cytoplasm was found to be toxic in cultured HEK-293 and PC12 cells (38). We could surmise that benefits from subcellular restriction of the SCA6 protein are context-or cell-type-specific, thus leading to reduced degeneration of retinal cells in the background of kap-α3 mutation, but not sufficiently helpful when α1ACT(Q70) is expressed in other tissues in the same background.…”

Proteolytic Cleavage of Polyglutamine Disease-Causing Proteins: Revisiting the Toxic Fragment Hypothesis

“…Concerning the putative effect of polyQ expansion on nuclear localization of Cav2.1 fragments, in transfected PC12 cells, while full-length Cav2.1 and C-terminal fragments starting at amino acid 1954 are mainly cytoplasmic, the fragments were found also in the nucleus as well and their nuclear levels increased with larger numbers of glutamines [160,169]. What is more, C-terminal truncations starting at residue 1954 [167] or 2096 were strikingly enriched in the nucleus of HEK 293 cells, an effect that in the latter case was contingent upon three functional NLSs located N-terminally of the polyQ sequence [163].…”

“…A recent study has nonetheless advocated the idea that the Cav2.1 fragments are more toxic when present in the cytoplasm [169]. In transfected PC12 cells, the 1954-recombinant C-terminal fragment of Cav2.1 elicited increased toxic when artificially targeted to the cytoplasm comparing with when directed at the nucleus, an effect that was more notorious in the case of the expanded protein.…”

Molecular mechanism of Spinocerebellar Ataxia type 6: glutamine repeat disorder, channelopathy and transcriptional dysregulation. The multifaceted aspects of a single mutation

“…The formation of insoluble aggregates which leads to the production of intranuclear inclusions is the principal pathogenic feature in most of the polyglutamine diseases (Paulson, 1999 ). In SCA6, aggregates are rare, preferentially located in the cytoplasm as in SCA2 (Huynh et al, 2000 ; Ishiguro et al, 2010 ; Takahashi et al, 2013 ) and very rarely localizing to the nucleus in Purkinje cells (Kordasiewicz et al, 2006 ; Ishiguro et al, 2010 ). In SCA6 the normal repeat size ranges from 4 to 18 units (Zhuchenko et al, 1997 ) while that of the expanded alleles is from 20 to 33 repeats (Jodice et al, 1997 ; Yabe et al, 1998 ).…”