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Human Molecular Genetics Advance Access originally published online on April 4, 2006
Human Molecular Genetics 2006 15(10):1587-1599; doi:10.1093/hmg/ddl080
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

C-termini of P/Q-type Ca2+ channel {alpha}1A subunits translocate to nuclei and promote polyglutamine-mediated toxicity

Holly B. Kordasiewicz1,2, Randall M. Thompson1,2, H. Brent Clark2,3 and Christopher M. Gomez1,2,*

1Department of Neuroscience, 2Department of Neurology and 3Section of Neuropathology, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA

* To whom correspondence should be addressed at: Department of Neurology, AMB S237, MC2030, The University of Chicago, 5841 S. Maryland, Chicago, IL 60637, USA. Tel: +1 7737026390; Fax: +1 7737025670; Email: gomez001{at}uchicago.edu

Received November 12, 2005; Accepted March 24, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
P/Q-type voltage-gated calcium channels are regulated, in part, through the cytoplasmic C-terminus of their {alpha}1A subunit. Genetic absence or alteration of the C-terminus leads to abnormal channel function and neurological disease. Here, we show that the terminal 60–75 kDa of the endogenous {alpha}1A C-terminus is cleaved from the full-length protein and is present in cell nuclei. Antiserum to the C-terminus (CT-2) labels both wild-type mouse and human Purkinje cell nuclei, but not leaner mouse cerebellum. Human embryonic kidney cells stably expressing ß3 and {alpha}2{delta} subunits and transiently transfected with full-length human {alpha}1A contain a 75 kDa CT-2 reactive peptide in their nuclear fraction. Primary granule cells transfected with C-terminally Green fluorescent protein (GFP)-tagged {alpha}1A exhibit GFP nuclear labeling. Nuclear translocation depends partly on the presence of three nuclear localization signals within the C-terminus. The C-terminal fragment bears a polyglutamine tract which, when expanded (Q33) as in spinocerebellar ataxia type 6 (SCA6), is toxic to cells. Moreover, polyglutamine-mediated toxicity is dependent on nuclear localization. Finally, in the absence of flanking sequence, the Q33 expansion alone does not kill cells. These results suggest a novel processing of the P/Q-type calcium channel and a potential mechanism for the pathogenesis of SCA6.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinocerebellar ataxia type 6 (SCA6) is a disorder of progressive cerebellar dysfunction and is one of at least three dominantly inherited neurological disorders caused by mutations in the CACNA1A gene (1Go,2Go). The CACNA1A gene encodes the {alpha}1A subunit, the transmembrane pore-forming subunit of the P/Q-type or CaV2.1 voltage-gated calcium channel (VGCC) (2Go). Whereas the other CACNA1A disorders are associated with simple missense, truncating or splicing mutations, SCA6, such as Huntington's disease (HD) (3Go) and other forms of SCA (4Go–6Go), is caused by abnormal expansion of a trinucleotide CAG repeat encoding an elongated tract of glutamine residues. In SCA6, the expansion is found in exon 47 of the CACNA1A gene, which normally contains a polymorphic CAG repeat tract (CAG)4–18 encoding 4–18 glutamines in the C-terminus of the {alpha}1A subunit, but is expanded to the pathological range of (CAG)19–33, encoding 19–33 glutamines (7Go,8Go).

VGCC are multimeric complexes composed of at least three protein subunits: a pore-forming subunit ({alpha}1) and two auxiliary subunits (ß and {alpha}2{delta}). Distinct genes encode more than 10 different {alpha}1 subunits ({alpha}1A–I,S), which confer different channel properties and are expressed in different cell types (9Go,10Go). Distinct phenotypic abnormalities result from mutations in the various VGCC genes (1Go,2Go). P/Q channels are involved in a diverse array of cell functions including neurotransmitter release, regulation of gene expression, release of calcium from internal stores and dendritic calcium transients (9Go–12Go). P/Q channels are highly expressed in cerebellar neurons and localize primarily to nerve terminals, dendrites and Purkinje cell soma (11Go,13Go).

The full-length {alpha}1A subunit contains four homologous transmembrane repeat domains (I–IV) flanked by three intracellular loops (LI–II, LII–III, LIII–IV) and cytoplasmic N- and C-termini (9Go). Complementary DNA sequencing and protein immunoblot studies with domain-specific antisera have demonstrated the presence of {alpha}1A polypeptides of a range of sizes and domain compositions arising from alternative splicing and possible proteolytic processing (14Go–16Go). Several studies have suggested the presence of N-terminal fragments of the {alpha}1A subunit that lack portions of the distal C-terminus ranging from 40 kDa to the entire last two transmembrane repeat domains (17Go–19Go). Conversely, a 75 kDa C-terminal fragment has been detected in protein extracts of full-length {alpha}1A-expressing human embryonic kidney (HEK) cells (17Go,20Go). These observations suggest that, at least in heterologous systems, the {alpha}1A C-terminus is cleaved and may form a stable {alpha}1A polypeptide.

The {alpha}1A C-terminus participates in a number of protein–protein interactions and plays a prominent role in modulating channel activity (21Go). Thus, its alteration by proteolytic cleavage or genetic mutation would have significant functional consequences. Mice homozygous for the leaner (Tgla) mutation, which express {alpha}1A subunits lacking the distal C-terminus, are severely ataxic, exhibit Purkinje cell degeneration and die if unattended at ~21 days (22Go).

In this study, we explored, in the cerebellum, the distribution of putative endogenous C-terminal polypeptides using an antibody we raised against an {alpha}1A C-terminal epitope. We found that the C-terminal {alpha}1A antiserum labels Purkinje cell nuclei and that a 60–75 kDa proteolytic fragment of the endogenous {alpha}1A C-terminus is present in cell nuclei. Using plasmid vectors expressing recombinant human {alpha}1A protein, we identified three nuclear localization signals (NLSs). Finally, because the cleaved C-terminal fragment bears a polyglutamine tract, which is expanded in SCA6, we tested whether expansion of the polyglutamine tract correlated with cell death. Cells expressing C-terminal proteins bearing polyglutamine tracts of 33 glutamines had more than twice the rate of cell death than those expressing unexpanded normal tracts. Furthermore, this polyglutamine-mediated cell death appears to be dependent on nuclear localization of the C-terminal fragment. These results suggest that the {alpha}1A C-terminal cleavage product may play a role in nuclear signaling and in the pathogenesis of SCA6.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The C-terminus of the {alpha}1A subunit is present in cell nuclei
To determine the subcellular distribution of the {alpha}1A subunit in human cerebellar cortex, we used affinity-purified, anti-peptide antibodies, specific for either the N-terminus (NT-1 antibody) or the C-terminus (CT-2 antibody) of the {alpha}1A subunit. Surprisingly, in paraffin-embedded human cerebellar tissue, the C-terminal antibody intensely stained Purkinje cell nuclei (Fig. 1A, inset). CT-2 nuclear staining was not observed in granule cells or any other cerebellar cell type. NT-1 and CT-2 antibody also labeled the Purkinje cell membrane, projections and cytoplasm as previously reported for other {alpha}1A antibodies (13Go,23Go,24Go) (Fig. 1A and B). NT-1 antibodies did not label cell nuclei (Fig. 1B), although they did intensely stain Purkinje cell membranes. A punctate staining pattern was observed at the cell membrane with both NT-1 and CT-2 antibodies (Fig. 1A and B, inset). Immunostaining by both NT-1 and CT-2 was specifically blocked by preincubation with their respective peptide immunogens (Fig. 1C and D), but not with other peptides (data not shown). An identical pattern of immunolabeling was seen in adult mouse cerebellar sections (Fig. 1E and F).


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  		Figure 1. The {alpha}1A C-terminus is present in Purkinje cell nuclei in mouse and human cerebellum. Cerebellar sections stained using immunoperoxidase (brown) with either anti-CT-2 (A, C, E, G and I) or anti-NT-1 (B, D, F, H and J). Blue nuclear counter stain is hematoxylin. Paraffin-embedded cerebellar sections were from adult human (A and B); adult human blocked by pre-incubation with the respective peptide (C and D), WT adult mouse (E and F), homozygous leaner mouse (G and H) and P18 WT mouse (I and J). Insets are enlargements of Purkinje cell staining. White arrows indicate Purkinje cell nuclei. Black arrows indicate Purkinje cell projections and cell soma. Scale bars: 75 µm.

 
As a further test of specificity, we compared immunostaining of cerebellar sections from homozygous leaner (Tgla) pups, which express mutant {alpha}1A subunits lacking a C-terminus and thus the CT-2 epitope, with age-matched post-natal day 18 (P18) control pups. CT-2 antibody failed to label Purkinje cells from leaner cerebellum, either in the cytoplasm or in the nucleus (Fig. 1G). NT-1 antibody stained leaner cerebellum in the same pattern as control cerebellum, although slightly less intensely (Fig. 1H and J). In control P18 cerebellum, CT-2 antibody labeled nuclei more intensely than wild-type (WT) adult nuclei, and both NT-1 and CT-2 antibody labeled WT P18 Purkinje cell soma (Fig. 1I and J, inset). Cell membranes and projections were stained relatively less intensely in control P18 cerebellum than in adults. These findings indicate that the C-terminus of the {alpha}1A subunit is localized to the nucleus as well as to the cell membrane where the full-length channel resides.

To investigate whether the nuclear labeling of the {alpha}1A C-terminal antibody can be attributed to expression and processing of the full-length {alpha}1A subunit, we transiently transfected HEK cells, stably expressing ß3 and {alpha}2{delta} (SHEKß3), with full-length human {alpha}1A cDNA (Fig. 2A). We hypothesized that the CT-2 nuclear labeling resulted from the cleavage product previously identified in full-length {alpha}1A-transfected HEK cells (20Go). HEK cells, although they do not express endogenous VGCCs, are able to form functional channels when {alpha}1, ß and {alpha}2{delta} are ectopically co-expressed. Auxiliary subunits (ß and {alpha}2{delta}) are required for proper trafficking of the {alpha}1 subunit (21Go); in the absence of the ß subunit {alpha}1 is retained in the endoplasmic reticulum, thus HEK cells stably expressing ß3 and {alpha}2{delta} were employed. Eighteen to twenty hours after transfection, CT-2 antibody stained the nucleus and cytoplasm, whereas NT-1 antibody solely labeled SHEKß3 cell cytoplasm (Fig. 3A).


Figure 0802
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  		Figure 2. Schematic diagram of {alpha}1A constructs used in this study. (A) Full-length {alpha}1A and various truncated forms. Amino acid numbers are displayed on the top. Constructs denoted Qn contain either Q4, Q11 or Q33 glutamine repeats. If the glutamine repeat size is not indicated, then Q11 was used. X indicates approximate location of NLS mutations. Constructs tagged with EGFP and/or an artificial NES are indicated. (B) A schematic representation of the C-terminal construct and approximate location of motifs of interest. Included are NLS1,2,3 and 4, the evolutionarily conserved histidine tract (HIS) and the CT-2 antibody recognition sequence (CT-2 Epitope).

 

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  		Figure 3. The C-terminal cleavage product localizes to cell nuclei in {alpha}1A transiently transfected stable ß3 and {alpha}2{delta} HEK293 (SHEKß3) cells, NIH3T3 cells and transfected cerebellar granule neurons. Confocal micrographs of cells transfected with human {alpha}1A cDNA ({alpha}1A, GFPNH3{alpha}1A or GFPCOOH{alpha}1A, as indicated). Cells were stained with CT-2 or NT-1 antibodies and the nucleic acid dye ToTo-3 (blue). Transmitted light images are provided. (A) Immunocytochemistry of SHEKß3 cells expressing {alpha}1A and stained with CT-2 or NT-1 antibody (green) and overlaid with ToTo-3 (blue). (B) Dual labeling of SHEKß3 cells with antibody (red) and EGFP (green). Cells transfected with GFPNH3{alpha}1A were stained with CT-2 antibody and cells transfected with GFPCOOH{alpha}1A were stained with NT-1 (constructs and antibodies are indicated in individual images). (C) The proportion (%) of SHEKß3 cells exhibiting nuclear labeling with N-terminal EGFP (GFPNH3{alpha}1A), C-terminal EGFP (GFPCOOH{alpha}1A), NT-1 antibody or CT-2 antibody (n=147, 114, 73 or 119, respectively). A significantly greater percentage of nuclei were labeled with C-terminal markers (CT-2 and C-terminal EGFP) than with N-terminal markers (NT-1 and N-terminal EGFP). EGFP (z=12.21, P<0.01) and antibody (z=9.86, P<0.01). (D) Dual labeling of NIH3T3 cells co-transfected with ß3, {alpha}2{delta} and {alpha}1A (GFPNH3{alpha}1A or GFPCOOH{alpha}1A) and stained with {alpha}1A antibodies (NT-1 or CT-2). (E) Primary cerebellar cultures expressing N-terminally GFP-tagged {alpha}1A (GFPNH3{alpha}1A), C-terminally GFP-tagged {alpha}1A (GFPCOOH{alpha}1A) or GFP-tagged {alpha}1A C-terminus (GFP2096). Scale bars: 10 µm.

 
To exclude artifacts due to antibody cross-reactivity, we generated fusion constructs that were tagged on either the N-terminus (GFPNH3{alpha}1A) or the C-terminus (GFPCOOH{alpha}1A) of the full-length protein with enhanced green fluorescent protein (EGFP) (Fig. 2A). When utilized in conjunction with our N- and C-terminal antibodies, the tagged {alpha}1A subunits allow for simultaneous localization of both ends of the protein in a single cell. In individual cells expressing N-terminally GFP-tagged {alpha}1A (GFPNH3{alpha}1A) and stained with C-terminal antibody (CT-2), signals for the two termini were spatially separated (Fig. 3B). The N-terminal GFP localized solely to the cytoplasm, indicating that the N-terminal portion of the protein resided outside the nucleus, as expected. In contrast, in these same cells, the C-terminal antibody stained both the cytoplasm and the nucleus, suggesting that some of the C-terminal {alpha}1A polypeptides localize to the cytoplasm with the N-terminus of the protein and a second population of C-terminal {alpha}1A polypeptide localizes to the nucleus. The converse experiment, with GFPCOOH{alpha}1A and NT-1 antibody exhibited similar results (Fig. 3B). Quantification of immunostain data showed that a significantly greater percentage of {alpha}1A expressing cells exhibited nuclear staining when stained with CT-2 antibody or transfected with C-terminally GFP-tagged {alpha}1A than with N-terminal markers (Fig. 3C). Similar results were obtained in the mouse fibroblast cell line, NIH3T3 (Fig. 3D), in which GFP-tagged {alpha}1A was co-expressed with ß3 and {alpha}2{delta}. This observation suggests that a portion of the C-terminus of the {alpha}1A subunit is cleaved and translocated to nuclei.

To determine whether {alpha}1A cleavage also occurs in neuronal cells, primary cerebellar granule cells were transfected with GFPNH3{alpha}1A or GFPCOOH{alpha}1A. In neurons transfected with GFPNH3{alpha}1A, GFP was localized to the cytoplasm, whereas in neurons transfected with GFPCOOH{alpha}1A, the GFP signal was present in both the nucleus and the cytoplasm (Fig. 3E). Finally, we generated a GFP fusion construct that expresses only the {alpha}1A C-terminus downstream of the predicted cleavage site, this comprises residues 2096–2510 (GFP2096) (Fig. 2A and B). The N-terminus of this {alpha}1A protein fragment was tagged with GFP. This C-terminal protein also localized to nuclei in transfected granule cells (Fig. 3E). Because of cross-reactivity, {alpha}1A specific antibodies were unable to distinguish recombinant {alpha}1A subunits from endogenous {alpha}1A in transfected granule cells. Nevertheless, these cell culture data suggest that the CT-2 nuclear staining observed in Purkinje cells arises from cleavage of the {alpha}1A C-terminus and its translocation to the nucleus.

The {alpha}1A C-terminus is cleaved from the full-length protein
Using antibody to the {alpha}1A C-terminus (CT-2), we identified three polypeptides of molecular weights 220, 170 and 60 kDa in western blots of mouse cerebellar lysates (Fig. 4A). The 220 and 170 kDa proteins correspond to previously reported full-length {alpha}1A subunit species (16Go,17Go). To test whether the 60 kDa polypeptide represents a C-terminal fragment of the {alpha}1A subunit, we utilized the spontaneous mouse mutant, leaner. Leaner mice have a splice site mutation in the CACNA1A gene that results in aberrant splicing in the region of the {alpha}1A C-terminus. This yields two predominant {alpha}1A splice forms that lack the C-terminus after residue 1967 including the CT-2 epitope (22Go). The CT-2 reactive 220, 170 and 60 kDa polypeptides are undetectable in cerebellar lysates of homozygous leaner mice (Tgla), compared with age-matched (P18) control pups under these experimental conditions (Fig. 4A), whereas the signal detected by antisera to other {alpha}1A polypeptide splice forms and antisera to other synaptic components, such as SNAP 25, synaptotagamin and synaptophysin, was identical in leaner and age-matched controls (data not shown).


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  		Figure 4. A C-terminal fragment of {alpha}1A is present in mouse cerebellum and in {alpha}1A-transfected SHEKß3 cells. (A) Cerebellar lysates from WT adult mice, P18 WT mice and homozygous leaner (Tgla–/–) mice were analyzed by western blot with anti-CT-2 antibody. Arrows indicate 220, 170 and 60 kDa CT-2-positive {alpha}1A proteins. Top panel of (A) is a longer exposure of CT-2 reactive 220 and 170 peptides. Anti-GAPDH was used as a loading control. (B) Immunoprecipitation (IP) of mouse cerebellar lysate with CT-2 antibody and staphylococcus protein A coated beads (SPA). Precipitated proteins were analyzed by western blot with anti-penta-HIS antibody (HIS). Arrow indicates 60 kDa band not present in negative controls, IP without antibody (WT) and IP without lysate (SPA). (C and D) SHEKß3 untransfected (UT), transfected with GFPNH3{alpha}1A or GFPCOOH{alpha}1A were analyzed by western blot with anti-CT-2 or anti-GFP. GFPNH3{alpha}1A cell lysates contained a 75 kDa CT-2-positive protein that was shifted ~30 kDa in GFPCOOH{alpha}1A cell lysates. (E) Immunoblot comparison of stable ß3 and {alpha}2{delta} HEK cells (SHEKß3) and untransfected HEK 293 cells with anti-ß3 and a loading control (anti-GAPDH). Molecular weights are indicated at the right of each panel in kDa. All experiments were replicated with similar results.

 
The C-terminus of the {alpha}1A subunit contains an evolutionarily conserved tract of 11 histidine residues. To verify the identity of the 60 kDa polypeptide as the C-terminus of {alpha}1A, we immunoprecipitated proteins from mouse cerebellar homogenates with CT-2 antibody, separated them using SDS–PAGE gel electrophoresis and reacted them with an antibody to penta-HIS (anti-penta-HIS, Sigma, St Louis, MO, USA). This reagent recognizes proteins possessing five or more consecutive histidines. Anti-penta-HIS antibody also recognized a 60 kDa species in the CT-2-precipitated lanes but not in control lanes (Fig. 4B). The 55 kDa polypeptide present in the experimental condition and the control without lysate (SPA and CT-2), corresponds to IgG heavy chain.

We next tested for the presence and size of a corresponding human {alpha}1A fragment in SHEKß3 cells transiently transfected with the cDNA encoding the full-length human {alpha}1A subunit. We detected a 75 kDa polypeptide, similar to that observed in mouse brain homogenates using CT-2 antibody, in lysates of {alpha}1A transfected SHEKß3 cells (Fig. 4C). Sequence alignment of the mouse {alpha}1A C-terminus containing the CT-2 epitope (MPII splice form) (25Go) and the known human {alpha}1A C-terminus predicts that the human form is 10–15 kDa larger than the mouse C-terminus.

To further confirm that the 75 kDa polypeptide corresponds to the {alpha}1A C-terminus, we performed western blot analysis of lysates of cells transfected with full-length {alpha}1A C-terminally labeled with GFP (GFPCOOH{alpha}1A). In these cell lysates, a novel 100 kDa protein was recognized by CT-2 antiserum but the 75 kDa protein was absent (Fig. 4D). The 100 kDa band was also reactive with GFP antibody, consistent with the presence of a 30 kDa GFP protein on the C-terminus of {alpha}1A extending the polypeptide (Fig. 4D). Thus, the C-termini of {alpha}1A subunits from two different species are cleaved in vivo and when expressed as recombinant proteins in cell culture. Finally, the identity of the 75 kDa protein as the {alpha}1A C-terminus was further confirmed using a second antibody to C-termini, CT-1 (24Go). Although the signal intensity was weaker, CT-1 antibody also recognized the 75 kDa protein (data not shown). These data confirm that there are two distinct populations of CT-2 reactive {alpha}1A proteins, the full-length {alpha}1A that contains the C-terminus and an {alpha}1A C-terminal fragment.

The {alpha}1A C-terminal fragment is present in nuclear fractions
To determine whether C-terminal labeling of nuclei (Fig. 1) corresponded to the 60 kD fragment in mouse homogenates (Fig. 4), we fractionated dissociated cerebellar neurons into nuclear and cytoplasmic compartments prior to electrophoresis and blotting with CT-2 antibody. As expected, the mouse 60 kDa polypeptide was enriched in nuclear fractions, whereas full-length polypeptides were nearly absent from nuclear fractions (Fig. 5A). In SHEKß3 cells transfected with the human {alpha}1A vector, the 75 kDa CT-2 positive peptide was also enriched in nuclear fractions (Fig. 5B). Controls for adequate fractionation confirmed that there was minimal contamination between the two fractions (Fig. 5A and B). Quantification of the endogenous mouse cerebellar 60 kDa polypeptide revealed that 84±1.2% of the cleaved C-termini were present in the nuclear fraction (Fig. 5C). The C-terminal fragment made up ~19% of the total CT-2 positive proteins (Fig. 5D). These data confirm that the 75 kDa C-terminal polypeptide is cleaved from the full-length protein and is present in cell nuclei. These findings can also account for the diffuse staining pattern observed with C-terminal markers in {alpha}1A-transfected cells (Fig. 3). Presumably, the cytoplasmic staining observed was the intact full-length protein, found here in the cytoplasmic fraction, whereas the nuclear staining corresponded to the cleaved C-terminus, shown here to be predominant in the nuclear fraction.


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  		Figure 5. C-terminal cleavage product is enriched in the nuclear fraction of mouse cerebellar lysates and {alpha}1A-transfected SHEKß3 cells. Nuclear (Nu) and cytoplasmic (Cy) fractions of (A) mouse cerebella and (B) SHEKß3 cells transfected with GFPNH3{alpha}1A were subjected to western blotting and probed with anti-CT-2. Anti-GAPDH (cytoplasmic marker) and anti-Histone 3 (nuclear marker) were used as fractionation controls. Molecular weights are indicated at the right of each panel in kDa. (C) Densitometer quantification of western blots of mouse cerebellar fractions probed with CT-2. Nuclear localization of the {alpha}1A C-terminus (60 kDa fragment) and the full-length {alpha}1A (full-length) is expressed as a proportion (%) of the total protein present in the nuclear fraction. (D) Quantification of the total CT-2 identified full-length peptides and the total 60 kDa fragment is expressed as total protein area (OD*mm). Asterisk indicates statistically significant differences (P<0.01, two-tailed, unpaired Student's t-test, n=6 cerebella). Error bars represent mean SE.

 
The {alpha}1A C-terminus contains NLSs
Analysis of the C-terminal protein sequence, using an online software package (www.psort.org), revealed four putative NLSs (Fig. 6A). To isolate the translocation element, we designed constructs that express only the {alpha}1A C-terminus downstream of the cleavage site as a fusion protein with an N-terminal GFP tag (GFP2096) (Fig. 2A and B). In SHEKß3 cells expressing GFP2096, 83.7% ±1.3 of the signal was present in the nucleus and only 17.3% ±1.3 in the cytoplasm (Fig. 6B). As shown in Figure 6, replacement of a lysine with an alanine in the first putative NLS of GFP2096 ({Delta}NLS1) caused a significant decrease in nuclear staining (64.3% ±2.8, P<0.01). Further mutation of the two arginines in NLS1 resulted in similar staining (data not shown). Mutation of NLS2 ({Delta}NLS2) and NLS3 ({Delta}NLS3) significantly decreased nuclear staining to 67.0% ±2.9, (P<0.01) and 72.7% ±2.8 (P<0.01), respectively (Fig. 6). Mutation of NLS4 did not significantly alter localization (83.9% ±1.0), suggesting that the alterations observed following mutation of NLS1, 2 or 3 were not simply due to replacement of basic residues, but rather due to alteration of specific targeting sequences. Finally, we mutated all three NLSs in the same GFP2096 construct ({Delta}NLS1,2,3) (Fig. 2A). The triple NLS mutation significantly decreased the nuclear staining (58.0% ±2.9) compared with the individual NLS mutants and the native GFP2096 (P<0.01, Fig. 6). The triple NLS mutant was not significantly different than the diffuse EGFP (63.9% ±1.6) staining pattern. This suggests that the three NLSs (NLS1–3) are necessary to maintain the selective nuclear localization of the {alpha}1A C-terminus.


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  		Figure 6. Mutations of three putative NLSs decrease nuclear compartmentalization of the C-terminus. (A) Diagram of the NLS sequences mutated. Bold underlined residues were mutated to alanine residues. Numbers correspond to location in amino acid sequence. (B) Proportion (%) of nuclear relative fluorescence in SHEKß3 cells transfected with GFP2096 NLS mutants. Asterisk indicates statistically significant differences compared with GFP2096 (P<0.01, two-tailed, unpaired Student's t-test, n=25 for each condition). Error bars represent mean SE. Statistically significant results were replicated on three separate occasions. (C) Representative confocal images from (B) transmitted image and ToTo-3 overlay is provided. A single plane was taken through the center of each nucleus. Scale bar: 10 µm.

 
Numerous proteins contain multiple targeting signals (26Go–28Go). In cell culture studies of several proteins containing multiple nucleocytoplasmic transport signals, the stronger targeting signal determines the localization of the protein (29Go,30Go). The mutations in NLS1–3 were unable to completely abolish nuclear localization, suggesting the presence of other nuclear targeting motifs (29Go,30Go). To further assess the nuclear targeting ability of the putative NLSs (NLS1–3), we attached two well-characterized nuclear export signals (NESs) to the C-terminus of GFP2096 (Fig. 2A). We found that the NES was unable to significantly alter nuclear localization of the native C-terminus (79.3% ±1.7, Fig. 7A). However, when the NES was attached to C-termini containing the three NLS mutations ({Delta}NLS1,2,3) nuclear localization was abolished (16.5% ±1.2, Fig. 7A). Thus, although the ‘artificial’ NES was unable to remove the C-terminus with intact NLSs from the nucleus, NES-directed export did occur following NLS elimination. Similar results were obtained in the fibroblast cell line NIH3T3 (GFP2096, 85.6% ±1.5; NES, 75.36% ±2.1; {Delta}NLS/NES, 28.8% ±2.8) (Fig. 7B). This is consistent with recent reports of ataxin-7 containing three NLSs and a native NES. The NES in ataxin-7 was only able to abolish nuclear localization when two of the NLSs were removed (31Go).


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  		Figure 7. Artificial NES is unable to alter nuclear localization of the {alpha}1A C-terminus with native NLSs intact. (A) Proportion (%) of nuclear fluorescence intensity in SHEKß3 cells transfected with GFP2096, GFP2096 tagged with an artificial NES (NES) or GFP2096 with triple NLS mutations and an NES ({Delta}NLS/NES). (B) Proportion (%) of nuclear fluorescence intensity in NIH3T3 cells transfected with GFP2096, NES and {Delta}NLS/NES. Asterisk indicates statistically significant differences compared with GFP2096 (P<0.01, two-tailed, unpaired Student's t-test, n=25 for each condition). Error bars represent mean SE. (C) Representative confocal images from (A), transmitted image and ToTo-3 overlay are provided. Statistically significant results were replicated on three separate occasions.

 
The cleaved human {alpha}1A C-terminus contains the polyglutamine tract
Purkinje cells preferentially express a splice form of the human {alpha}1A subunit containing an extended C-terminus encoded by exon 47, and a small tract of glutamine residues (polyglutamine), normally polymorphic in length from four to 18 glutamines (8Go,23Go). Expansion of this polyglutamine tract to the range of 19–33 glutamine residues is associated with the dominantly inherited degenerative cerebellar disease, SCA6 (7Go,8Go). On the basis of the size of the proteolytic fragment (~400 amino acids), we predicted the region of cleavage and hypothesized that the C-terminal cleavage product contains the polyglutamine tract.

In western blots of cells expressing {alpha}1A subunits, containing either Q11 ({alpha}1AQ11) or Q33 ({alpha}1AQ33) repeats, the size of the proteolytic fragment correlated with the predicted size of the polyglutamine tract (Fig. 8A). Furthermore, the CT-2 antibody reacted in western blots with the C-terminal cleavage product generated from full-length {alpha}1AQ11 and {alpha}1AQ33 with relatively equal intensity (density relative to GAPDH: Q11, 0.25±0.06; Q33, 0.28±0.05) (Fig. 8B). Finally, polyglutamine tracts do not alter the ratio of full-length to cleaved C-terminal {alpha}1A (Q11, 0.57±0.17; Q33, 0.62±0.10; Fig. 8B). Similarly, expression of {alpha}1A C-terminal constructs (GFP2096) containing either Q4, Q11 or Q33 repeats were not significantly different from each other (density relative to GAPDH: Q4, 2.35±0.21; Q11, 2.41±0.27; Q33, 2.78±0.32) (Fig. 8C and D). These findings suggest that there is no significant difference in expression between the unexpanded (Q4 or Q11) and expanded (Q33) cleavage products in these experimental paradigms. This contradicts previous findings that suggest C-terminal proteolytic fragments bearing expanded polyglutamine tracts are more stable than those with normal polyglutamine tracts (20Go).


Figure 0808
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  		Figure 8. The {alpha}1A C-terminus contains the polyglutamine tract. (A) The {alpha}1A C-terminal cleavage product contains the polyglutamine tract and shifts ~5 kDa in SHEKß3 cells transfected with {alpha}1AQ33 compared with {alpha}1AQ11, as analyzed by CT-2 probed western blot. (B) Left graph represents quantification of 75 kDa band with densitometer. Intensity is expressed as the area (mm*OD) of 75 kDa divided by the area of GAPDH (n=6). Right graph represents the ratio of full-length (240 kDa) peptide to cleaved (75 kDa) peptide. (C) GFP-tagged {alpha}1A C-termini shift relative to polyglutamine expansion length. (D) Quantification of tail constructs relative to GAPDH (n=6). Error bars represent mean SE. None of the conditions tested were significantly different.

 
Polyglutamine expansions in {alpha}1A C-termini increase toxicity
Previous studies have suggested that the polyglutamine-expanded {alpha}1A in SCA6 may lead to cell death by altering calcium channel kinetics (23Go,32Go–34Go). Because C-termini containing normal or expanded polyglutamine tracts are cleaved from the full-length protein, we hypothesized that SCA6 C-termini themselves are toxic, independent of channel function. We transfected SHEKß3 cells with GFP2096 containing Q4, (GFP2096Q4), Q11 (GFP2096Q11) or Q33 (GFP2096Q33) and assessed cell viability with propidium iodide (PI) exclusion analyzed by fluorescence-activated cell sorting (FACS). In this assay, cell death is expressed as a proportion of dead transfected cells above baseline, where baseline is the proportion of dead untransfected cells. Seventy-two hours post-transfection, cells transfected with GFP2096Q33 exhibited a 2-fold greater rate of cell death (20.0% ±1.3) compared with those transfected with GFP2096Q11 (8.4% ±1.6, P<0.01) or GFP2096Q4 (3.0% ±1.1, P<0.01) and significantly greater than untransfected cells (P<0.01) (Fig. 9A).


Figure 0809
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  		Figure 9. Cell death in expanded {alpha}1A C-termini-expressing cells. (A) Cell death as determined by FACS analyzed PI exclusion of SHEKß3 cells transfected with GFP-tagged {alpha}1A C-termini containing glutamine tracts of 33 (GFP2096Q33), Q11 (GFP2096Q11), Q4 (GFP2096Q4) or Q33 alone (GFPQ33). C-termini with 50% ({Delta}NLS1,2,3) or 20% nuclear localization ({Delta}NLS/NES) were also assessed containing Q4, Q11 or Q33 glutamines. Percent of transfected dead cells was normalized to the percent of untransfected cells dead. Asterisks indicate statistically different results from respective GFP2069Qn (P<0.05, one-way ANOVA). Twenty thousand events were recorded per n (n=12). Statistically significant results were replicated on three separate occasions. (B) FACS analyzed cell death in primary granule cells after 7 DIV. Fifteen thousand events were recorded per mouse for GFPQ33, GFP2096Q4, GFP2096Q11 and GFP2096Q33 (n=4, 4, 5 and 6, respectively). (C) Representative plot of FACS controls. Included are populations of dead untransfected cells, healthy untransfected cells and healthy transfected cells. (D) Representative confocal images of SHEKß3 cells and granule cells expressing GFP2096Q33, GFP2096Q11, GFP2096Q4 or GFPQ33. Error bars represent mean SE. Scale bar: 10 µm.

 
It has been reported that expanded glutamine tracts (Q80) alone may be toxic to cells independent of protein context (35Go). To test for a direct effect of 33 glutamine residues on cell viability, we deleted the remainder of the {alpha}1A C-terminus, except the glutamine tract and the flanking 30 amino acids that include NLS3 (GFPQ33), and compared the toxicity of this polypeptide. SHEKß3 cells transfected with GFPQ33 did not exhibit any more cell death than untransfected cells (0.8% ±1.9, Fig. 9A), consistent with previous studies using Q35 tracts (35Go).

In other polyglutamine diseases, including dentatorubral-pallidoluysian atrophy and SCA1, cell death is dependent on nuclear localization of the polyglutamine containing protein (36Go,37Go). We hypothesized that, like other polyglutamine diseases, the toxicity of expanded (Q33) C-termini is dependent on nuclear localization of the C-terminal fragment. To test this hypothesis, we utilized the constructs {Delta}NLS1,2,3 and {Delta}NLS/NES containing Q4, Q11 or Q33 glutamines. {Delta}NLS1,2,3 is ~50% nuclear, whereas {Delta}NLS/NES is almost entirely excluded from the nucleus (20% nuclear) and the native C-terminal protein (GFP2096) is nearly exclusively localized to the nucleus (90% nuclear, Fig. 7). SHEKß3 cells expressing expanded {Delta}NLS1,2,3 ({Delta}NLS1,2,3Q33) exhibit a 37% decrease in cell death (11.7% ±1.4) compared with unmodified C-termini containing Q33 (GFP2096Q33, 20.0% ±1.3, P<0.01). Cells expressing unexpanded {Delta}NLS1,2,3, containing Q4 (4.1% ±1.7) or Q11 (8.7% ±0.6), were not significantly different than their respective unmodified counterparts (GFP2096Q4, 3.0% ±1.1; Q11, 8.4% ±1.6). Similarly, cell death displayed in cells expressing {Delta}NLS/NES, containing Q4, Q11 or Q33, was not significantly different from one another ({Delta}NLS/NESQ4, 3.6% ±0.4; Q11, 2.8% ±0.7; Q33, 4.2% ±0.8), although {Delta}NLS/NESQ11 and {Delta}NLS/NESQ33 were significantly less toxic than their unmodified counterparts, GFP2096Q11 and GFP2096Q33, respectively (P<0.05). These results suggest that polyglutamine-mediated cell death is dependent on localization of the C-terminal fragment to the nucleus.

The results obtained with unmodified C-termini (GFP2096) were replicated in transfected cerebellar granule cells (Fig. 9B). Granule cells expressing GFP2096Q33 exhibited significantly more death (10.4% ±1.7) than unexpanded GFP2096Q4 (5.9% ±4.2, P<0.05) or GFP2096Q11 (6.0% ±0.2, P<0.01). Interestingly, in SHEKß3 cells, cell death was significantly different between cells expressing GFP2096Q11 and GFP2096Q4. This difference was not observed, however, in granule cells. Similar to SHEKß3 cells, death in granule cells expressing the expanded glutamine tract alone (GFPQ33), lacking the C-terminal sequences, was not significantly different than baseline (1.3% ±1.6). SHEKß3 cells and granule cells transfected with any of the GFP2096 constructs exhibited nearly 90% nuclear staining (Fig. 9D). GFP2096Q33, GFP2096Q11 and GFP2096Q4 all exhibited a speckled distribution pattern in both SHEKß3 and granule cells (Fig. 9D). Quantification of images confirmed that GFP2096Q4, GFP2096Q11 and GFP2096Q33 populations did not have significantly different average relative fluorescence intensities (ARFIs) (GFP2096Q4, 139.5±6.1; GFP2096Q11, 135.1±8.3; GFP2096Q33, 135.3±7.2). GFPQ33 did have a significantly higher (P<0.05) ARFI than GFP2096Q33, Q11 or Q4 (GFPQ33, 170.33±14.7).

These data suggest that the C-terminus of the {alpha}1A subunit is toxic to cells when harboring an expanded polyglutamine tract, and as suggested in other polyglutamine disorders (4Go), this toxic effect may be dependent on the protein context of the surrounding polypeptide and nuclear localization of the protein.


   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
These studies demonstrate, for the first time, that the C-terminus of the {alpha}1A subunit of the P/Q-type VGCC is cleaved from the full-length protein and translocated to the nucleus. This post-translational modification is robust and occurs in Purkinje cells of multiple species and cultured SHEKß3 cells, NIH3T3 cells or neurons transfected with recombinant human {alpha}1A. The C-terminal cleavage product contains three NLSs that promote its localization to the nucleus. Finally, the polyglutamine tract in these C-termini is polymorphic in length and expanded forms associated with SCA6, when localized to nuclei, are toxic to cultured cells and neurons. These findings may indicate a novel role for the P/Q-type calcium channel and suggest a possible mechanism in the pathogenesis of SCA6.

Previous studies have sought to determine the effect of the expanded polyglutamine tract in the C-terminus on P/Q channel kinetics. Expression studies in cultured cells or oocytes have demonstrated that the SCA6 polyglutamine expansion alters the P/Q channel gating and currents (24Go,32Go,34Go), although the exact mutant channel phenotype appears to be highly dependent on splice form and experimental conditions. Moreover, expression of full-length channels bearing expanded polyglutamine tracts has yet to be linked to cell death. Given the present findings that cleavage of the polyglutamine-containing C-terminus occurs in the expression systems used to measure channel function, this post-translational modification may impact the interpretation of these electrophysiological studies. Specifically, the ratio of cleaved to uncleaved {alpha}1A subunits in previous P/Q channel recordings remains to be determined.

Until now, the {alpha}1A subunit has been considered a solely cytoplasmic protein (7Go,8Go,38Go). Expression studies at the level of both RNA and protein suggest that the {alpha}1A C-terminus splice form bearing the polyglutamine tract is preferentially expressed in cerebellar Purkinje cells (13Go,23Go). In this study, shown using CT-2 antibody, a significant proportion of the cleaved C-terminus is present in Purkinje cell nuclei. Similarly, nuclear accumulation appears to be required for cell death. This provides a potential explanation for the selective degeneration of Purkinje cells in SCA6, regardless of disease mechanism.

In other polyglutamine diseases, aside from SCA2, disease pathogenesis requires entry of the mutant protein or a proteolytic fragment containing the expanded polyglutamine tract into the nucleus (3Go,4Go,6Go). Mice expressing expanded ataxin-1 do not develop disease when the NLS in ataxin-1 is mutated (37Go). Similarly, Drosophila expressing spinal and bulbar muscular atrophy causing mutations in the androgen receptor do not exhibit toxicity when the mutated receptor is tagged with an NES (39Go). Finally, expanded ataxin-7 is unable to kill cultured granule cells when its nuclear targeting signals are compromised (31Go). Our results are consistent with these mechanisms of pathogenesis. The cellular toxicity of a C-terminal {alpha}1A polypeptide bearing the expanded polyglutamine tract of SCA6 and requiring nuclear localization suggests that the mechanism of pathogenesis of SCA6 may overlap, in part, with that of other polyglutamine diseases.

In our system, toxicity is not only dependent on nuclear localization, but also appears to occur in a dose-dependent manner. Expanded C-termini with only 50% nuclear localization ({Delta}NLSQ33) exhibit significantly attenuated levels of toxicity compared with unmodified expanded (Q33) C-termini (GFP2096), yet toxicity is still significantly elevated when compared with non-expanded controls (Fig. 9). This suggests that the amount of expanded C-terminus in the nucleus affects the level of toxicity. This is also a plausible explanation for why cell death has not been observed in unstressed cells expressing full-length expanded {alpha}1A under the time frame of these experiments, but rather requires decades of expression in most SCA6 patients. Only cleaved C-terminal fragments translocate to nuclei, the unprocessed full-length protein prevents nuclear entry of the polyglutamine-containing C-terminus by retaining it in the cytoplasm, thus reducing the amount of C-terminus present in nuclei and reducing toxicity. This prevention of nuclear entry may account for the delayed time course of toxicity observed in patients and our inability to observe cell death with full-length {alpha}1A.

Although nuclear translocation may be a common feature for SCA6 and most other polyglutamine diseases, SCA6 differs in several ways from other members of this disease family (40Go). First, proteolytic cleavage appears to be a constitutive process for WT {alpha}1A as well as expanded alleles and may reflect a normal signaling process. Thus, unlike in HD, SCA1 or SCA3, therapeutic measures to prevent cleavage or translocation of the C-terminus may interfere with normal regulatory processes. This outcome might, in fact, be predicted by the case of the leaner mouse mutant, in which the absence of the {alpha}1A C-terminus results in selective Purkinje cell degeneration (22Go).

Secondly, the pathogenic size of the polyglutamine expansion in SCA6 is small compared with other polyglutamine diseases, and the largest identified expansion (Q33), although toxic as part of the {alpha}1A C-terminus, is in the normal range for all other polyglutamine diseases (3Go,4Go,6Go). Together with the finding that simple Q33 (Fig. 9) or Q35 (35Go) polyglutamine tracts located in the nucleus have no effect on cell viability, our finding that over-expressed WT {alpha}1A C-termini are slightly toxic implies that the protein context of the {alpha}1A C-terminus may mediate the effect of the pathogenic polyglutamine tract. Our results are consistent with other studies that show that expression of both expanded and unexpanded {alpha}1A C-termini result in cell death, although the expanded tail is significantly more toxic than the unexpanded (20Go). It is tempting to speculate that the motifs involved in the normal nuclear function of the {alpha}1A C-terminus may facilitate the pathogenicity of the {alpha}1A polyglutamine tract. This conclusion is further supported by the observation that in SCA6 the age of onset correlates better with the combined size of expanded and unexpanded polyglutamine alleles than with expanded tracts alone (41Go). Moreover, one study suggests that patients with another form of polyglutamine-mediated ataxia, SCA2, develop ataxia at an earlier age if they bear normal unexpanded {alpha}1A polyglutamine tracts in the larger, rather than the smaller range (42Go).

Paradoxically, the leaner allele of the mouse CACNA1A mutant lacks {alpha}1A splice forms bearing the same cleaved region of the C-terminus and as homozygotes give rise to marked Purkinje cell degeneration (22Go). Because cultured cerebellar neurons from homozygote leaner mice exhibit 40% reduced P/Q current density, one interpretation of this finding is that Purkinje cell death results from reduced VGCC currents (43Go). However, mice heterozygous for other CACNA1A mutant alleles, such as the targeted {alpha}1A disruption (44Go), exhibit similar reductions in P/Q current density, but have normal cerebellar function and morphology. Moreover, heterologous expression of truncated {alpha}1A subunits, predicted by the leaner mutation, reveals that they exhibit minimally altered channel kinetics (43Go). Thus, a plausible explanation for the pronounced cell death phenotype in leaner is that the C-terminus plays an added role in Purkinje cell viability. Our data illustrating pronounced nuclear C-terminal staining in WT P18 Purkinje cell nuclei, coincident with the initiation of neuronal degeneration in leaner mice, is consistent with a role for the C-terminus in Purkinje cell viability.

Preliminary deletion studies suggest that the cleavage site is above residue 2044 of the C-terminus near domains known to interact with several cytoplasmic proteins (9Go,21Go,45Go,46Go). Online sequence analysis using databases for protease sites and other motifs reveals a putative PEST site downstream of the predicted region of cleavage, but does not reveal discrete protease cleavage site in this region of the C-terminus (47Go). Nevertheless, PEST sites are potential calpain recognition site (47Go,48Go). Calpain has also been identified as the protease that cleaves the C-terminus of the L-type calcium channel (49Go). However, when expressed in SHEKß3 cells neither calpain inhibitors nor co-expressed human calpastatin, a natural calpain inhibitor, were able to block {alpha}1A cleavage (unpublished data). This suggests that the putative protease was resistant or spatially isolated from the protease inhibitors.

Cleavage and nuclear translocation of a portion of several cytoplasmic proteins is a recently recognized cellular process, shown in at least some cases to mediate nuclear signaling. For example, ßAPP, MAPK, p75 and Notch are cleaved outside the nucleus, in direct or indirect response to membrane signaling, to generate a polypeptide that is translocated to the nucleus (50Go,51Go). More importantly, it has been reported that the C-terminus of the {alpha}1C subunit of L-type calcium channels is cleaved (49Go), and recently it has been suggested that the {alpha}1C C-terminal fragment is also present in cell nuclei and may act in transcription regulation (52Go).

Finally, although there is substantial evidence to indicate that the C-terminus of the intact WT {alpha}1A subunit has a role at the neuronal membrane (1Go,9Go,13Go), the potential role of the cleaved C-terminus in the nucleus is as yet unknown. From a structural standpoint, the {alpha}1A C-terminus contains domains that associate with intracellular signaling proteins to modulate channel activity and synaptic transmission, such as calmodulin (CaM), ß4 auxiliary VGCC subunits and G protein ß{gamma} (21Go,45Go,53Go), but no previous studies have identified an association with any nuclear proteins.

In addition to the four NLS sequences, online motif analysis using a sequence analysis package, Expasy (www.expasy.org), predicts several potential interactions and post-translational modifications that could reveal the function of {alpha}1A in the nucleus. Further study will be necessary to explore the role of these and other motifs in the biological activity of cleavage, nuclear translocation and cell death in SCA6. Identification of the biological activity of this translocation event may provide valuable insight into the control of cell-specific gene expression and mechanisms of neurodegenerative disease.


   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture and transfection
HEK293 cells and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM)-F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma), 2 mM L-glutamine and 50 µg/ml gentamicin (Invitrogen) in 5% CO2 at 37°C. Cells were transfected when they were ~60% confluent with 0.4 µg of DNA per 35 mm dish using Effectine reagent (Quiagen, Valencia, CA, USA), 0.4 µg of DNA per 35 mm dish was used in each transfection. Stable HEK293 cells were made using linear bi-cistronic pBudCE4.1 vector (Invitrogen) that expresses {alpha}2{delta} and ß3 subunits. Cells were selected with 250 µg/ml Zeocin (Invitrogen) and cloned. Primary cerebellar cultures were obtained from P7 mice. Cerebella were dissociated in 0.05% trypsin DMEM (Invitrogen) containing DNAase. Cultures were maintained in DMEM-F12 (Invitrogen) supplemented with 5% FBS, 5% horse serum, 25 mM KCl, 2 mM glutamine and 30 mM glucose. For each condition, 5x106 granule cells were transfected with 5 µg of DNA by electroporation (Nucleofector II; Amaxa, Koelin, Germany). Granule cells were plated at a density of 2x105 cells/cm2.

Cloning
All clones were made from {alpha}1A VGCC subunit cDNA (GenBank accession no. AF004884, MERCK bioscience). All GFP clones were generated with EGFP-C2 (Clontech, Palo Alto, CA, USA). NLS and EGFP mutations were performed via PCR (Quick-Change II XL Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA, USA). EGFP-C2 was mutated to a fainter GFP (mGFP) for FACS by the mutations L64F and T65S. GFPQ33 was generated by digestion of two Q33 flanking Kpn1 sites and ligation into EGFP-C1. NES tags were generated from custom oligonucleotides (Sigma) encoding an NES derived from MAPKK and HIV-Rev (LQKKLEELLPPLERLTG) and flanked by restriction sites (54Go).

Immunolabeling
Immunohistochemistry was performed as previously reported except as modified below (24Go). Briefly, paraffin-embedded sections of perfused brains were dewaxed and rehydrated, then steamed for 20 min in antigen retrieval solution (Reveal; Biocare Medical, Walnut Creek, CA, USA). Sections were blocked and exposed to primary antibody both NT-1 or CT-2 (1 µg/ml) for 12 h at 4°C, washed with phosphate-buffered saline (PBS), incubated in biotinylated anti-rabbit (Vector Labs, Burlingame, CA) secondary, reacted with ABC (Vector Labs), washed and then counterstained with diaminobenzidine (Vector Labs). Images were acquired with Zeiss light microscope and digital SPOT camera.

Immunocytochemistry of cell cultures was performed ~18–20 h after transfection of HEK cells or NIH3T3 cells on poly-D-lysine coated cover slips and after 7 days in vitro of primary neurons. Cells were fixed for 20 min in paraformaldehyde (PFA), washed three times in Tris-buffered saline (TBS), permeabilized with 0.01% Tween, blocked 30 min in 10% donkey serum/TBS, incubated in NT-1 or CT-2 primary antibody, washed three times in TBS, then incubated in secondary either Texas-Red donkey anti-rabbit (Jackson Immunoresearch, Westgrove, PA, USA) or Alexafluor 488 goat anti-rabbit (Molecular probes, Eugene, OR, USA). Images were acquired with a single-laser confocal microscope (MRC 1024; Olympus, Melville, NY, USA) and LazerSharp software (BIO-RAD, Hercules, CA, USA). Analysis was performed with MetaMorph software. All cells were imaged at the same settings. Intensity was determined with the brightest condition exhibiting little saturation (<255). Percent of nuclear staining was obtained by dividing the ARFI of the cytoplasm by the sum of the ARFI for both the nucleus and cytoplasm. Data are expressed as mean±standard error (SE). Data were compared using Student's t-test and differences were considered significant if P<0.05. Comparison of populations of cells labeled with either N-terminal or C-terminal markers (Fig. 3C) were done with a significance test for the estimation of a population proportion.

Immunoblotting
Forty-eight hours after transfection, cell cultures were lysed with ice-cold cell lyse buffer (MPER reagent; Pierce, Rockford, IL, USA) and mouse cerebellum were homogenized in ice-cold tissue lyse buffer (TBS, 1% Triton X-100), both buffers were supplemented with 10 mM EDTA, 10 mM EGTA and protease inhibitor tabs (Roche, Indianapolis, IN, USA). Cell fractionation was performed with NE-PER Nuclear and cytoplasmic extraction reagent kit (Pierce). Fifty micrograms of total protein or fraction were subjected to SDS–PAGE (8% Tris–Glycine gel, Invitrogen) and transferred to a nitrocellulose membrane. Membranes were probed with either {alpha}1A-specific custom antibodies CT-2 or NT-1 raised against RHGRRLPNGYYAGHGAPR or MARFGDEMPARYGGGGSG (15Go), respectively (Sigma), anti-ß3 (Alamone, Haifa, Israel), anti-Flag-M2 (Sigma), anti-penta-HIS (Sigma), monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA), anti-GFP Living colors monoclonal (Clontech) or anti-Histone 3 (Cell-Signaling, Beverly, MA, USA). This was followed by incubation with a secondary antibody, either anti-mouse or anti-rabbit IgG horseradish peroxidase (Amersham Biosciences, Piscataway, NJ, USA). Affinity purification was performed as previously reported (24Go).

Cell death assay
FACS was performed 72 h after transfection. SHEKß3 cells were trypsinized and incubated in 2 µg/ml PI (Molecular Probes) for 30 min at room temperature, washed in PBS and fixed in 1% PFA/PBS at physiological pH. Cells were sorted on FACS (FacsCalibur, BD Biosciences, San Jose, CA, USA) and data analysis was performed with FACS quantification software (Cellquestpro, BD Biosciences). Twenty thousand events were recorded for each n, where n is equivalent to one well in a six-well plate. mGFP constructs were used because EGFP overlapped with FL2 filters. The percent of transfected dead cells was expressed as the ratio of both GFP- and PI-positive cells by the total number of GFP-positive cells. This was normalized to untransfected by subtracting the percent of untransfected dead cells. Percent untransfected dead was determined by dividing the number of PI-positive cells by the total number of cells not GFP positive. Multiple groups of FACS data were analyzed using one-way ANOVA and both Bonferroni and Tukey post hoc tests. Results were plotted as mean±SE and differences were considered significant if P<0.05.


   ACKNOWLEDGEMENTS
 
We thank Colleen Forester, Katie M. Wiens and Anna McDowell for their technical assistance. We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the University of Minnesota Cancer Center, a comprehensive cancer center designated by the National Cancer Institute, supported in part by P30 CA77598. This work was supported by NIH NS38332, the National Ataxia Foundation and the Bob Alison Ataxia Research Center.

Conflict of Interest statement. There is no conflict of interest.


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