Introduction
Spinocerebellar ataxia type 7 (SCA7) is a rare neurodegenerative disease caused by a trinucleotide repeat expansion in the ataxin-7 gene [
1‐
3]. SCA7 is one of nine known polyglutamine (polyQ) diseases characterized by aggregation of polyQ-expanded protein, nuclear inclusions and repeat-dependent neuronal toxicity [
4,
5].
Ataxin-7 protein is ubiquitously expressed in the brain with cytoplasmic and nuclear staining [
6,
7]. Ataxin-7 functions as part of two known transcriptional repressor complexes: the Spt/Ada/Gcn5 acetylase (STAGA) chromatin remodeling complex with histone acetyltransferase (HAT) activity [
8]; and the TATA-BP free TAF-containing complex (TFTC) [
9,
10]. In SCA7 patients there is increased localization of ataxin-7 to discrete nuclear inclusions [
6,
7,
11,
12]. Aggregation of polyglutamine-expanded ataxin-7 into the nucleus, concomitant with neurodegeneration and transcriptional deregulation, is also a feature of SCA7 transgenic mouse models [
13‐
16].
Transcriptional deregulation in polyglutamine diseases has been associated with acetylation state changes, by alteration of the HAT activity of member complexes [
17] or by direct interaction of ataxins with histone deacetylases (HDACs), in particular HDAC3 [
18,
19]. HDAC3 is an integral constituent of multiple co-repressor complexes that activate HDAC3 and are recruited by transcription factors to alter histone acetylation and repress transcription [
20‐
22]. HDAC3 shuttles between the cytoplasm and nucleus [
23], altering activity of transcriptional repressor complex target genes [
24]. HDAC3 is highly expressed in the brain [
25] and is essential for viability [
26]. In cell studies, high levels of HDAC3 are neurotoxic whereas HDAC3 suppression protects against neuronal cell death [
27]. Further, this appears to be a modular switch in which HDAC3 interaction with HDAC1 promotes survival or death [
28]. This is consistent with the proposed neuroprotective nature of HDAC inhibitors, including those targeting HDAC3, in reducing pathological behavioral phenotypes and neurodegeneration in mouse models [
29‐
32].
Previous work by our lab and others has shown that ataxin-7 posttranslational modification (PTM) increases ataxin-7 stability, effecting aggregation and toxicity of the polyQ-expanded protein [
16,
33], with deacetylation influencing stability of ataxin-7 although the enzyme that was responsible for this effect was not determined. In this study we found that HDAC3 enhanced PTMs and the stability of ataxin-7, with evidence that ataxin-7 and HDAC3 physically interact and colocalize. Their association enhances cellular toxicity in a polyQ-dependent manner. HDAC3 displays robust expression in neurons and glia in the mouse cerebellum. We detect increased levels of HDAC3 in SCA7 mice as well as altered lysine acetylation levels and deacetylase activity in the brains of SCA7 transgenic mice, indicating that the association between HDAC3 and ataxin-7 may be a pathological feature of the disease.
Discussion
Many polyglutamine diseases, including SCA7, feature transcriptional deregulation associated with altered acetylation state [
17,
34,
35] and cellular toxicity that is reversible by HDAC inhibition [
35]. We have previously shown that acetylation and deacetylation affect ataxin-7 stability [
16]. As ataxin-7 functions in a transcriptional repressor complex, one proposed mechanism for deregulation is aberrant interaction of the polyQ-expanded protein with factors that regulate acetylation/deacetylation. In this study we focused on a role for HDAC family members in ataxin-7 regulation. This is a critical study given that HDAC inhibitors have been found to ameliorate symptoms in many models of trinucleotide repeat diseases, including Huntington’s disease (HD) and Freidrich’s ataxia [
31,
32]. Our study indicates that HDAC inhibitors may also be a target for SCA7 treatment.
In this study we identified HDAC3 as a protein binding-partner of ataxin-7. We showed that HDAC3 modifies ataxin-7 stability, PTM and subcellular localization. Enhanced stability of ataxin-7 could be due to increased production or to reduced degradation. Our previous study supports the latter conclusion, as we have previously found that modification of ataxin-7 at lysine-257, by SUMOylation or acetylation, enhances ataxin-7 stability [
16]. Consistent with this study, the PTM-deficient mutant K257R does not have increased protein levels or modifications with HDAC3 co-expression. Future studies may uncover the role of modifications at lysine-257 on HDAC3 effects. Interestingly, we did not find a differential effect on ataxin-7 stability or PTMs using a catalytically-inactive mutant of HDAC3 compared to the wild type HDAC3. This indicates that the protein-protein interaction between ataxin-7 and HDAC3, not its deacetylase activity, may be involved in enhancing ataxin-7 levels and modifications. An alternate explanation is that another HDAC family member that is in complex with HDAC3 is responsible for modifying ataxin-7.
A major effect of the interaction between ataxin-7 and HDAC3 is increased levels of the mutant ataxin-7 protein, with enhanced nuclear expression in cells overexpressing HDAC3, concomitant with increased cell toxicity. Subcellular redistribution may be due to ataxin-7 shuttling with HDAC3, which is known to undergo nuclear/cytoplasmic shuttling as part of a transcriptional corepressor complex [
24]. While this effect on localization may occur via PTM regulation, previous studies have shown that SUMOylation does not effect subcellular localization of ataxin-7 [
33] and our study further indicates that it is not a result of HDAC enzymatic activity.
As with many neurodegenerative disease proteins, ataxin-7 is ubiquitously expressed in the brain and yet the disease manifests in specific brain regions. One proposition is that regional or cell-specific expression of interacting proteins, such as HDAC family members, contributes to selectivity of disease progression [
36,
37]. Given the varied effects of HDAC3 interaction on ataxin-7
in vitro, we explored the region-specificity of HDAC3 in the mouse brain. One previous study analyzed HDAC3 mRNA in the brain, with high expression noted in granular cells of the cerebellum and hippocampus [
25]. Our study in normal mouse brain found HDAC3 protein highly expressed in the cerebellum, with moderate expression in pons and hippocampus, in particular the subgranular zone and also caudate putamen, particularly the subventricular zone. HDAC3 knockout mice are embryonic lethal due to cardiomyopathy [
26]. However, conditional knockout mice have indicated a role for HDAC3 in brain function as specific deletion in the hippocampus implicated HDAC3 as a critical negative regulator of long-term memory formation [
38], consistent with our findings of marked HDAC3 expression in the hippocampus. Our expression analysis suggests HDAC3 may also play a role in neurogenesis, which is consistent with previous studies showing that HDAC3 is expressed in developing rat brain and that HDAC activity is crucial for determining cell progenitor fate [
39].
Given the high expression of HDAC3 in the cerebellum, and the major degeneration of this region in SCA7, we determined the cell-specific expression of HDAC3. Neuronal and glial-cell markers showed HDAC3 to be highly expressed in the Purkinje cell layer, with distinct colocalization with Bergmann glial cell soma and processes. SCA7 animal models have revealed that polyQ-expanded ataxin-7 dysfunction in either neurons or glia of the cerebellum leads to behavioural phenotypes and neurodegeneration, but that rescue of ataxin-7 function in both cell types is required to reverse the SCA7 phenotype [
40,
41]. This non-cell autonomous feature indicates that SCA7 degeneration resulting from ataxin-7 dysfunction results from interactions in purkinje cells and Bergmann glia, both of which our study shows express HDAC3.
Given this remarkable expression pattern we next assessed HDAC3 expression in a well-characterized SCA7 transgenic mouse model. In the cerebellum we found increased HDAC3 protein levels in the SCA7 polyQ-expanded transgenic mice. In cells we showed that HDAC3 and ataxin-7 interaction was enhanced with polyQ-expanded ataxin-7, which we hypothesize could lead to increased stability of the HDAC3 protein in SCA7. Interestingly, increased expression of HDAC3 protein has recently been shown to be highly toxic to cerebellar and cortical neurons but not to non-neuronal cells [
27], which may explain why neurons selectively die despite our evidence for high expression of HDAC3 in glia.
In the retina we found decreased acetyl-lysine levels in photoreceptor cell body layers, concomitant with increased deacetylase activity. These changes are consistent with previous studies that evaluated promoter occupancy of the SCA7-92Q mice relevant to histone H3 acetylation and found a significant decrease with disease progression at 6 and 11 weeks of age [
8]. Further, in HD mouse models histone H3 acetylation is decreased during disease [
42]. However, our study is particularly interesting as it is known that HDAC activity requires binding to cofactors [
22] and previous studies have show that HDAC3 interaction with a transcriptional corepressor, N-CoR, increased HDAC activity [
21]. HDAC3 interaction with ataxin-7, a constituent component of transcriptional corepressor complexes, could also potentially increase HDAC activity, enhancing toxicity.
From our findings we hypothesize that HDAC3 interaction with polyQ-expanded ataxin-7, particularly in the pathological regions in SCA7, increases HDAC3 stability and HDAC activity, leading to transcriptional deregulation, toxicity and neurodegeneration. Future studies would directly analyze this role by looking at the affect of altered HDAC3 levels in a SCA7 transgenic model, particularly on behavioural phenotypes, cerebellar degeneration and morbidity. A pathogenic role for HDAC3 in SCA7 must be further assessed in transgenic mouse models before any conclusions are drawn, although it is consistent with previous studies that HDAC inhibitors with high affinity to HDAC3 complexes [
43] may ameliorate deficits in polyQ disease models. In a mouse model of Freidich’s Ataxia, treatment with HDAC inhibitors with an HDAC3 preference enhanced motor coordination and ameliorated multiple pathological deficits [
32]. Using a similar compound another study found that HDAC inhibition reduced behavioural phenotypes and pathology as well as partially correcting transcriptional deregulation in a mouse model of HD [
31] although other studies have questioned the specificity of the drug or did not reproduce all the beneficial effects found in the first study [
44,
45]. Furthermore, HDAC3 knockdown in a
C. elegans HD model suppressed neurotoxicity [
30], consistent with our findings of HDAC3 enhancing cellular toxicity. In contrast to these latter reports, one study found no benefit of partial HDAC3 knockdown in a genetic cross with an HD mouse model [
46]. However, recent studies suggest that the wild-type Htt binds to HDAC3 and dissociation of HDAC3 when mutant Htt is present is essential for toxicity [
47]. We propose that the physical interaction of HDAC3 with ataxin-7, and other expansion disease proteins resulting in cellular toxicity (perhaps with distinct mechanisms), makes HDAC3 a target for intervention in these diseases. In support of a role for HDAC3 in trinucelotide repeat diseases, a recent report found that HDAC3 promotes instability, leading to expansions of trinucleotide repeats in cell and animal models [
48].
In summary, we found that HDAC3 and ataxin-7 physically interact, leading to increased stability, enhancing post-translational modifications and increased subcellular localization of ataxin-7. Furthermore, we detected altered HDAC3 levels, acetyl-lysine levels and deacetylase activity in a transgenic mouse model of SCA7 and found HDAC3 to be highly expressed in both neurons and glia in the cerebellum of nontransgenic and SCA7 transgenic mice. Further studies will elucidate the physical nature of the interaction between ataxin-7 and HDAC3, the effect of polyQ-expansion on the interaction and levels of HDAC3 and on SCA7 pathogenesis, and whether alteration of HDAC3 levels may be a viable pharmacological target. With reports of HDAC3 binding to three different ataxin proteins [
18,
19,
47], and HDAC3-specific inhibition ameliorating disease in animal models [
31,
32,
34], our study adds to growing evidence for HDAC3 modulation as a target for multiple neurodegenerative diseases.
Methods
SCA7 transgenic mice
Production and characterization of SCA7 transgenic mice, with ataxin-7 containing 10 or 92 CAG repeats expressed under the direction of the prion promoter (PrP) has been described previously [
14]. Expression levels for PrP-mycFlag-SCA7-10Q-Line A305 and PrP-mycFlag-SCA7-92Q-Line B306 were determined in the initial characterization [
16]. Transgenic mice in the PrP-mycFlag-SCA7-92Q-Line 306 overexpress ataxin-7 two-fold relative to endogenous levels, have retinal pathology by 12-weeks of age and have a shortened lifespan (9 months). At the Buck Institute for Research on Aging, an AAALAC international B306 accredited institution (Unit #001070), all procedures were approved by the Institutional Animal and Use Committee (A4213-01).
Plasmid constructs
Ataxin-7 cDNA was cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA), with inserts containing either 10 or 92 CAG repeats, and a stop mutant created by site-directed mutagenesis at amino acid position 266, as previously published [
16]. Site-directed mutagenesis was performed to generate a K257R amino acid substitution as previously described [
16]. All constructs were sequenced to confirm that the appropriate mutation was introduced and CAG repeat length was not altered. For co-expression studies, plasmids encoding HDACs 2–8 were purchased from Origene (Rockville, MD): HDAC2 (#SC110918), HDAC3 (#SC112704), HDAC6 (#SC111132), HDAC8 (#SC321829) or Addgene (Cambridge, MA): HDAC4 (#13821), HDAC5 (#13822). HDAC7 plasmid was a gift from Dr. Hung-Yin Kao and the HDAC3 catalytically-inactive mutant construct (H134A/H135A substitutions) was a gift from Dr. Eric Verdin.
Cell culture, transfection and harvesting
HEK293T cells were cultured in DMEM (Mediatech, Manassas, VA) containing 1% penicillin/streptomycin (25 units/mL each) and 10% heat-inactivated fetal bovine serum (DMEM complete) unless otherwise specified. Transient transfections were performed using lipofectamine 2000 (L2000, Invitrogen) according to manufacturer’s instructions. For total protein extractions, cells were seeded in 6-well dishes at approximately 5 × 105 cells/well. Each well was transfected with 4 μg total DNA by L2000 in DMEM lacking serum and antibiotics. Following 72 h incubation at 37°C, cells were harvested, lysed in Mammalian Protein Extraction Reagent (MPER; Thermo Scientific, Rockford, IL) containing protease inhibitors (Complete Mini, Roche Applied Science, Mannheim, Germany), sonicated and spun at 12,000 g for 15 min to remove undigested cellular components. Protein concentration was determined using a BCA assay (Pierce, Rockford, IL). For subcellular fractionation assays, 6 × 106 cells were seeded in 10 cm plates, transfected with 25 μg DNA by L2000 in serum-free DMEM and incubated for 72 h at 37°C. For immunocytochemistry, including cellular toxicity assays, cells were seeded in 8-well slides at 6–10 × 104 cells/well. Each well was transfected with 1.2 μg total DNA in serum-free DMEM and processed following 48 h incubation at 37°C.
Subcellular fractionation
For subcellular fractionation protein assays, transfected HEK293T cells were harvested and nuclear and cytoplasmic fractions were separated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Rockford Illinois) according to the manufacturer’s instructions. Briefly, cells were harvested in trypsin-EDTA (Mediatech) and approximately 6 × 106 cells were spun at 500 g for 3 min. Supernatant was removed and approximately 50 μL of cells recovered. Cytoplasmic Extraction Reagents (CERI and CERII) were added to these cells, which were then vortexed at high-speed and spun at 16,000 g for 5 min at 4°C. The cytoplasmic protein fraction was recovered from the supernatant fraction. The pellet was resuspended in Nuclear Extraction buffer (NER) by intermittent high-speed vortexing over 40 min and the sample was centrifuged at 16,000 g for 10 min at 4°C. The nuclear protein was separated into the supernatant fraction. Protein concentrations of the nuclear and cytoplasmic fractions were determined using a BCA assay and 10 μg of protein were subjected to western blotting.
Immunoprecipitation
Pull-down assays of transfected HEK293T cells were undertaken using Protein G Sepharose beads (GE Healthcare, Uppsla, Sweden), washed and used according to the manufacturer’s instructions. Lysates (500 μg) were pre-cleared by incubation with beads without the antibody for 1 h at 4°C with rotation. Pre-cleared lysates were incubated with primary antibody, 3 μL ataxin-7 (PA1-749) or 5 μL HDAC3 (sc-17795), overnight with rotation at 4°C. Antibody-incubated lysates were transferred to the bead slurry and immunoprecipitated for 2 h at 4°C with rotation. Immunodepleted sample was removed after centrifugation at high-speed for 3 min. Bound beads were then washed rigorgously in lysis buffer (MPER with proteinase inhibitors, as described above) and once in HEPES made up in lysis buffer without proteinase inhibitors to remove salts. Beads were precipitated by boiling in water with loading buffer and DTT (10 mM) for 10 min and spun briefly to collect beads. Half of the precipitate was used for western blotting, along with immunodepleted samples and 10 μg of input protein.
Western blotting of transfected cells
For western blotting of transfected HEK293T cells, 10 μg of protein containing DTT (10 mM) was heated at 95°C for 10 min. Samples were subjected to SDS-PAGE using 4-12% NuPage Bis-Tris (Invitrogen) gels under reducing conditions in MES SDS running buffer (Invitrogen). Gels were run at 200 V for 70 min, transferred to 0.45 μM nitrocellulose membrane (Whatman, Dassel, Germany) in NuPage transfer buffer (Invitrogen) with 10% methanol and western transfer undertaken at a constant 350 mA for 1 h. Membranes were blocked in TBS with 0.1% Tween 20 (TBS-T) containing 5% milk for 1 h. Primary antibodies were diluted in blocking solution at 1:1000 for ataxin-7 (PA1-749, Thermo Scientific, Rockford, IL), 1:100 for HDAC3 (sc-17795, Santa Cruz Biotechnology), 1:5000 for GAPDH (MAB734; Chemicon, Temecula, CA), 1:1000 for β-actin (#4967S; Millipore, Billerica, MA) and 1:500 for poly-ADP ribose (PARP; SA-253, Enzo Life Science, Farmingdale, NY). Immunoblots were developed with a peroxidase-conjugated secondary antibody and enhanced chemiluminescence.
Immunocytochemistry
Cells were fixed by adding 8% paraformaldehyde directly to culture medium to a final concentration of 4%. Cells were incubated for 20 min with gentle agitation then washed with PBS at room temperature (RT). Cells were permeabilized in 0.25% Triton in TBS for 15 min, washed in TBS, and blocked in 10% NDS in TBS for 1 h. Primary antibody was incubated with cells overnight at 4°C after dilution in TBS containing 1% BSA at 1:200 for ataxin-7 (K antibody: [
14,
16] and 1:20 for HDAC3 (sc-17795, Santa Cruz Biotechnology). Cells were then washed in TBS and secondary antibody in TBS containing 1% BSA was applied for 1 h at RT. Ataxin-7 was labeled with 1:500 Alexa 488 donkey anti-rabbit and HDAC3 was labeled with 1:500 Alexa 555 donkey anti-mouse. For the cell toxicity assay, 1:5000 Hoescht 33342 (10 mg/ml; H21492, Invitrogen) was included in the secondary antibody incubation. A no primary antibody control was included in each experiment to detect background fluorescence. Cells were washed, dried and mounted with ProlongGold +/− DAPI (Invitrogen). Slides were cured overnight. Confocal images were obtained using a Zeiss LSM 510 NLO microscope and Zeiss LSM Image Browser software. Laser settings used were 488 Ar to capture ataxin-7, 543 He/Ne for HDAC3 and Chameleon Ultra tuned to 780 nm for DAPI acquisition. Following capture, all images were compiled in Adobe Photoshop using identical parameters for each channel. For the cellular toxicity assays, 100 cells/field were counted in two separate fields for each transfection condition and this was duplicated in a separate transfection.
Protein extraction and western blotting of SCA7 transgenic mouse tissue
Protein was extracted from the cerebellum of 9-month old mice: PrP-mycFlag-SCA7-10Q-Line A305, PrP-mycFlag-SCA7-92Q-Line B306 and non-transgenic littermate controls. Cerebella were homogenized in 10 ml/g tissue of Tissue Protein Extraction Reagent (TPER; Thermo Scientific) containing protease inhibitors, DNase (10 U/mL), MgCl2 (1.2 mM), epoxymycin (1 μM) phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set II, Calbiochem, La Jolla, CA) and HDAC inhibitors (50 μM TSA, 30 μM sodium butyrate, 30 mM nicotinamide). Tissue was homogenized, snap frozen at −80°C overnight, defrosted at RT to enhance cell lysis, sonicated and spun at 12,000 g for 15 min. Supernatant and pellets were kept for analysis. Protein concentration was determined using a BCA assay (Pierce, Rockford, IL) and 40 μg of proteins were run on polyacrylamide gels in MES buffer for 70 min at 200 V. Proteins were transferred to a 0.45 μm nitrocellulose (Whatman, Dassel, Germany) membrane at 20 V overnight (14 h) at 4°C in NuPAGE tranfer buffer with 10% methanol. Band intensity was quantified using NIH ImageQuant TL v2005. Membranes were blocked in TBS with 0.1% Tween 20 (TBS-T) containing 5% milk for 1 h. Primary antibodies were diluted in blocking solution at 1:1000 for ataxin-7 (PA1-749; Affinity Bioreagents), 1:2500 for HDAC3 (ab7030, Abcam, Cambridge, MA) and 1:1000 tubulin (T4026; Sigma-Aldrich, St Louis, MI) Immunoblots were developed with a peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Pierce).
Immunohistochemistry of SCA7 transgenic mouse brain sections
SCA7 mice were perfused with PBS and then 4% PFA in PBS. The eyes and brain were removed and embedded in paraffin blocks. Brain and retinal tissue was cut into 7 μM sections on a microtome. Paraffin-embedded sections were washed with xylene twice for 5 min to deparaffinize the tissue. Sections were re-hydrated in consecutive ethanol washes (100% to 70%) before resuspension in 1X Tris-buffered saline for 15 min. All washes were performed at RT. For antigen retrieval, sections were microwaved in 10 mM citrate buffer, pH 6.0, for 5 min at 40% power in a 1100 W microwave oven (Sanyo). Sections were allowed to cool in the same buffer for 20 min at RT and were then transferred to TBS for 10 min. For immunostaining, sections were blocked in 10% normal donkey serum in TBS for 1 h at RT. Ataxin-7 (1:200; K antibody), HDAC3 (1:20 for retinal tissue and 1:500 for cerebellar tissue; Abcam ab7030), acetyl-lysine (1:250; Abcam ab21623), glial fibrillary acidic protein (GFAP; 1:500; Sigma G3893), neuronal nuclear marker (NeuN; 1:200; Millipore MAB377), calbindin (1:500; C9848 Sigma) and S100 calcium binding protein beta (S100β; 1:1000; Abcam ab11178) primary antibodies were diluted in 1% BSA in TBS and incubated overnight at 4°C. All subsequent steps were performed at RT. Sections were washed in TBS. Alexa 488 donkey anti-rabbit secondary antibody (1:500 in 1% BSA in TBS) was applied to sections for 1 h. Following incubation in secondary antibody, sections were washed in TBS and mounted in ProlongGold with DAPI. Slides were cured overnight. Epifluorescence images were captured on a Nikon Eclipse E800 microscope. Confocal images were obtained using a Zeiss LSM 510 NLO microscope and Zeiss LSM Image Browser software.
HDAC activity assay
In situ HDAC activity was determined in retinal sections using the Fluor-de- Lys™ HDAC flurometric activity assay (Enzo Life Sciences International Inc, Plymouth Meeting, PA) previously modified for use in tissue [
49]. Briefly, paraformaldehyde-fixed retinal sections (7 μM) were incubated with deacetylase substrate (200 μM) in assay buffer (50 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) for 3 h at RT. HDAC I/II -specific inhibitor, trichostatin A (100 μM; Sigma, Steinham, Germany), was added to some sections to determine the influence of sirtuin activity. Buffer-only controls were included in all experiments. After incubation, sections were washed briefly in PBS and then fixed in methanol at −80°C for 20 min. Freshly prepared Fluor-de- Lys™ developer (0.5X) in assay buffer was added and sections were coverslipped and immediately visualized on a Nikon Eclipse E800 microscope in the DAPI range (excitation 350–380 nm; emission 440–460 nm).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CED carried out the experiments, participated in the study design, performed statistical analysis and drafted the manuscript. MCA, CR, TP and CV carried out experiments and performed statistical analysis. LME conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the manuscript.