Huntington's disease (HD) is caused by a polyglutamine expansion within the huntingtin (Htt) protein. Both loss of function of normal Htt and gain of a toxic function by the polyglutamine-expanded mutant Htt protein have been proposed to be responsible for HD, although the molecular mechanisms involved are unclear. We show that Htt is a neuroprotective protein in both HD-related and unrelated model systems. Neuroprotection by Htt is mediated by its sequestration of histone deacetylase-3 (HDAC3), a protein known to promote neuronal death. In contrast to the normal Htt, mutant Htt interacts poorly with HDAC3. However, expression of mutant Htt liberates HDAC3 from Htt, thus de-repressing its neurotoxic activity. Indeed, mutant Htt neurotoxicity is inhibited by the knockdown of HDAC3 and markedly reduced in HDAC3-deficient neurons. A reduction in Htt–HDAC3 interaction is also seen in neurons exposed to other apoptotic stimuli and in the striatum of R6/2 HD mice. Our results suggest that the robust interaction between Htt and HDAC3 along with the ability of mutant Htt to disrupt this association while not itself interacting with HDAC3 provides an explanation for both the loss-of-function and gain-of-toxic-function mechanisms proposed for HD. Moreover, our results identify HDAC3 as an essential player in mutant Htt-induced neurodegeneration.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (Htt) gene that produces a mutant protein with an elongated polyglutamine (polyQ) stretch near the N terminus (Zuccato et al., 2010; Crook and Housman, 2011; Ross and Tabrizi, 2011). The disease is characterized by selective and progressive degeneration of striatal GABAergic neurons and is manifested by adult-onset motor dysfunction, cognitive decline, and psychiatric disturbances. The polyQ expansion in mutant Htt (mut-Htt) is believed to confer a novel toxic gain-of-function effect, although the pathogenic mechanisms involved are not well understood (Zuccato et al., 2010; Crook and Housman, 2011; Ross and Tabrizi, 2011). Several mechanisms have been proposed to be involved, including transcriptional dysregulation, excitotoxicity, impaired energy metabolism, abnormal protein cleavage and aggregation, defects in axonal transport, and abnormal protein–protein interactions (Zuccato et al., 2010; Crook and Housman, 2011; Ross and Tabrizi, 2011). Loss of normal Htt function has also been proposed to contribute to HD. Indeed, heterozygous Htt knock-out mice as well as mice in which the Htt gene is selectively ablated in postmitotic neurons exhibit progressive neurodegeneration during adulthood (Nasir et al., 1995; Dragatsis et al., 2000). Disease-associated loss of normal Htt has also been shown in mouse models of ischemia, trauma, and spinal cord injury (Zhang et al., 2003).
In this study, we examined the possibility that Htt has a broader role in maintaining neuronal survival, functioning by directly acting on proteins regulating neuronal death. We find that Htt not only protects against mut-Htt-induced toxicity but can also protect neurons in paradigms unrelated to HD. We present evidence showing that Htt acts by sequestration of histone deacetylase-3 (HDAC3), a protein with established neurotoxic activity (Bardai and D'Mello, 2011). Although interacting poorly with HDAC3 itself, we find that mut-Htt promotes the disassociation of HDAC3 from Htt. Mut-Htt neurotoxicity is greatly reduced in the absence of HDAC3, suggesting that HDAC3 is a key player in HD pathogenesis.
Materials and Methods
Flag-tagged HDAC3 expression plasmid was bought from Addgene. GFP- and RFP-tagged Htt plasmids were a kind gift from Dr. Troy Littleton (Massachusetts Institute of Technology, Cambridge, MA). The untagged Htt plasmids were a kind gift from Dr. Dimitri Krainc (Harvard Medical School, Boston, MA). All cell culture media and reagents were purchased from Invitrogen. Huntingtin antibody (catalog #5656) was from Cell Signaling Technology. HDAC3 antibody (catalog #H3034) used for immunoprecipitation was from Sigma-Aldrich and that for Western blot analysis was bought from Santa Cruz Biotechnology (catalog #sc-11417). HDAC3 shRNA plasmids (TRCN0000039391 and TRCN0000039392), referred to here as HDAC3–shRNA1 and HDAC3–shRNA2, respectively, and a control plasmid (pLKO.1) were purchased from Sigma-Aldrich. The ability of these shRNA constructs to suppress HDAC3 expression was shown previously (Bardai and D'Mello, 2011).
Culturing, treatment, and transfection of neurons.
Cerebellar granule neurons (CGNs) were cultured from 7–8 day old Wistar rats as described previously (D'Mello et al., 1993). All transfections were done on day 5 using the calcium phosphate method as described previously (Bardai and D'Mello, 2011; Dastidar et al., 2011) and treated with either high potassium [HK; serum-free basal medium Eagle (BME) medium with 25 mm KCl] or low potassium (LK; serum-free BME medium) 8 h later unless the transfection was with shRNA plasmid, in which case treatment was done after 48 h. Serum-free culture medium is particularly important for experiments with HDAC3 in CGNs because neurotoxicity is not observed in the presence of serum, presumably because of IGF-1 in serum. Viability of transfected neurons (identified by immunocytochemistry) was assessed 24 h after treatment by staining cell nuclei with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI). When shRNA constructs were used, a GFP-expressing plasmid was cotransfected in a ratio of 1:6.5 to identify neurons expressing the shRNA. Cortical cultures were prepared from Wistar rats at embryonic day 16 or 17 as described previously (Majdzadeh et al., 2008). Cultures were kept in Neurobasal medium with 1% B27 supplement, 0.25% l-glutamine, 1% penicillin/streptomycin, 0.1% HEPES, and 1.1% sodium pyruvate without serum to minimize glial proliferation. Transfections were done on day 8 and treated with 1 mm homocysteic acid (HCA) 6 h later. Viability of transfected neurons was assessed after 14 h.
Immunoprecipitation and Western blot.
HEK293 cells were transfected with plasmids as indicated using Lipofectamine 2000. For cotransfection experiments, the amount of DNA was kept constant by using a “filler” plasmid, pLKO.1, in the control samples. For experiments using primary CGN cultures, the cells were treated with HK or LK media for 6 h on day 7. For R6/2 samples, mice were killed at 6 or 13 weeks, striatum, cortex, and cerebella were separated, and protein samples were prepared. For all endogenous samples, 1000 μg of total protein was used for immunoprecipitation using 5 μg of the appropriate antibody. Incubations for immunoprecipitation were done for 12–14 h with rocking at 4°C. The immunoprecipitated proteins were subjected to SDS-PAGE and probed with the antibody as indicated.
HDAC3 shRNA or control plasmids were cotransfected with GFP-tagged mut-Htt in 2:1 ratio for 48 h. CGNs were then switched to HK media, whereas cortical cultures were left untreated. Viability of transfected neurons (GFP-positive) was assessed 24 h later.
Generation of Hdac3−/− conditional knock-out mice and cultures.
Hdac3−/− conditional knock-out mice were generated as described previously (Bardai et al., 2012). Briefly, mice homozygous for loxP sites in the HDAC3 locus (Hdac3neo-loxP) were bred to Nes–Cre transgenic mice (The Jackson Laboratory) that express the Cre recombinase throughout the developing CNS, allowing for the generation of CNS-specific Hdac3+/− mice. These Hdac3+/− mice were then interbred, and genotyping of pups was performed on the day of birth. Cortical neuron cultures were prepared from individual Hdac3−/− and Hdac3+/+ pups on the day of birth (P0). The cultures were transfected with either GFP or mut-Htt plasmid on day 7 after plating, and viability was assessed 20 h later.
The R6/2 transgenic mouse model of HD.
Female C57BL/6J (B6) mice hemizygous for the ovarian transplant of the truncated mutant Htt transgene containing 160± glutamines in the polyQ region were bred with wild-type CBA/J (CBA) male mice. Both mice were purchased from The Jackson Laboratory. Only F1 pups were used in the experiments. Genotyping of pups was performed using the primers recommended by The Jackson Laboratory. At 6 and 13 weeks after birth, mice with the transgene (R6/2) or their wild-type littermates of the same gender were killed, the brains were dissected, and the striatum, cortex, and cerebellum were separated. Lysates were made from the brain tissue and used for immunoprecipitation assays.
TUNEL assays were performed using the fluorometric in situ cell death detection kit, TMR red from Roche Applied Science, according to the instructions of the manufacturer. Briefly, transfected neurons were fixed in 4% paraformaldehyde. After washing with PBS, the neurons were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate solution at 4°C and incubated with fluorescein-conjugated TUNEL reaction mixture at 37°C for 1 h in the dark. After reaction termination, the cells were washed with PBS and stained with DAPI before visualization under a fluorescent microscope.
Ectopic expression of Htt protects neurons from apoptosis
To examine whether Htt has neuroprotective functions, we expressed it in cultured CGNs, a neuronal type from a brain region not affected in HD. CGNs can be maintained in medium containing depolarizing levels of potassium (HK), which mimics the survival-promoting effect of neuronal activity in vivo. Treatment with non-depolarizing medium (LK) results in cell death of these neurons (D'Mello et al., 1993). Expression of Htt588–Q15 (henceforth referred to as Htt), composed of the N-terminal 588 aa of Htt and containing 15 glutamine repeats, completely protects against LK-induced death (Fig. 1A,C). In contrast, expression of a polyQ-expanded form of the protein, Htt588–Q138 (henceforth referred to as mut-Htt) promoted death of otherwise healthy neurons. A similar protection by Htt was observed in cortical neurons induced to die by treatment with HCA (Fig. 1B). These results demonstrate that elevated Htt expression protects neurons even in experimental models not directly related to HD pathogenesis. Results obtained using DAPI staining of the nuclei were confirmed using the TUNEL assay (Fig. 1F).
Htt interacts with HDAC3, and this interaction is reduced in apoptotic conditions
LK-induced death of CGNs as well as apoptosis in cortical neurons and HT22 cells is dependent on HDAC3 (Bardai and D'Mello, 2011). We tested the possibility that neuroprotection by Htt is mediated by suppressing the neurotoxic activity of HDAC3. As shown in Figure 1, D and E, HDAC3-induced neurotoxicity in both CGNs and cortical neurons is substantially reduced when Htt is coexpressed. To examine how Htt inhibits HDAC3 neurotoxicity, we investigated whether the two proteins interact. Coimmunoprecipitation analysis revealed robust interaction between HDAC3 and Htt (Fig. 2A). In comparison, interaction with mut-Htt was weak. HDAC3 and Htt colocalize in the cytoplasm of CGNs, cortical neurons, and HT22 cells when the two proteins are expressed, confirming interaction (Fig. 2B). Additionally, interaction between endogenous Htt and HDAC3 is observed in CGNs.
Interestingly, although Htt and HDAC3 levels are similar in healthy and dying neurons, interaction between them can only be detected in healthy neurons (Fig. 2C,D; Bardai and D'Mello, 2011). Reduction in HDAC3–Htt interaction was also observed under conditions of neurodegeneration in the R6/2 transgenic mouse model of HD, which express an N-terminal fragment of mut-Htt. As seen in Figure 2E, whereas interaction was similar in the striatum of presymptomatic R6/2 mice (6 weeks old), it was markedly reduced at 13 weeks when neuropathology and behavioral deficits are obvious. A discernible reduction was observed in the R6/2 cortex, which is also affected in HD but not in the cerebellum. In fact, Htt–HDAC3 interaction is elevated in the cerebellum, possibly explaining its relative resistance to degeneration in HD. Together, these results suggest that Htt protects neurons by sequestering HDAC3 through physical interaction and that the liberation of HDAC3 from Htt de-represses its neurotoxic activity leading to cell death.
Mut-Htt neurotoxicity is dependent on HDAC3
HDAC3 neurotoxicity is inhibited by treatment with IGF-1 or the expression of Akt, a kinase activated by IGF-1 treatment (Bardai and D'Mello, 2011). We showed previously that protection by IGF-1/Akt against HDAC3 neurotoxicity is mediated by the inhibition of glycogen synthase kinase-3β (GSK3β), a kinase that is widely implicated in promoting neuronal death and that phosphorylates HDAC3 (Bardai and D'Mello, 2011). Coincidentally, toxicity by mut-Htt is also inhibited by IGF-1 and Akt (Humbert et al., 2002; Rangone et al., 2005). Additionally, and as demonstrated previously using chemical inhibitors (Carmichael et al., 2002; Valencia et al., 2012), suppression of GSK3β protects against mut-Htt toxicity (Fig. 3A). These results suggest that mut-Htt requires HDAC3 for its neurotoxic effect. Consistent with this conclusion, shRNA-mediated knockdown of HDAC3 reduces mut-Htt toxicity in both CGNs and cortical neurons (Fig. 3B,C). Additionally, HDAC3-deficient neurons display reduced vulnerability to mut-Htt toxicity (Fig. 3D). Because mut-Htt promotes death and given that HDAC3 is necessary for its neurotoxic effect, it could be predicted that mut-Htt disrupts the association between Htt and HDAC3. This is what is observed when mut-Htt is coexpressed with Htt and HDAC3 in HEK293 cells (Fig. 3F). Conversely, the overexpression of Htt reduces mut-Htt toxicity when coexpressed in CGNs (Fig. 3G). Although the protection by Htt in neurons appears to contradict the disruption of Htt–HDAC3 interaction by mut-Htt observed in HEK293 cells, it is possible that liberation of HDAC3 depends also on additional factors that are present in HEK293 cells but not in neurons. While additional studies to identify such factors are necessary, one candidate is GSK3β activity, which is normally low in healthy neurons but the elevation of which causes neurodegeneration.
Our report is significant for several reasons. First, we show that normal Htt is a neuroprotective protein. Other studies have reached a similar conclusion. However, in general, these studies have used neurons cultured from Htt-expressing transgenic mice or cell lines stably expressing Htt (Rigamonti et al., 2000; Leavitt et al., 2001, 2006; Van Raamsdonk et al., 2005). Given that Htt has diverse functional actions, the protection seen in these cells could conceivably be attributable to secondary effects resulting from long-term Htt expression. Similarly, the degeneration seen in mice lacking Htt could be the consequence of developmental defects, general biochemical abnormalities such as reduced energy metabolism, or defects in processes such as axonal transport. We show that acute expression of Htt protects primary neurons against death by mut-Htt toxicity as well as in paradigms unconnected to HD, demonstrating that it has direct and broad neuroprotective activity. This supports the results of Ho et al. (2001) who showed that Htt protected against mut-Htt when the two proteins were coexpressed in cell lines. In a separate study, Zhang et al. (2003) found that siRNA-mediated knockdown of endogenous Htt induced death in a neuronal cell line but not in non-neuronal cells.
Second, we show for the first time that Htt interacts with HDAC3, a protein that is selectively toxic to neurons (Bardai and D'Mello, 2011). Our results suggest that Htt protects neurons through the sequestration of HDAC3. It deserves mention that a recent publication reported that R6/2 HD mice deficient in HDAC3 do not display reduction in disease severity or progression (Moumné et al., 2012). However, that study had used Hdac3+/− heterozygotes in which expression of HDAC3 was reduced by only 20%, with no reduction observed in cytoplasmic HDAC3 (Moumné et al., 2012). Moreover, compensatory effects by other HDAC proteins cannot be excluded in this total knock-out line. In our experiments, expression of HDAC3 was virtually undetectable using both shRNA and neurons from Hdac3−/− conditional knock-out mice.
Third, our results provide a possible explanation for both the loss-of-function and gain-of-function mechanisms proposed for HD pathogenesis. Loss of Htt function would de-repress the neurotoxic activity of HDAC3. HD neuropathology is also observed in mouse models in which mut-Htt is expressed in the context of normal levels of Htt. This gain-of-function effect can be explained by the ability of mut-Htt to promote disassociation of HDAC3 from Htt. Exactly how this is achieved remains to be clarified. It is possible that mut-Htt activates GSK3β, reducing its affinity for Htt. Phosphorylation of HDAC3 by GSK3β is necessary for its neurotoxicity (Bardai and D'Mello, 2011). Consistent with the idea that liberation of HDAC3 from Htt is a key event in neurotoxicity by mut-Htt is the finding that the level of interaction is reduced in the striatum of symptomatic R6/2 mice but not in presymptomatic mice. Htt–HDAC3 interaction was not reduced in the cerebellum. Because mut-Htt is expressed in both brain regions, it is not clear why the interaction with Htt is not disrupted in the cerebellum. This suggests that, although necessary, the presence of mut-Htt is not sufficient for the disassociation of HDAC3 from Htt and that other factors are also involved. These may include extrinsic factors, such as IGF-1, which is highly produced in the cerebellum and through which activation of Akt can inhibit GSK3β.
Most importantly, our results provide a unifying model (Fig. 4) that explains how a seemingly disparate group of pharmacological agents and molecules, namely normal Htt (Rigamonti et al., 2000; Leavitt et al., 2001, 2006), IGF-1(Humbert et al., 2002; Rangone et al., 2005), pan-HDAC and HDAC3-selective inhibitors (Thomas et al., 2008; Aiken et al., 2009; Jia et al., 2012), HDAC3 knockdown (in Caenorhabditis elegans) (Bates et al., 2006), and GSK3β inhibitors (Carmichael et al., 2002; Valencia et al., 2012), all protect against mut-Htt-induced neurodegeneration. Once HDAC3 dissociates from Htt, it is possible that the resulting neurotoxicity is attributable to transcriptional alterations involving genes regulated by HDAC3. Indeed, transcriptional dysregulation has been implicated in the pathogenesis of HD (Cha, 2000; Steffan et al., 2001). We could speculate that one such downstream target of HDAC3-mediated repression could be BDNF, a neurotrophic factor necessary for the survival of striatal and cortical neurons. This would be consistent with the finding that Htt promotes the expression of BDNF in cortical neurons (by inhibiting HDAC3-mediated repression of its transcription) and that this activity is lost in the mutant form of the protein (Zuccato et al., 2001).
This research was supported by National Institutes of Health Grant NS040408 (S.R.D.). We thank Jason Pfister and Sathi Mallick for performing confirmatory experiments. We are grateful to Troy Littleton and Dimitri Krainc for providing us with Htt and mutant-Htt plasmids.
- Correspondence should be addressed to Santosh R. D'Mello, Department of Molecular and Cell Biology, University of Texas at Dallas, 800 West Campbell, Richardson, TX 75080.