Taken together, our findings reveal a novel molecular mechanism by which cocaine and cAMP signaling regulate HDAC5 nuclear accumulation to limit adaptations that increase the rewarding impact of cocaine (). Our findings support the noted role for HDAC5 in limiting cocaine reward behavior (Renthal et al., 2007
); however, our observations that cocaine induces transient, delayed dephosphorylation and nuclear import of HDAC5 to suppress cocaine reward is a significant departure from previous ideas of how cocaine regulates HDAC5 function in vivo
(Renthal et al., 2007
). We observed a significant regulation of HDAC5 phosphorylation and nuclear levels that strongly suggest that dynamic regulation of this epigenetic factor plays a crucial role in limiting the impact of cocaine reward in vivo
Working model for how cocaine and cAMP signaling regulate HDAC5 nuclear import and limit cocaine reward behavior.
Several studies have reported that cocaine exposure increases P-S259 HDAC5 levels by Western blotting or immunohistochemistry (Dietrich et al., 2011
; Host et al., 2011
; Renthal et al., 2007
), but with the near perfect conservation of amino acids spanning the P-S259 site in HDAC4, HDAC5, HDAC7 and HDAC9, it is important to note that the P-S259 antibody recognizes multiple class IIa HDAC proteins, not only HDAC5. In contrast to these reports, our study revealed a robust decrease in P-S259 and P-S498 levels on HDAC5 (). Our analysis of the P-S279 HDAC5 site, which is also highly conserved in HDAC4 and HDAC9, revealed that total P-S279 immunoreactivity was not specific to HDAC5 (i.e.
HDAC5 knockout mouse tissues had significant residual P-S279 immunoreactivity). To achieve HDAC5-specific analysis of these conserved sites, we had to first IP total HDAC5 protein prior to Western blotting with the phosphorylation site-specific antibodies (e.g. Figure S1C
). In the future, it will be important to determine whether the reported increases in P-S259 signal after cocaine exposure reflect specific regulation of HDAC5 or might instead represent regulation of other class IIa HDAC(s).
The binding of HDAC5 to 14-3-3 proteins is mediated by phosphorylation of S259 and S498 sites, and this association is thought to be important for HDAC5 cytoplasmic localization (Chawla et al., 2003
; McKinsey et al., 2000a
; McKinsey et al., 2000b
; Sucharov et al., 2006
; Vega et al., 2004
). Similar to previous work, we observe that the HDAC5 S259A/S498A mutant protein is largely localized within the nucleus or evenly distributed between nucleus and cytoplasm. However, this mutant has significantly reduced P-S279 levels (Figure S4D
), which suggests that the increase in nuclear localization of this mutant may be due, at least in part, to reduced P-S279 levels. This conclusion is strengthened by the observation that combining the S279E phosphomimetic mutation with the S259A/S498A mutations results in increased cytoplasmic distribution of HDAC5 and resistance to cAMP-induced nuclear import. The S259A/S498A/S279E HDAC5 mutant does not bind to 14-3-3 (data not shown), which strongly suggests that P-S279 exerts its effect on HDAC5 nuclear import through a 14-3-3-independent mechanism. Several studies have reported that phosphorylation close to, or within, an NLS can mask a protein’s interaction with other proteins or inactivate its NLS function (Jans et al., 1991
; Moll et al., 1991
). Due to the strong concentration of positively charged residues within the HDAC5 NLS, we speculate that the introduction of three negative charges by organic phosphate at S279 might neutralize the NLS charge or induce a conformational change that reduces association with nuclear import proteins.
During review of our manuscript, a study reported regulation of P-S279 HDAC5 by PKA in COS7 cells (Ha et al., 2010
), and provided evidence that P-S279 promoted nuclear retention in these cells. Similar to this study, we had also found that purified PKA phosphorylates HDAC5 S279 in vitro (Figure S2A
); however, we found that basal phosphorylation at this site, at least in striatal neurons, did not require PKA activity (Figure S2C
). In addition, our direct measurements of endogenous HDAC5 P-S279 levels revealed that forskolin treatment of COS7 cells, striatal neurons, cortical neurons, or acute, adult striatal slices actually decreased P-S279 HDAC5 levels ( and S2
, and data not shown), which seems incompatible with the proposed role for P-S279 in the COS7 cells. We speculate that the expression of constitutively-active PKA in COS7 cells may regulate additional HDAC5 sites that influence nuclear localization and require P-S279 or that over-expressed HDAC5-EGFP is regulated differently than endogenous HDAC5 in COS7 cells. Additional experiments will be required to help resolve the different conclusions drawn by these two studies, but in striatal neurons, it seems clear that HDAC5 P-S279 does not promote nuclear accumulation, but quite the opposite.
Our observations about the role and regulation of HDAC5 P-S279 in cocaine-induced behavioral plasticity raise a number of interesting questions for future study. For example, what is the nuclear function of HDAC5 that limits cocaine reward? Nestler and colleagues (Renthal et al., 2007
) reported that the enzymatic HDAC domain of HDAC5 is required for reducing cocaine reward, suggesting that the ultimate substrate is histone deacetylation and indirect suppression of HDAC5 target genes. Indeed, many hundreds of genes were aberrantly increased or decreased by cocaine in the HDAC5 KO mice at 24 hrs after repeated cocaine injections. Since these were total HDAC5 KO mice, lacking HDAC5 expression throughout the lifetime of the animal, it is difficult to know whether these are direct effects of HDAC5 on the identified genes. Moreover, the time point analyzed (i.e.
24 hrs) is during a phase when HDAC5 phosphorylation and nucleocytoplasmic localization are similar to saline control conditions. In the future, it will be interesting to determine the target genes that are bound and regulated by HDAC5 after cocaine, particularly at those time points when enhanced HDAC5 nuclear function is observed following cocaine exposure. It is possible, and perhaps likely, that regulation of multiple HDAC5 gene targets contributes to the reduction of cocaine reward behavior, and dissecting out the relative contributions of each gene target will represent a major challenge going forward.
It is interesting to note that the HDAC5 S279A mutant suppressed cocaine reward to a greater extent than WT HDAC5 (). There are several possible explanations for this difference, including: (1) The HDAC5 S279A mutant in vivo resides constitutively in the nucleus, whereas the WT HDAC5 is only transiently localized in nucleus upon cocaine exposure. In this case, the levels of the P-S259/P-S498 would presumably be low such that P-S279 plays the dominant major role in subcellular localization (unlike the striatal cultures). (2) The HDAC5 S279A mutant has reduced nuclear export kinetics compared to WT HDAC5, and as a result, resides in the nucleus for a longer time after cocaine exposure. We found that P-S279 HDAC5 increases nuclear export kinetics (). If HDAC5 S279A cannot be rephosphorylated following nuclear import, then HDAC5 S279A may remain in the nucleus and exert longer-lasting effects following cocaine exposure. (3) HDAC5 P-S279 may regulate not only its nuclear/cytoplasmic localization, as documented in our study, but might also regulate its function as a transcriptional co-repressor in the nucleus. As such, the HDAC5 S279A mutant may be a more effective co-repressor via unknown mechanisms. Due to technical limitations, we were unable to visualize the subcellular distribution of the HDAC5 mutants in vivo. Nevertheless, our findings in this study reveal an important role for dephosphorylation of P-S279 HDAC5 in the regulation of cocaine reward behavior.
Our findings in striatal cultured neurons revealed a high degree of co-localization of HDAC5-EGFP with endogenous MEF2A and MEF2D, two of the well-studied transcription factor proteins that interact with HDAC5, suggesting MEF2 as a possible mediator of HDAC5 function in reducing cocaine reward sensitivity after repeated cocaine experience. Consistent with this idea, we reported recently that expression of constitutively-active MEF2 in the NAc enhances cocaine reward behavior (Pulipparacharuvil et al., 2008
), which is opposite of the effect of HDAC5 expression in this region. In the future, it will be important to determine whether HDAC5 exerts its effects on cocaine reward through binding to MEF2 proteins, or whether the critical nuclear target of HDAC5 in the mediation of cocaine reward may be one or more previously undescribed transcription factors. The identification of HDAC5 target genes after cocaine exposure may help determine whether MEF2 and HDAC5 bi-directionally regulate cocaine reward through a common pathway or whether these proteins regulate cocaine behavior through distinct transcriptional mechanisms in vivo
Similar to our observed regulation of HDAC5 P-S279, previous studies in striatal neurons have reported that cAMP signaling increases PP2A activity (Ahn et al., 2007
), which then dephosphorylates the Cdk5 substrates, Wave1 (Ceglia et al., 2010
) and dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) (Bibb et al., 1999
; Nishi et al., 2000
). Acute cocaine does not alter the levels or activity of Cdk5 or levels of p35 in striatum (Kim et al., 2006
; Takahashi et al., 2005
), suggesting that the decrease in P-S279 is due to increased phosphatase activity rather than decreased Cdk5 activity. Interestingly, cocaine and cAMP signaling has been shown to induce transient DARPP-32 nuclear accumulation via dephosphorylation in striatal neurons (Stipanovich et al., 2008
). Similar to our findings with HDAC5, nuclear accumulation of DARPP-32 attenuates cocaine reward behavior, which is proposed to involve epigenetic gene regulation (Stipanovich et al., 2008
). Together, these findings indicate that while cocaine induces rewarding effects, the striatum stimulates negative feedback processes, such as enhanced HDAC5 and DARPP-32 nuclear levels, to attenuate the reward impact of future cocaine exposures. As such, these proteins may represent critical intrinsic mechanisms for counteracting the maladaptive changes in reward circuit function, and understanding these negative feedback processes may reveal new avenues for the treatment of drug addiction.
Taken together, our findings reveal that cocaine regulates the transient nuclear accumulation of HDAC5, and this likely occurs through a newly discovered molecular mechanism involving PP2A phosphatase-dependent dephosphorylation of HDAC5 at three critical phosphoserines, S259, S279, and S498. The removal of phosphate from these sites likely increases the NLS function and decreases binding to 14-3-3 proteins, and promotes the repression of HDAC5 target genes in the nucleus. Importantly, our findings reveal that dephosphorylation of S279 HDAC5 is critical for its ability to limit the development of cocaine reward-related behavioral adaptations, but not natural reward behavior. Since cocaine-experienced HDAC5 KO mice have enhanced place preference to cocaine, and this effect is rescued by NAc expression of WT HDAC5 (Renthal et al., 2007
), our combined findings suggest that HDAC5 provides a delayed braking mechanism on gene expression programs that support the development, but not expression, of cocaine reward behaviors. As such, deficits in this process may contribute to the development of maladaptive behaviors associated with addiction following repeated drug use in humans.