We show that levels of dimethylK9/K27-H3, a marker of gene repression, are highly increased in both the social defeat and social isolation models. Despite the fact that one model involves an active form of stress and the other a type of passive stress, we have shown some common depressive-like symptoms (e.g., anhedonia-like symptoms) and anxiety-related behavior in the two paradigms (Krishnan et al., 2007
; Wallace et al., 2009
). The increased H3 methylation observed would reduce the ability of the affected genes to be transcribed either at baseline or in response to other stimuli, and the consequences of such increased methylation may be far-reaching. Indeed, a key advantage of this ChIP-chip analysis is that it provides a novel look at genes that are largely repressed or silenced.
Among the regulated genes are several involved in inflammatory pathways and regulation of redox state. One possibility is that the repression of such genes may be protective (e.g., by limiting the consequences of the initial activation of these biochemical pathways), but that this comes at the cost of decreased ability to activate the pathways later in the face of further challenges. Sep-15
, which shows differential H3 methylation between stressed and control animals, has been shown to have antioxidant properties, and its dysregulation is implicated in neurodegenerative disorders, such as Alzheimer's Disease (Chen and Berry, 2003
; Ashrafi et al., 2007
). Activity of the Na+
ATPase, as well as activity of enzymes in the mitochondrial respiratory chain, are also disrupted in animal models of stress (Rezin et al., 2008
). This corresponds with our findings in that we see regulation of Cox6c
, subunits of cytochrome c oxidase. Likewise, the repression of genes involved in NF-κB signaling is interesting in that we have recently demonstrated a pro-depressive effect of inhibiting the NF-κB pathway in the NAc (LaPlant et al., 2009
). This is in contrast to the effect of NF-κB in hippocampus, where activation of the pathway decreases neurogenesis and depressive-like phenotypes (Pace et al., 2006
; Koo and Duman, 2008
). This work adds to an evolving literature that has implicated a range of inflammatory, cell stress, and cytokine pathways in depression-related phenomena. Chronic inflammation, through cytokine release, induces depression-like behavior in animals, and treatment of hepatitis C with interferon α induces depression in a large subset of patients (Dantzer et al., 2008
). Conversely, behavioral stress, as shown here, activates cytokine signaling in the brain, which may contribute to a range of stress-related behavioral abnormalities (Shintani et al., 1995
; Maier et al., 1999
; Anisman et al., 2008
Another class of genes highly regulated in common in the social defeat and social isolation models are those involved in transcriptional regulation. Jdp2
shows greatly induced H3 methylation in stressed animals. The product of this gene binds to c-Jun, with the resulting complex inducing transcriptional repression by binding to AP-1 sites and preventing the binding of active AP-1 transcription factor complexes. Therefore, repression of Jdp2
would lead to decreased repression of c-Jun and hence to an increase in AP-1 mediated transcription. Moreover, Jdp2 protein binds to ATF-2 and causes transcriptional repression via HDAC3 (Eitoku et al., 2008
), and we have recently demonstrated antidepressant-like effects of ATF-2 when overexpressed in the NAc (Green et al., 2008
), making this protein an attractive target for future investigations.
For phospho-CREB, our results demonstrate that, unlike H3 methylation state, binding of phospho-CREB to gene promoters is differentially regulated in the social defeat and social isolation models of depression. It is interesting, however, that the same general categories of genes showed altered levels of phospho-CREB binding as found for H3 methylation. Additionally, prominent among the genes that displayed opposite regulation of phospho-CREB binding in the two stress models are those related to actin remodeling. It is well documented that hippocampal volume is decreased in depressed patients (Egger et al., 2008
), and that there is hippocampal dendritic atrophy in chronic stress paradigms (Magarinos et al., 1996
). Identification of stress-induced alterations in several genes involved in actin remodeling in the NAc raises the novel possibility that related morphological changes may occur in NAc neurons. Our data suggest that CREB activity may function as an overall switch that coordinates such regulation in the context of chronic stress.
The clinical efficacy of antidepressants, all of which alter monoaminergic neurotransmission, is well-documented, however, it is unclear how chronic antidepressant administration changes the behavioral state of the organism. Our analyses provide new insight into the long-term effects of antidepressant treatments on the brain. Specifically, our data demonstrate that chronic imipramine treatment dramatically reverses many of the effects of chronic social defeat stress on H3 methylation and on phospho-CREB binding in the NAc. While such actions would not be expected to account for the complete behavioral effects elicited by antidepressant treatment, they provide a beginning framework and suggest several novel molecular pathways to pursue. Among the genes that show reversal of stress regulation by imipramine is Chrh2
, which encodes the corticotropin releasing hormone receptor, which has been highly implicated in stress responses (Nemeroff and Vale, 2005
One of the most intriguing aspects of the social defeat paradigm is its consistent ability to generate animals, which have undergone equivalent levels of defeat stress, but do not exhibit a depression-like phenotype (Krishnan et al., 2007
). Consistent with previous studies where we demonstrated that such resilient animals display some unique changes in mRNA expression in the NAc compared to susceptible animals (Krishnan et al., 2007
), we now show that resiliency is likewise associated with a set of unique chromatin modifications in the NAc with respect to H3 methylation and phospho-CREB binding, further supporting the notion that resilience is an active process and not simply the absence of changes that occur in vulnerable animals. The identification of such “resilience genes,” therefore, defines a novel drug discovery pathway whereby new antidepressant treatments can be developed to promote resiliency. Given that susceptible and resilient responses are seen among inbred mice raised in identical environmental settings, the factors responsible for these differential adaptations to chronic defeat stress remain unknown. One possibility is that pre-existing epigenetic modifications contribute to these distinct responses (Krishnan and Nestler, 2008
). Regardless, the deleterious adaptations in susceptible animals may involve “allostatic overload.” Stress in the short-term normally elicits adaptive responses that are beneficial to the animal; in a more chronic context, the system may be overtaxed in some individuals, resulting in dysregulation of multiple neural processes (McEwen et al., 2008
One of the most striking findings of the present study is the significant overlap in the chromatin changes in the NAc associated both with chronic imipramine treatment and with resiliency. This observation supports the highly novel view that resilient individuals are less vulnerable to the deleterious effects of chronic stress in part by mounting naturally some of the same changes in gene expression that occur in the NAc. Such overlapping genes provide a rich array of possible mediators of resiliency and antidepressant responses. However, since less than half of all patients treated with available antidepressants show a complete response, the genes that show chromatin regulation in resilient animals, and not after antidepressant treatment, may provide an even better set of novel targets for the development of new agents with a broader range of clinical efficacy.
The experiments undertaken in this study examined adult male mice only. Previous work has shown that there are important effects of both gender and age that should be considered going forward. Females are more susceptible to depression and more likely exhibit atypical symptoms of depression (Gorman, 2006
). Furthermore, a better understanding of childhood and geriatric depression, and improved treatments, are sorely needed (Lenze et al., 2008
; Tsapakis et al., 2008
). It would also be interesting in future studies to examine individual differences in chromatin mechanisms in susceptible and resilient mice, something not yet feasible due to the current need to pool tissue from multiple animals for ChIP analysis.
The results of the present study illustrate the power of epigenetic investigations in psychiatric illness. ChIP-chip techniques provide novel insight into the detailed transcriptional mechanisms underlying the regulation of previously known gene targets in depression models. They also reveal many previously unknown gene targets and thereby provide a rational framework for the development of new treatment agents. Ultimately, a detailed step by step manipulation of individual genes, or sets of genes, identified by these methods will be needed to validate them as bona fide mediators of depression or antidepressant action. In the meantime, this work enables a more complete appreciation of the molecular changes induced in the brain that underlie vulnerability or resistance to depression and its effective treatment.