Targeted Epigenetic Remodeling of the Cdk5 Gene in Nucleus Accumbens Regulates Cocaine- and Stress-Evoked Behavior.

UNLABELLED: Recent studies have implicated epigenetic remodeling in brain reward regions following psychostimulant or stress exposure. It has only recently become possible to target a given type of epigenetic remodeling to a single gene of interest, and to probe the functional relevance of such regulation to neuropsychiatric disease. We sought to examine the role of histone modifications at the murine Cdk5 (cyclin-dependent kinase 5) locus, given growing evidence of Cdk5 expression in nucleus accumbens (NAc) influencing reward-related behaviors. Viral-mediated delivery of engineered zinc finger proteins (ZFP) targeted histone H3 lysine 9/14 acetylation (H3K9/14ac), a transcriptionally active mark, or histone H3 lysine 9 dimethylation (H3K9me2), which is associated with transcriptional repression, specifically to the Cdk5 locus in NAc in vivo We found that Cdk5-ZFP transcription factors are sufficient to bidirectionally regulate Cdk5 gene expression via enrichment of their respective histone modifications. We examined the behavioral consequences of this epigenetic remodeling and found that Cdk5-targeted H3K9/14ac increased cocaine-induced locomotor behavior, as well as resilience to social stress. Conversely, Cdk5-targeted H3K9me2 attenuated both cocaine-induced locomotor behavior and conditioned place preference, but had no effect on stress-induced social avoidance behavior. The current study provides evidence for the causal role of Cdk5 epigenetic remodeling in NAc in Cdk5 gene expression and in the control of reward and stress responses. Moreover, these data are especially compelling given that previous work demonstrated opposite behavioral phenotypes compared with those reported here upon Cdk5 overexpression or knockdown, demonstrating the importance of targeted epigenetic remodeling tools for studying more subtle molecular changes that contribute to neuropsychiatric disease. SIGNIFICANCE STATEMENT: Addiction and depression are highly heritable diseases, yet it has been difficult to identify gene sequence variations that underlie this heritability. Gene regulation via epigenetic remodeling is an additional mechanism contributing to the neurobiological basis of drug and stress exposure. In particular, epigenetic regulation of the Cdk5 gene alters responses to cocaine and stress in mouse and rat models. In this study, we used a novel technology, zinc-finger engineered transcription factors, to remodel histone proteins specifically at the Cdk5 gene. We found that this is sufficient to regulate the expression of Cdk5 and results in altered behavioral responses to cocaine and social stress. These data provide compelling evidence of the significance of epigenetic regulation in the neurobiological basis of reward- and stress-related neuropsychiatric disease.

Targeted GAS6 delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis.

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.

Inflammatory pain in mice induces light cycle-dependent effects on sleep architecture.

As a syndrome, chronic pain comprises physical, emotional, and cognitive symptoms such as disability, negative affect, feelings of stress, and fatigue. A rodent model of long-term inflammatory pain, induced by complete Freund's adjuvant (CFA) injection, has previously been shown to cause anhedonia and dysregulated naturalistic behaviors, in a manner similar to animal models of stress. We examined whether this extended to alterations in circadian rhythms and sleep, such as those induced by chronic social defeat stress, using actigraphy and wireless EEG. CFA-induced inflammatory pain profoundly altered sleep architecture in male and female mice. Injection of the hind paw, whether with CFA or saline, reduced some measures of circadian rhythmicity such as variance, period, and amplitude. CFA increased sleep duration primarily in the dark phase, while sleep bout length was decreased in the light and increased in the dark phase. Additionally, CFA reduced wake bout length, especially during the dark phase. Increases in REM and SWS duration and bouts were most significant in the dark phase, regardless of whether CFA had been injected at its onset or 12 hours prior. Taken together, these results indicate that inflammatory pain acutely promotes but also fragments sleep.

Behavioral outcomes of complete Freund adjuvant-induced inflammatory pain in the rodent hind paw: a systematic review and meta-analysis.

ABSTRACT: Many analgesics inadequately address the psychiatric comorbidities of chronic and persistent pain, but there is no standard preclinical model of pain-altered behavior to support the development of new therapies. To explore this conflicting and inconclusive literature, we conducted a focused systematic review and meta-analysis on the effect of complete Freund adjuvant-induced (CFA) rodent hind paw inflammation on multiple classical indicators of exploratory behavior, stress coping, and naturalistic behavior. Our primary objective was to define CFA's effect on assays including, but not limited to, the elevated plus maze and forced swim test. Our secondary objective was to discover how variables such as species and strain may influence outcomes in such assays. We searched Ovid MEDLINE, Embase, Scopus, and Web of Science in April and October 2020 for studies with adult rodents injected with CFA into the hind paw and subsequently tested for aspects of "anxiety-like" or "depressive-like" behaviors. Forty-four studies evaluated performance in the elevated plus or zero maze, open field test, light-dark box, place escape and avoidance paradigm, forced swim test, tail suspension test, sucrose preference test, wheel running, and burrowing assay. Complete Freund adjuvant modestly but significantly decreased exploratory behavior, significantly increased passive stress coping in the tail suspension test but not the forced swim test, and significantly decreased preference for sucrose and naturally rewarding activity. Subgroup analyses revealed significant differences between species and animal sourcing. Based on the evidence provided here, we conclude future studies should focus on CFA's effect on natural rewards and naturalistic behaviors.

Long-term inflammatory pain does not impact exploratory behavior and stress coping strategies in mice.

ABSTRACT: Pain puts patients at risk for developing psychiatric conditions such as anxiety and depression. Preclinical mouse models of pain-induced affective behavior vary widely in methodology and results, impairing progress towards improved therapeutics. To systematically investigate the effect of long-term inflammatory pain on exploratory behavior and stress coping strategy, we assessed male C57BL/6J mice in the forced swim test (FST), elevated zero maze, and open field test at 4 and 6 weeks postinjection of Complete Freund's Adjuvant, while controlling for testing order and combination. Inflammatory pain did not induce a passive stress coping strategy in the FST and did not reduce exploratory behavior in the elevated zero maze or the open field test. Using systematic correlational analysis and composite behavioral scores, we found no consistent association among measures for mice with or without inflammatory pain. A meta-analysis of similar studies indicated a modest, significant effect of Complete Freund's Adjuvant on exploratory behavior, but not immobility in the FST, and high heterogeneity among effect sizes in all 3 paradigms. Given the urgency for understanding the mechanisms of pain comorbidities and identifying novel therapies, these findings support the reallocation of our limited resources away from such unreliable assays and toward motivated and naturalistic behaviors. Future studies in pain and psychiatric translational research may benefit by considering outcomes beyond binary categorization, quantifying the associations between multiple measured behaviors, and agnostically identifying subtle yet meaningful patterns in behaviors.

A regulatory pathway linking caffeine action, mood and the diurnal clock.

Depression is a leading cause of disability worldwide. Circadian abnormalities and mood changes are symptoms of depression. The psychostimulant caffeine alters wakefulness and alleviates other depression-related symptoms during chronic intake, but the underlying mechanisms are unclear. It is not known, whether and how acute caffeine administration affects mood. Molecular approaches, transgenic mouse models, pharmacological intervention and behavioral analysis were combined to uncover a regulatory pathway, which connects caffeine action with diurnal signaling via the key dopaminergic protein DARPP-32 and alters mood-related phenotypes in mice, which are often assessed in the context of antidepressant action. We observed that Thr75-DARPP-32 binds to the circadian regulator CLOCK and disrupts CLOCK:BMAL1 chromatin binding, thereby affecting gene expression. T75A-DARPP-32 mutant mice show reduced caffeine effects on CLOCK:BMAL1 and lack caffeine-induced effects on mood. This study provides a link between caffeine, diurnal signaling and mood-related behaviors, which may open new perspectives for our understanding of antidepressant mechanisms in the mouse brain.

Cell-Type-Specific Epigenetic Editing at the Fosb Gene Controls Susceptibility to Social Defeat Stress.

Chronic social defeat stress regulates the expression of Fosb in the nucleus accumbens (NAc) to promote the cell-type-specific accumulation of ΔFosB in the two medium spiny neuron (MSN) subtypes in this region. ΔFosB is selectively induced in D1-MSNs in the NAc of resilient mice, and in D2-MSNs of susceptible mice. However, little is known about the consequences of such selective induction, particularly in D2-MSNs. This study examined how cell-type-specific control of the endogenous Fosb gene in NAc regulates susceptibility to social defeat stress. Histone post-translational modifications (HPTMs) were targeted specifically to Fosb using engineered zinc-finger proteins (ZFPs). Fosb-ZFPs were fused to either the transcriptional repressor, G9a, which promotes histone methylation or the transcriptional activator, p65, which promotes histone acetylation. These ZFPs were expressed in D1- vs D2-MSNs using Cre-dependent viral expression in the NAc of mice transgenic for Cre recombinase in these MSN subtypes. We found that stress susceptibility is oppositely regulated by the specific cell type and HPTM targeted. We report that Fosb-targeted histone acetylation in D2-MSNs or histone methylation in D1-MSNs promotes a stress-susceptible, depressive-like phenotype, while histone methylation in D2-MSNs or histone acetylation in D1-MSNs increases resilience to social stress as quantified by social interaction behavior and sucrose preference. This work presents the first demonstration of cell- and gene-specific targeting of histone modifications, which model naturally occurring transcriptional phenomena that control social defeat stress behavior. This epigenetic-editing approach, which recapitulates physiological changes in gene expression, reveals clear differences in the social defeat phenotype induced by Fosb gene manipulation in MSN subtypes.

Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2.

BACKGROUND: Exposure to drugs of abuse alters the epigenetic landscape of the brain's reward regions, such as the nucleus accumbens. We investigated how combinations of chromatin modifications affect genes that regulate responses to cocaine. We focused on Auts2, a gene linked to human evolution and cognitive disorders, which displays strong clustering of cocaine-induced chromatin modifications in this brain region. METHODS: We combined chromosome conformation capture, circularized chromosome conformation capture, and related approaches with behavioral paradigms relevant to cocaine phenotypes. Cell type-specific functions were assessed by fluorescence-activated cell sorting and viral-mediated overexpression in Cre-dependent mouse lines. RESULTS: We observed that Auts2 gene expression is increased by repeated cocaine administration specifically in D2-type medium spiny neurons in the nucleus accumbens, an effect seen in male but not female mice. Auts2 messenger RNA expression was also upregulated postmortem in the nucleus accumbens of male human cocaine addicts. We obtained evidence that chromosomal looping, bypassing 1524 kb of linear genome, connects Auts2 to the Caln1 gene locus under baseline conditions. This looping was disrupted after repeated cocaine exposure, resulting in increased expression of both genes in D2-type medium spiny neurons. Cocaine exposure reduces binding of CCCTC-binding factor, a chromosomal scaffolding protein, and increases histone and DNA methylation at the Auts-Caln1 loop base in the nucleus accumbens. Cell type-specific overexpression of Auts2 or Caln1 in D2-type medium spiny neurons demonstrated that both genes promote cocaine reward. CONCLUSIONS: These findings suggest that cocaine-induced alterations of neuronal three-dimensional genome organization destabilize higher order chromatin at specific loci that regulate responses to the drug.