Targeted Epigenetic Remodeling of the \u3cem\u3eCdk5\u3c/em\u3e Gene in Nucleus Accumbens Regulates Cocain- and Stress-Evoked Behavior

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 gound 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

Phenotyping and Preclinical Meta-Analysis of Behavioral Outcomes from Complete Freund’s Adjuvant-Induced Inflammatory Pain in the Rodent Hind-paw

Pain and opioid use disorder prevalence, diagnoses of mental health disorders, and rate of suicide have all skyrocketed over the last 30 years as a matrix of public health crises. Each reduces quality of life and installs risk of the others; together, they have burdened our country with nearly unmanageable healthcare challenges. Although the critical demand for novel and more effective therapeutics has been clear for decades, relatively little progress has been made to address pain and its comorbidities. Repeated clinical trial failures have called into question the preclinical evidence upon which they are based. Preclinical models of pain-induced negative affect have been adapted from those of chronic stress and their corresponding classical tests of behavior, which featured predictive validity for antidepressants and anxiolytics. Now, animal models of pain and pain-induced behavioral comorbidities are described in extensive, highly variable, and inconclusive literature. The work presented in this dissertation aims to address these limitations by defining the behavioral outcomes of hind-paw inflammatory pain, using extensive phenotyping, systematic review, and meta-analysis. I hypothesized that a rigorous and reproducible model of inflammatory pain-induced negative affect would have similar behavioral outcomes to preclinical models of chronic stress. In the first study presented in this dissertation, I use systematic behavioral phenotyping and analysis following hind-paw injection of Complete Freund’s Adjuvant (CFA) in male C57BL/6J mice. CFA did not reduce exploratory behavior in the open field test (OFT) or elevated zero maze (EZM) and did not induce a passive stress coping strategy in the forced swim test (FST). Behavioral differences between saline controls and CFA mice were not apparent at four or six weeks post-injection, regardless of testing order and combination. Behavioral measures did not correlate between phenotyping assays, and composite behavioral scores did not correlate to severity of thermal hyperalgesia. A focused and limited meta-analysis of similar studies with male C57BL/6 mice revealed a modest but significant effect of CFA on exploratory behavior in the OFT and EPM/EZM, but not on stress coping in the FST. In the second study presented in this dissertation, I conduct a comprehensive systematic review and meta-analysis of the effect of CFA-induced hind-paw inflammation in rodents on multiple indicators of exploratory, stress coping, naturalistic, and rewarding behaviors. This meta-analysis broadens the one previously described by extending the considered population to include male and female mice and rats of all strains, and by expanding the considered outcomes to include anhedonic and ethologically relevant behavior. I hypothesized that CFA exposure in rodents would have little to no significant effect on exploratory behavior and stress coping, and moderate effect on naturalistic and rewarding behaviors, when evaluating dozens of studies together. Secondarily, I hypothesized that any statistically significant global summary effects would be moderated by methodological variables, such as species, strain, sex, injection laterality, age, amount of CFA injected, and interval between injection and testing. A systematic search of four databases in 2020 returned 46 studies evaluating performance in the elevated plus or zero maze (EPM/EZM), open field test (OFT), light/dark box (LDB), place escape/avoidance paradigm (PEAP), forced swim test (FST), tail suspension test (TST), sucrose preference test (SPT), wheel running, and burrowing assay after hind-paw CFA injection. Discrete meta-analyses for each of these nine behavioral measures indicated that CFA (relative to saline exposure) modestly but significantly decreases exploratory behavior, increases passive stress coping in the TST but not the FST, and significantly decreases preference for sucrose and naturally rewarding activity. Heterogeneity among included studies was moderate to high across all nine behavioral tests. Sub-group analyses and meta-regressions revealed that species and animal sourcing were significant moderators of those effects, but not duration of inflammation, sex, or paw laterality. Quality assessments identified multiple aspects of internal validity that future studies can improve on to achieve consistency and reproducibility in this field. These meta-analyses also identified significant publication bias for the most commonly used tests, the EPM/EZM and OFT. These findings together indicate that CFA-induced hind-paw inflammation does not have consistent or substantial effects on exploratory behavior and stress coping, and suggest that tests such as the EPM and FST exhibit poor face and construct validity for modeling pain and mental health comorbidities. The results of this dissertation provoke thoughtful reflection on whether such tests are appropriate for modeling any aspect of mental health, as they evoke the same symptoms they aim to measure and rely on subjective interpretations. Future research agendas may leverage the information presented in this dissertation to optimize the models of pain-induced negative affect used in mechanistic studies. Subsequent studies on the effects of inflammatory pain on central nervous system physiology are encouraged to focus on naturalistic and ethologically relevant, hedonically rewarding, and motivated behaviors, which will better align with the current paradigm shift of studying individual symptoms and ethologically relevant behaviors

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

International audienceDepression 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

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.This work was supported by the National Multiple Sclerosis Society (Grant RG 4046-A6) and the National Institutes of Health (Grant R21 NS079144-01, Neuropathology Training Grant T32 NS007098, and Cellular and Molecular Biology and Genetics Training Grant T32 GM007491).Peer Reviewe