CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model.

Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Chromatin-mediated alternative splicing regulates cocaine-reward behavior.

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Nr4a1 suppresses cocaine-induced behavior via epigenetic regulation of homeostatic target genes.

Endogenous homeostatic mechanisms can restore normal neuronal function following cocaine-induced neuroadaptations. Such mechanisms may be exploited to develop novel therapies for cocaine addiction, but a molecular target has not yet been identified. Here we profiled mouse gene expression during early and late cocaine abstinence to identify putative regulators of neural homeostasis. Cocaine activated the transcription factor, Nr4a1, and its target gene, Cartpt, a key molecule involved in dopamine metabolism. Sustained activation of Cartpt at late abstinence was coupled with depletion of the repressive histone modification, H3K27me3, and enrichment of activating marks, H3K27ac and H3K4me3. Using both CRISPR-mediated and small molecule Nr4a1 activation, we demonstrated the direct causal role of Nr4a1 in sustained activation of Cartpt and in attenuation of cocaine-evoked behavior. Our findings provide evidence that targeting abstinence-induced homeostatic gene expression is a potential therapeutic target in cocaine addiction.

Sex-Specific Regulation of Fear Memory by Targeted Epigenetic Editing of Cdk5.

BACKGROUND: Sex differences in the expression and prevalence of trauma- and stress-related disorders have led to a growing interest in the sex-specific molecular and epigenetic mechanisms underlying these diseases. Cyclin-dependent kinase 5 (CDK5) is known to underlie both fear memory and stress behavior in male mice. Given our recent finding that targeted histone acetylation of Cdk5 regulates stress responsivity in male mice, we hypothesized that such a mechanism may be functionally relevant in female mice as well. METHODS: We applied epigenetic editing of Cdk5 in the hippocampus and examined the regulation of fear memory retrieval in male and female mice. Viral expression of zinc finger proteins targeting histone acetylation to the Cdk5 promoter was paired with a quantification of learning and memory of contextual fear conditioning, expression of CDK5, and enrichment of histone modifications of the Cdk5 gene. RESULTS: We found that male mice exhibit stronger long-term memory retrieval than do female mice, and this finding was associated with male-specific epigenetic activation of hippocampal Cdk5 expression. Sex differences in behavior and epigenetic regulation of Cdk5 occurred after long-term, but not short-term, fear memory retrieval. Finally, targeted histone acetylation of hippocampal Cdk5 promoter attenuated fear memory retrieval and increased tau phosphorylation in female but not male mice. CONCLUSIONS: Epigenetic editing uncovered a female-specific role of Cdk5 activation in attenuating fear memory retrieval. This finding may be attributed to CDK5 mediated hyperphosphorylation of tau only in the female hippocampus. Sex-specific epigenetic regulation of Cdk5 may reflect differences in the effect of CDK5 on downstream target proteins that regulate memory.

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.

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.