Cannabis and synaptic reprogramming of the developing brain.

Recent years have been transformational in regard to the perception of the health risks and benefits of cannabis with increased acceptance of use. This has unintended neurodevelopmental implications given the increased use of cannabis and the potent levels of Δ9-tetrahydrocannabinol today being consumed by pregnant women, young mothers and teens. In this Review, we provide an overview of the neurobiological effects of cannabinoid exposure during prenatal/perinatal and adolescent periods, in which the endogenous cannabinoid system plays a fundamental role in neurodevelopmental processes. We highlight impaired synaptic plasticity as characteristic of developmental exposure and the important contribution of epigenetic reprogramming that maintains the long-term impact into adulthood and across generations. Such epigenetic influence by its very nature being highly responsive to the environment also provides the potential to diminish neural perturbations associated with developmental cannabis exposure.

Human mediator subunit MED26 functions as a docking site for transcription elongation factors.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

Δ9-Tetrahydrocannabinol inhibits Hedgehog-dependent patterning during development.

Many developmental disorders are thought to arise from an interaction between genetic and environmental risk factors. The Hedgehog (HH) signaling pathway regulates myriad developmental processes, and pathway inhibition is associated with birth defects, including holoprosencephaly (HPE). Cannabinoids are HH pathway inhibitors, but little is known of their effects on HH-dependent processes in mammalian embryos, and their mechanism of action is unclear. We report that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) induces two hallmark HH loss-of-function phenotypes (HPE and ventral neural tube patterning defects) in Cdon mutant mice, which have a subthreshold deficit in HH signaling. THC therefore acts as a 'conditional teratogen', dependent on a complementary but insufficient genetic insult. In vitro findings indicate that THC is a direct inhibitor of the essential HH signal transducer smoothened. The canonical THC receptor, cannabinoid receptor-type 1, is not required for THC to inhibit HH signaling. Cannabis consumption during pregnancy may contribute to a combination of risk factors underlying specific developmental disorders. These findings therefore have significant public health relevance.

Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity.

Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.

Overcoming addiction stigma: Epigenetic contributions to substance use disorders and opportunities for intervention.

Substance use disorders (SUDs) are frequently stigmatized by society. However, overcoming stigmas to effectively treat SUDs requires acknowledging the developmental path of neural vulnerability. These vulnerabilities begin long before adulthood, providing opportunities to change the trajectory before the disorder becomes entrenched. This article raises attention to reversible epigenetic underpinnings of this vulnerability.

Prenatal Δ9-Tetrahydrocannabinol Exposure in Males Leads to Motivational Disturbances Related to Striatal Epigenetic Dysregulation.

BACKGROUND: Cannabis remains one of the most widely abused drugs during pregnancy. In utero exposure to its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), can result in long-term neuropsychiatric risk for the progeny. This study investigated epigenetic signatures underlying these enduring consequences. METHODS: Rat dams were exposed daily to THC (0.15 mg/kg) during pregnancy, and adult male offspring were examined for reward and depressive-like behavioral endophenotypes. Using unbiased sequencing approaches, we explored transcriptional and epigenetic profiles in the nucleus accumbens (NAc), a brain area central to reward and emotional processing. An in vitro CRISPR (clustered regularly interspaced short palindromic repeats) activation model coupled with RNA sequencing was also applied to study specific consequences of epigenetic dysregulation, and altered molecular signatures were compared with human major depressive disorder transcriptome datasets. RESULTS: Prenatal THC exposure induced increased motivation for food, heightened learned helplessness and anhedonia, and altered stress sensitivity. We identified a robust increase specific to males in the expression of Kmt2a (histone-lysine N-methyltransferase 2A) that targets H3K4 (lysine 4 on histone H3) in cellular chromatin. Normalizing Kmt2a in the NAc rescued the motivational phenotype of prenatally THC-exposed animals. Comparison of RNA- and H3K4me3-sequencing datasets from the NAc of rat offspring with the in vitro model of Kmt2a upregulation revealed overlapping, significant disturbances in pathways that mediate synaptic plasticity. Similar transcriptional alterations were detected in human major depressive disorder. CONCLUSIONS: These studies provide direct evidence for the persistent effects of prenatal cannabis exposure on transcriptional and epigenetic deviations in the NAc via Kmt2a dysregulation and associated psychiatric vulnerability.

Molecular mechanisms of maternal cannabis and cigarette use on human neurodevelopment.

Prenatal development is highly sensitive to maternal drug use due to the vulnerability for disruption of the fetal brain with its ongoing neurodevelopment, resulting in lifelong consequences that can enhance risk for psychiatric disorders. Cannabis and cigarettes are the most commonly used illicit and licit substances, respectively, among pregnant women. Although the behavioral consequences of prenatal cannabis and cigarette exposure have been well-documented in epidemiological and clinical studies, only recently have investigations into the molecular mechanisms associated with the developmental impact of early drug exposure been addressed. This article reviews the literature relevant to long-term gene expression disturbances in the human fetal brain in relation to maternal cannabis and cigarette use. To provide translational insights, we discuss animal models in which protracted molecular consequences of prenatal cannabis and cigarette exposure can be better explored and which enable future evaluation of epigenetic pathways, such as DNA methylation and histone modification, that could potentially maintain abnormal gene regulation and related behavioral disturbances. Altogether, this information may help to address the current gaps of knowledge regarding the impact of early drug exposure that set in motion lifelong molecular disturbances that underlie vulnerability to psychiatric disorders.

The role of enhancers as centres for general transcription factor recruitment.

Activation of eukaryotic genes requires a tight temporal control of trans-acting-factor binding to different types of sequence elements. General transcription factors (GTFs) have a central role in the regulation of RNA polymerase II (Pol II) function because they are involved in the initiation of transcription at all class II promoters. Recent studies have shown that GTFs and Pol II are recruited to enhancer elements and that this binding is an early event in gene activation. We propose that an important role of some enhancers is to function as nucleation centres for the assembly of the pre-initiation complex to regulate the timing of gene activation during development, differentiation and the cell cycle.

Formation of an active tissue-specific chromatin domain initiated by epigenetic marking at the embryonic stem cell stage.

The differentiation potential of stem cells is determined by the ability of these cells to establish and maintain developmentally regulated gene expression programs that are specific to different lineages. Although transcriptionally potentiated epigenetic states of genes have been described for haematopoietic progenitors, the developmental stage at which the formation of lineage-specific gene expression domains is initiated remains unclear. In this study, we show that an intergenic cis-acting element in the mouse lambda5-VpreB1 locus is marked by histone H3 acetylation and histone H3 lysine 4 methylation at a discrete site in embryonic stem (ES) cells. The epigenetic modifications spread from this site toward the VpreB1 and lambda5 genes at later stages of B-cell development, and a large, active chromatin domain is established in pre-B cells when the genes are fully expressed. In early B-cell progenitors, the binding of haematopoietic factor PU.1 coincides with the expansion of the marked region, and the region becomes a center for the recruitment of general transcription factors and RNA polymerase II. In pre-B cells, E2A also binds to the locus, and general transcription factors are distributed across the active domain, including the gene promoters and the intergenic region. These results suggest that localized epigenetic marking is important for establishing the transcriptional competence of the lambda5 and VpreB1 genes as early as the pluripotent ES cell stage.

High times for cannabis: Epigenetic imprint and its legacy on brain and behavior.

Extensive debates continue regarding marijuana (Cannabis spp), the most commonly used illicit substance in many countries worldwide. There has been an exponential increase of cannabis studies over the past two decades but the drug's long-term effects still lack in-depth scientific data. The epigenome is a critical molecular machinery with the capacity to maintain persistent alterations of gene expression and behaviors induced by cannabinoids that have been observed across the individual's lifespan and even into the subsequent generation. Though mechanistic investigations regarding the consequences of developmental cannabis exposure remain sparse, human and animal studies have begun to reveal specific epigenetic disruptions in the brain and the periphery. In this article, we focus attention on long-term disturbances in epigenetic regulation in relation to prenatal, adolescent and parental germline cannabinoid exposure. Expanding knowledge about the protracted molecular memory could help to identify novel targets to develop preventive strategies and treatments for behaviors relevant to neuropsychiatric risks associated with developmental cannabis exposure.

A chromosome 3-encoded repressor of the human telomerase reverse transcriptase (hTERT) gene controls the state of hTERT chromatin.

Telomerase is crucial for human carcinogenesis. The limiting component of telomerase activity is telomerase reverse transcriptase (hTERT), undetectable in differentiated somatic cells but present in most tumor cells. There is evidence that hTERT transcription is shut down by a repressor in normal cells, but the mechanisms that turn on or maintain expression in tumor cells are not understood. To identify cis-acting regulatory elements, we scanned the hTERT gene for nuclease sensitive sites. In tumor cells and in in vitro transformed fibroblasts that contain hTERT mRNA, we detected a pattern of nuclease-sensitive sites in the second intron different from that in normal fibroblasts. To test whether the chromatin state characterized by the increased nuclease sensitivity plays a role in tumor-specific hTERT expression, we used a telomerase-positive breast carcinoma line, 21NT. Introduction of a normal chromosome 3 into these cells is known to down-regulate hTERT expression, probably through transcriptional silencing. 21NT cells displayed a similar pattern of micrococcal nuclease (MNase) sensitivity to other telomerase-positive lines, whereas the hTERT chromatin of the chromosome 3-hybrids resembled that of normal fibroblasts. In segregants that had lost the normal chromosome 3, the MNase sensitivity pattern characteristic of telomerase-positive cells was restored, and some (but not all) re-expressed the hTERT gene. The simplest model compatible with these results, and with data on the mapping of an hTERT repressor on chromosome 3, is that hTERT expression in tumor cells depends on an open state of intron 2-chromatin. We propose that, during the development of the breast carcinoma from which the 21NT cell line was derived, loss of function of this repressor led to chromatin remodeling necessary (but probably not sufficient) for expression of the hTERT gene. An improved understanding of the precise mechanism of hTERT dysregulation in human cancer may well find applications in the development of antitelomerase cancer therapy.

Epigenetic Effects of Cannabis Exposure.

The past decade has witnessed a number of societal and political changes that have raised critical questions about the long-term impact of marijuana (Cannabis sativa) that are especially important given the prevalence of its abuse and that potential long-term effects still largely lack scientific data. Disturbances of the epigenome have generally been hypothesized as the molecular machinery underlying the persistent, often tissue-specific transcriptional and behavioral effects of cannabinoids that have been observed within one's lifetime and even into the subsequent generation. Here, we provide an overview of the current published scientific literature that has examined epigenetic effects of cannabinoids. Though mechanistic insights about the epigenome remain sparse, accumulating data in humans and animal models have begun to reveal aberrant epigenetic modifications in brain and the periphery linked to cannabis exposure. Expansion of such knowledge and causal molecular relationships could help provide novel targets for future therapeutic interventions.

Cross-generational THC exposure alters the developmental sensitivity of ventral and dorsal striatal gene expression in male and female offspring.

Cannabis (Cannabis sativa, Cannabis indica) is the illicit drug most frequently abused by young men and women. The growing use of the drug has raised attention not only on the impact of direct exposure on the developing brain and behavior later in life, but also on potential cross-generational consequences. Our previous work demonstrated that adolescent exposure to Δ9-tetrahydrocannabinol (THC), the main psychoactive component of cannabis, affects reward-related behavior and striatal gene expression in male offspring that were unexposed to the drug during their own lifespan. The significant sex differences documented for most addiction and psychiatric disorders suggest that understanding the perturbation of the brain in the two sexes due to cannabis could provide insights about neuronal systems underpinning vulnerability to psychiatric illnesses. In the current study, we expanded our previous observations in males by analyzing the female brain for specific aberrations associated with cross-generational THC exposure. Based on the impact of adolescent development on subsequent adult behavioral pathology, we examined molecular patterns during both adolescence and adulthood. The results revealed a switch from the ventral striatum during adolescence to the dorsal striatum in adulthood in alterations of gene expression related to synaptic plasticity in both sexes. Females, however, exhibited stronger correlation patterns between genes and also showed locomotor disturbances not evident in males. Overall, the findings demonstrate cross-generational consequences of parental THC exposure in both male and female offspring.

Regulation of the human telomerase reverse transcriptase gene.

Most somatic human cells lack telomerase activity because they do not express the telomerase reverse transcriptase (hTERT) gene. Conversely, most cancer cells express hTERT and are telomerase positive. For most tumors it is not clear whether hTERT expression is due to their origin from telomerase positive stem cells or to reactivation of the gene during tumorigenesis. Telomerase negative cells lack detectable cytoplasmic and nuclear hTERT transcripts; in telomerase positive cells 0.2 to 6 mRNA molecules/cell can be detected. This suggests that expression is regulated by changes in the rate of hTERT gene transcription. In tumor cell lines hTERT expression behaves like a recessive trait, indicating that lack of expression in normal cells is due to one or several repressors. Studies with monochromosomal hybrids indicate that several chromosomes may code for such repressors. A number of transcription factors, tumor suppressors, cell cycle inhibitors, cell fate determining molecules, hormone receptors and viral proteins have been implicated in the control of hTERT expression; but these studies have not yet provided a clear explanation for the tumor specific expression of the hTERT gene, and the cis-acting elements which are the targets of repression in normal cells still have to be identified.

Genome-Wide DNA Methylation Profiling Reveals Epigenetic Changes in the Rat Nucleus Accumbens Associated With Cross-Generational Effects of Adolescent THC Exposure.

Drug exposure during critical periods of development is known to have lasting effects, increasing one's risk for developing mental health disorders. Emerging evidence has also indicated the possibility for drug exposure to even impact subsequent generations. Our previous work demonstrated that adolescent exposure to Δ(9)-tetrahydrocannabinol (THC), the main psychoactive component of marijuana (Cannabis sativa), in a Long-Evans rat model affects reward-related behavior and gene regulation in the subsequent (F1) generation unexposed to the drug. Questions, however, remained regarding potential epigenetic consequences. In the current study, using the same rat model, we employed Enhanced Reduced Representation Bisulfite Sequencing to interrogate the epigenome of the nucleus accumbens, a key brain area involved in reward processing. This analysis compared 16 animals with parental THC exposure and 16 without to characterize relevant systems-level changes in DNA methylation. We identified 1027 differentially methylated regions (DMRs) associated with parental THC exposure in F1 adults, each represented by multiple CpGs. These DMRs fell predominantly within introns, exons, and intergenic intervals, while showing a significant depletion in gene promoters. From these, we identified a network of DMR-associated genes involved in glutamatergic synaptic regulation, which also exhibited altered mRNA expression in the nucleus accumbens. These data provide novel insight into drug-related cross-generational epigenetic effects, and serve as a useful resource for investigators to explore novel neurobiological systems underlying drug abuse vulnerability.

Parental THC exposure leads to compulsive heroin-seeking and altered striatal synaptic plasticity in the subsequent generation.

Recent attention has been focused on the long-term impact of cannabis exposure, for which experimental animal studies have validated causal relationships between neurobiological and behavioral alterations during the individual's lifetime. Here, we show that adolescent exposure to Δ(9)-tetrahydrocannabinol (THC), the main psychoactive component of cannabis, results in behavioral and neurobiological abnormalities in the subsequent generation of rats as a consequence of parental germline exposure to the drug. Adult F1 offspring that were themselves unexposed to THC displayed increased work effort to self-administer heroin, with enhanced stereotyped behaviors during the period of acute heroin withdrawal. On the molecular level, parental THC exposure was associated with changes in the mRNA expression of cannabinoid, dopamine, and glutamatergic receptor genes in the striatum, a key component of the neuronal circuitry mediating compulsive behaviors and reward sensitivity. Specifically, decreased mRNA and protein levels, as well as NMDA receptor binding were observed in the dorsal striatum of adult offspring as a consequence of germline THC exposure. Electrophysiologically, plasticity was altered at excitatory synapses of the striatal circuitry that is known to mediate compulsive and goal-directed behaviors. These findings demonstrate that parental history of germline THC exposure affects the molecular characteristics of the striatum, can impact offspring phenotype, and could possibly confer enhanced risk for psychiatric disorders in the subsequent generation.

Maternal cannabis use alters ventral striatal dopamine D2 gene regulation in the offspring.

BACKGROUND: Prenatal cannabis exposure has been linked to addiction vulnerability, but the neurobiology underlying this risk is unknown. METHODS: Striatal dopamine and opioid-related genes were studied in human fetal subjects exposed to cannabis (as well as cigarettes and alcohol). Cannabis-related gene disturbances observed in the human fetus were subsequently characterized with an animal model of prenatal Δ-9-tetrahydrocannabinol (THC) (.15 mg/kg) exposure. RESULTS: Prenatal cannabis exposure decreased dopamine receptor D2 (DRD2) messenger RNA expression in the human ventral striatum (nucleus accumbens [NAc]), a key brain reward region. No significant alterations were observed for the other genes in cannabis-exposed subjects. Maternal cigarette use was associated with reduced NAc prodynorphin messenger RNA expression, and alcohol exposure induced broad alterations primarily in the dorsal striatum of most genes. To explore the mechanisms underlying the cannabis-associated disturbances, we exposed pregnant rats to THC and examined the epigenetic regulation of the NAc Drd2 gene in their offspring at postnatal day 2, comparable to the human fetal period studied, and in adulthood. Chromatin immunoprecipitation of the adult NAc revealed increased 2meH3K9 repressive mark and decreased 3meH3K4 and RNA polymerase II at the Drd2 gene locus in the THC-exposed offspring. Decreased Drd2 expression was accompanied by reduced dopamine D2 receptor (D(2)R) binding sites and increased sensitivity to opiate reward in adulthood. CONCLUSIONS: These data suggest that maternal cannabis use alters developmental regulation of mesolimbic D(2)R in offspring through epigenetic mechanisms that regulate histone lysine methylation, and the ensuing reduction of D(2)R might contribute to addiction vulnerability later in life.

The proteasome restricts permissive transcription at tissue-specific gene loci in embryonic stem cells.

The ability of stem cells to activate different gene expression programs requires the choreographed assembly of trans-acting factors at enhancers and promoters during cell differentiation. In this study, we show that the proteasome acts on specific regulatory regions in embryonic stem (ES) cells to prevent incorrect transcriptional initiation. Chemical or siRNA-mediated inhibition of proteasome activity results in increased transcription factor and RNA polymerase II binding and leads to activation of cryptic promoters. Analysis of the binding profiles of different proteasome subunits in normal ES cells and following RNAi knockdown of individual subunits provides evidence for a targeted assembly of the 26S proteasome at specific regulatory elements. Our results suggest that the proteasome promotes a dynamic turnover of transcription factor and Pol II binding at tissue-specific gene domains in ES cells, thereby restricting permissive transcriptional activity and keeping the genes in a potentiated state, ready for activation at later stages.