Low Dopamine D2 Receptor Expression Drives Gene Networks Related to GABA, cAMP, Growth and Neuroinflammation in Striatal Indirect Pathway Neurons.

Background: A salient effect of addictive drugs is to hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Reduced dopamine type 2 receptor availability in the striatum is an important pathophysiological mechanism for addiction that is both consequential and causal for other molecular, cellular, and neuronal network differences etiologic for this disorder. Here, we sought to identify gene expression changes attributable to innate low expression of the Drd2 gene in the striatum and specific to striatal indirect medium spiny neurons (iMSNs). Methods: Cre-conditional, translating ribosome affinity purification (TRAP) was used to purify and analyze the translatome (ribosome-bound messenger RNA) of iMSNs from mice with low/heterozygous or wild-type Drd2 expression in iMSNs. Complementary electrophysiological recordings and gene expression analysis of postmortem brain tissue from human cocaine users were performed. Results: Innate low expression of Drd2 in iMSNs led to differential expression of genes involved in GABA (gamma-aminobutyric acid) and cAMP (cyclic adenosine monophosphate) signaling, neural growth, lipid metabolism, neural excitability, and inflammation. Creb1 was identified as a likely upstream regulator, among others. In human brain, expression of FXYD2, a modulatory subunit of the Na/K pump, was negatively correlated with DRD2 messenger RNA expression. In iMSN-TRAP-Drd2HET mice, increased Cartpt and reduced S100a10 (p11) expression recapitulated previous observations in cocaine paradigms. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice. Conclusions: This study provides strong molecular evidence that, in addiction, inhibition by the indirect pathway is constitutively enhanced through neural growth and increased GABA signaling.

Single nucleus transcriptomics of ventral midbrain identifies glial activation associated with chronic opioid use disorder.

Dynamic interactions of neurons and glia in the ventral midbrain mediate reward and addiction behavior. We studied gene expression in 212,713 ventral midbrain single nuclei from 95 individuals with history of opioid misuse, and individuals without drug exposure. Chronic exposure to opioids was not associated with change in proportions of glial and neuronal subtypes, however glial transcriptomes were broadly altered, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes. Genes associated with activation of the immune response including interferon, NFkB signaling, and cell motility pathways were upregulated, contrasting with down-regulated expression of synaptic signaling and plasticity genes in ventral midbrain non-dopaminergic neurons. Ventral midbrain transcriptomic reprogramming in the context of chronic opioid exposure included 325 genes that previous genome-wide studies had linked to risk of substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

Cell type specific transcriptomic differences in depression show similar patterns between males and females but implicate distinct cell types and genes.

Major depressive disorder (MDD) is a common, heterogenous, and potentially serious psychiatric illness. Diverse brain cell types have been implicated in MDD etiology. Significant sexual differences exist in MDD clinical presentation and outcome, and recent evidence suggests different molecular bases for male and female MDD. We evaluated over 160,000 nuclei from 71 female and male donors, leveraging new and pre-existing single-nucleus RNA-sequencing data from the dorsolateral prefrontal cortex. Cell type specific transcriptome-wide threshold-free MDD-associated gene expression patterns were similar between the sexes, but significant differentially expressed genes (DEGs) diverged. Among 7 broad cell types and 41 clusters evaluated, microglia and parvalbumin interneurons contributed the most DEGs in females, while deep layer excitatory neurons, astrocytes, and oligodendrocyte precursors were the major contributors in males. Further, the Mic1 cluster with 38% of female DEGs and the ExN10_L46 cluster with 53% of male DEGs, stood out in the meta-analysis of both sexes.

IUPHAR - invited review - Ibogaine - A legacy within the current renaissance of psychedelic therapy.

Ibogaine is a powerful psychoactive substance that not only alters perception, mood and affect, but also stops addictive behaviors. Ibogaine has a very long history of ethnobotanical use in low doses to combat fatigue, hunger and thirst and, in high doses as a sacrament in African ritual contexts. In the 1960's, American and European self-help groups provided public testimonials that a single dose of ibogaine alleviated drug craving, opioid withdrawal symptoms, and prevented relapse for weeks, months and sometimes years. Ibogaine is rapidly demethylated by first-pass metabolism to a long-acting metabolite noribogaine. Ibogaine and its metabolite interact with two or more CNS targets simultaneously and both drugs have demonstrated predictive validity in animal models of addiction. Online forums endorse the benefits of ibogaine as an "addiction interrupter" and present-day estimates suggest that more than ten thousand people have sought treatment in countries where the drug is unregulated. Open label pilot studies of ibogaine-assisted drug detoxification have shown positive benefit in treating addiction. Ibogaine, granted regulatory approval for human testing in a Phase 1/2a clinical trial, joins the current landscape of psychedelic medicines in clinical development.

Single Nucleus Transcriptomics Reveals Pervasive Glial Activation in Opioid Overdose Cases.

Dynamic interactions of neurons and glia in the ventral midbrain (VM) mediate reward and addiction behavior. We studied gene expression in 212,713 VM single nuclei from 95 human opioid overdose cases and drug-free controls. Chronic exposure to opioids left numerical proportions of VM glial and neuronal subtypes unaltered, while broadly affecting glial transcriptomes, involving 9.5 - 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes, with prominent activation of the immune response including interferon, NFkB signaling, and cell motility pathways, sharply contrasting with down-regulated expression of synaptic signaling and plasticity genes in VM non-dopaminergic neurons. VM transcriptomic reprogramming in the context of opioid exposure and overdose included 325 genes with genetic variation linked to substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain's reward circuitry.

Convergent abnormalities in striatal gene networks in human cocaine use disorder and mouse cocaine administration models.

Cocaine use disorder (CUD) is an intractable syndrome, and rising overdose death rates represent a substantial public health crisis that exacts tremendous personal and financial costs on patients and society. Sharp increases in cocaine use drive the urgent need for better mechanistic insight into this chronic relapsing brain disorder that currently lacks effective treatment options. To investigate the transcriptomic changes involved, we conducted RNA sequencing on two striatal brain regions that are heavily implicated in CUD, the nucleus accumbens and caudate nucleus, from men suffering from CUD and matched controls. Weighted gene coexpression analyses identified CUD-specific gene networks enriched in ionotropic receptors and linked to lowered neuroinflammation, contrasting the proinflammatory responses found in opioid use disorder. Integration of comprehensive transcriptomic datasets from mouse cocaine self-administration models revealed evolutionarily conserved gene networks in CUD that implicate especially D1 medium spiny neurons as drivers of cocaine-induced plasticity.

Heterozygote Dopamine Transporter Knockout Rats Display Enhanced Cocaine Locomotion in Adolescent Females.

Cocaine is a powerful psychostimulant that is one of the most widely used illicit addictive. The dopamine transporter (DAT) plays a major role in mediating cocaine's reward effect. Decreases in DAT expression increase rates of drug abuse and vulnerability to comorbid psychiatric disorders. We used the novel DAT transgenic rat model to study the effects of cocaine on locomotor behaviors in adolescent rats, with an emphasis on sex. Female rats showed higher response rates to cocaine at lower acute and chronic doses, highlighting a higher vulnerability and perceived gender effects. In contrast, locomotor responses to an acute high dose of cocaine were more marked and sustained in male DAT heterozygous (HET) adolescents. The results demonstrate the augmented effects of chronic cocaine in HET DAT adolescent female rats. Knockout (KO) DAT led to a level of hyperdopaminergia which caused a marked basal hyperactivity that was unchanged, consistent with a possible ceiling effect. We suggest a role of alpha synuclein (α-syn) and PICK 1 protein expressions to the increased vulnerability in female rats. These proteins showed a lower expression in female HET and KO rats. This study highlights gender differences associated with mutations which affect DAT expression and can increase susceptibility to cocaine abuse in adolescence.

Structure-Activity Relationships of Dopamine Transporter Pharmacological Chaperones.

Mutations in the dopamine transporter gene (SLC6A3) have been implicated in many human diseases. Among these is the infantile parkinsonism-dystonia known as Dopamine Transporter Deficiency Syndrome (DTDS). Afflicted individuals have minimal to no functional dopamine transporter protein. This is primarily due to retention of misfolded disease-causing dopamine transporter variants. This results in a variety of severe motor symptoms in patients and the disease ultimately leads to death in adolescence or young adulthood. Though no treatment is currently available, pharmacological chaperones targeting the dopamine transporter have been shown to rescue select DTDS disease-causing variants. Previous work has identified two DAT pharmacological chaperones with moderate potency and efficacy: bupropion and ibogaine. In this study, we carried out structure-activity relationships (SARs) for bupropion and ibogaine with the goal of identifying the chemical features required for pharmacological chaperone activity. Our results show that the isoquinuclidine substituent of ibogaine and its analogs is an important feature for pharmacological chaperone efficacy. For bupropion, the secondary amine group is essential for pharmacological chaperone activity. Lastly, we describe additional ibogaine and bupropion analogs with varying chemical modifications and variable pharmacological chaperone efficacies at the dopamine transporter. Our results contribute to the design and refinement of future dopamine transporter pharmacological chaperones with improved efficacies and potencies.

Single nucleus multi-omics identifies human cortical cell regulatory genome diversity.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.

Convergence of case-specific epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid overdose.

Opioid use disorder is a highly heterogeneous disease driven by a variety of genetic and environmental risk factors which have yet to be fully elucidated. Opioid overdose, the most severe outcome of opioid use disorder, remains the leading cause of accidental death in the United States. We interrogated the effects of opioid overdose on the brain using ChIP-seq to quantify patterns of H3K27 acetylation in dorsolateral prefrontal cortical neurons isolated from 51 opioid-overdose cases and 51 accidental death controls. Among opioid cases, we observed global hypoacetylation and identified 388 putative enhancers consistently depleted for H3K27ac. Machine learning on H3K27ac patterns predicted case-control status with high accuracy. We focused on case-specific regulatory alterations, revealing 81,399 hypoacetylation events, uncovering vast inter-patient heterogeneity. We developed a strategy to decode this heterogeneity based on convergence analysis, which leveraged promoter-capture Hi-C to identify five genes over-burdened by alterations in their regulatory network or "plexus": ASTN2, KCNMA1, DUSP4, GABBR2, ENOX1. These convergent loci are enriched for opioid use disorder risk genes and heritability for generalized anxiety, number of sexual partners, and years of education. Overall, our multi-pronged approach uncovers neurobiological aspects of opioid use disorder and captures genetic and environmental factors perpetuating the opioid epidemic.

Methylation of the tyrosine hydroxylase gene is dysregulated by cocaine dependence in the human striatum.

Cocaine dependence is a chronic, relapsing disorder caused by lasting changes in the brain. Animal studies have identified cocaine-related alterations in striatal DNA methylation; however, it is unclear how methylation is related to cocaine dependence in humans. We generated methylomic profiles of the nucleus accumbens using human postmortem brains from a cohort of individuals with cocaine dependence and healthy controls (n = 25 per group). We found hypermethylation in a cluster of CpGs within the gene body of tyrosine hydroxylase (TH), containing a putative binding site for the early growth response 1 (EGR1) transcription factor, which is hypermethylated in the caudate nucleus of cocaine-dependent individuals. We replicated this finding and found it to be specific to striatal neuronal nuclei. Furthermore, this locus demonstrates enhancer activity which is attenuated by methylation and enhanced by EGR1 overexpression. These results suggest that cocaine dependence alters the epigenetic regulation of dopaminergic signaling genes.

BMAA, Methylmercury, and Mechanisms of Neurodegeneration in Dolphins: A Natural Model of Toxin Exposure.

Dolphins are well-regarded sentinels for toxin exposure and can bioaccumulate a cyanotoxin called ß-N-methylamino-l-alanine (BMAA) that has been linked to human neurodegenerative disease. The same dolphins also possessed hallmarks of Alzheimer's disease (AD), suggesting a possible association between toxin exposure and neuropathology. However, the mechanisms of neurodegeneration in dolphins and the impact cyanotoxins have on these processes are unknown. Here, we evaluate BMAA exposure by investigating transcription signatures using PCR for dolphin genes homologous to those implicated in AD and related dementias: APP, PSEN1, PSEN2, MAPT, GRN, TARDBP, and C9orf72. Immunohistochemistry and Sevier Münger silver staining were used to validate neuropathology. Methylmercury (MeHg), a synergistic neurotoxicant with BMAA, was also measured using PT-GC-AFS. We report that dolphins have up to a three-fold increase in gene transcription related to Aß+ plaques, neurofibrillary tangles, neuritic plaques, and TDP-43+ intracytoplasmic inclusions. The upregulation of gene transcription in our dolphin cohort paralleled increasing BMAA concentration. In addition, dolphins with BMAA exposures equivalent to those reported in AD patients displayed up to a 14-fold increase in AD-type neuropathology. MeHg was detected (0.16-0.41 µg/g) and toxicity associated with exposure was also observed in the brain. These results demonstrate that dolphins develop neuropathology associated with AD and exposure to BMAA and MeHg may augment these processes.

Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.

Epigenetic mechanisms, like those involving DNA methylation, are thought to mediate the relationship between chronic cocaine dependence and molecular changes in addiction-related neurocircuitry, but have been understudied in human brain. We initially used reduced representation bisulfite sequencing (RRBS) to generate a methylome-wide profile of cocaine dependence in human post-mortem caudate tissue. We focused on the Iroquois Homeobox A (IRXA) gene cluster, where hypomethylation in exon 3 of IRX2 in neuronal nuclei was associated with cocaine dependence. We replicated this finding in an independent cohort and found similar results in the dorsal striatum from cocaine self-administering mice. Using epigenome editing and 3C assays, we demonstrated a causal relationship between methylation within the IRX2 gene body, CTCF protein binding, three-dimensional (3D) chromatin interaction, and gene expression. Together, these findings suggest that cocaine-related hypomethylation of IRX2 contributes to the development and maintenance of cocaine dependence through alterations in 3D chromatin structure in the caudate nucleus.

Assessment of stress markers in restrained individuals following physical stress with and without sham CED activation.

INTRODUCTION: Law enforcement and pre-hospital care personnel often confront individuals who must be physically restrained. Many are under the influence of illicit substances, and law enforcement officers may need to use a controlled electrical device (CED) to gain control of the individual and they are often placed into the prone maximum restraint (PMR) position. These techniques have previously been evaluated for their physiologic effects. The purpose of this study was to investigate the psychological effects of anticipating and experiencing a sham CED activation in healthy human subjects who were exercised and restrained compared with no sham activation by assessing the differences in a panel of several known biomarkers of stress. METHODS: We performed a randomized, crossover controlled human subject trial to study the stress associated with exercise, physical exhaustion, and restraint with and without an added psychological stress simulating the field use of a CED. Twenty five total subjects; each subject performed two different trials each consisting of a brief period of intense exercise on a treadmill to exhaustion followed by placement in the PMR with and without induced psychological stress. Blood samples were collected for analysis pre and post exercise, as well as 10 min after completion of the exercise. A panel of hormones and stress markers were measured. RESULTS: We found no significant differences in any of the stress biomarkers measured between the two study groups. A trend towards higher levels of copeptin was measured in the sham CED activation arm. CONCLUSION: During a brief period of intense exercise followed by the psychological stress of anticipated CED application, there did not appear to be statistically significant changes in the stress panel of biomarkers measured, only a trend towards significance for higher copeptin levels in the patients exposed to the psychological stress.

Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome.

Long interspersed element-1 retrotransposons (LINE-1 or L1) are ∼6 kb mobile DNA elements implicated in the origins of many Mendelian and complex diseases. The actively retrotransposing L1s are mostly limited to the L1 human specific (L1Hs) transcriptional active (Ta) subfamily. In this manuscript, we present REBELseq as a method for the construction of Ta subfamily L1Hs-enriched next-generation sequencing libraries and bioinformatic identification. REBELseq was performed on DNA isolated from NeuN+ neuronal nuclei from postmortem brain samples of 177 individuals and empirically-driven bioinformatic and experimental cutoffs were established. Putative L1Hs insertions passing bioinformatics cutoffs were experimentally validated. REBELseq reliably identified both known and novel Ta subfamily L1Hs insertions distributed throughout the genome. Differences in the proportion of individuals possessing a given reference or non-reference retrotransposon insertion were identified. We conclude that REBELseq is an unbiased, whole genome approach to the amplification and detection of Ta subfamily L1Hs retrotransposons.

l-Serine Reduces Spinal Cord Pathology in a Vervet Model of Preclinical ALS/MND.

The early neuropathological features of amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) are protein aggregates in motor neurons and microglial activation. Similar pathology characterizes Guamanian ALS/Parkinsonism dementia complex, which may be triggered by the cyanotoxin ß-N-methylamino-l-alanine (BMAA). We report here the occurrence of ALS/MND-type pathological changes in vervets (Chlorocebus sabaeus; n = 8) fed oral doses of a dry powder of BMAA HCl salt (210 mg/kg/day) for 140 days. Spinal cords and brains from toxin-exposed vervets were compared to controls fed rice flour (210 mg/kg/day) and to vervets coadministered equal amounts of BMAA and l-serine (210 mg/kg/day). Immunohistochemistry and quantitative image analysis were used to examine markers of ALS/MND and glial activation. UHPLC-MS/MS was used to confirm BMAA exposures in dosed vervets. Motor neuron degeneration was demonstrated in BMAA-dosed vervets by TDP-43+ proteinopathy in anterior horn cells, by reactive astrogliosis, by activated microglia, and by damage to myelinated axons in the lateral corticospinal tracts. Vervets dosed with BMAA + l-serine displayed reduced neuropathological changes. This study demonstrates that chronic dietary exposure to BMAA causes ALS/MND-type pathological changes in the vervet and coadministration of l-serine reduces the amount of reactive gliosis and the number of protein inclusions in motor neurons.

Assessment of the Association of D2 Dopamine Receptor Gene and Reported Allele Frequencies With Alcohol Use Disorders: A Systematic Review and Meta-analysis.

Importance: The association between the D2 dopamine receptor gene (DRD2) Taq1A locus (rs1800497) and alcohol use disorder (AUD) is enduring but the subject of long-standing controversy; meta-analysis of studies across 3 decades shows an association between rs1800497 and AUD, but genome-wide analyses have detected no role for rs1800497 in any phenotype. No evidence has emerged that rs1800497, which is located in ANKK1, perturbs the expression or function of DRD2. Objective: To resolve contradictions in previous studies by identifying hidden confounders and assaying for functional effects of rs1800497 and other loci in the DRD2 region. Data Sources: PubMed (882 studies), Embase (1056 studies), and Web of Science (501 studies) databases were searched through August 2018. Three clinical populations-Finnish, Native American, and African American participants-were genotyped for 208 to 277 informative single-nucleotide polymorphisms (SNPs) across the DRD2 region to test the associations of SNPs in this region with AUD. Study Selection: Eligible studies had diagnosis of AUD made by accepted criteria, reliable genotyping methods, sufficient genotype data to calculate odds ratios and 95% CIs, and availability of control allele frequencies or genotype frequencies. Data Extraction and Synthesis: After meta-analysis of 62 studies, metaregression was performed to detect between-study heterogeneity and to explore the effects of moderators, including deviations of cases and controls from allele frequencies in large population databases (ExAC and 1000 Genomes). Linkage to AUD and the effect on gene expression of rs1800497 were evaluated in the context of other SNPs in the DRD2 region. Data analysis was performed from August 2018 to March 2019. This study follows the Preferred Reporting Items for Systematic Reviews and Meta-analyses reporting guideline. Main Outcomes and Measures: The effects of rs1800497 and other SNPs in the DRD2 region on gene expression were measured in human postmortem brain samples via differential allelic expression and evaluated in other tissues via publicly available expression quantitative locus data. Results: A total of 62 studies of DRD2 and AUD with 16 294 participants were meta-analyzed. The rs1800497 SNP was associated with AUD (odds ratio, 1.23; 95% CI, 1.14-1.31; P < .001). However, the association was attributable to spuriously low allele frequencies in controls in positive studies, which also accounted for some between-study heterogeneity (I2 = 43%; 95% CI, 23%-58%; Q61 = 107.20). Differential allelic expression of human postmortem brain and analysis of expression quantitative loci in public data revealed that a cis-acting locus or loci perturb the DRD2 transcript level; however, rs1800497 does not and is not in strong disequilibrium with such a locus. Across the DRD2 region, other SNPs are more strongly associated with AUD than rs1800497, although no DRD2 SNP was significantly associated in these 3 clinical samples. Conclusions and Relevance: In this meta-analysis, the significant association of DRD2 with AUD was reassessed. The DRD2 association was attributable to anomalously low control allele frequencies, not function, in positive studies. For genetic studies, statistical replication is not verification.

Genome-wide brain DNA methylation analysis suggests epigenetic reprogramming in Parkinson disease.

OBJECTIVE: Given the known strong relationship of DNA methylation with environmental exposure, we investigated whether brain regions affected in Parkinson disease (PD) were differentially methylated between PD cases and controls. METHODS: DNA chip arrays were used to perform a genome-wide screen of DNA methylation on the dorsal motor nucleus of the vagus (DMV), substantia nigra (SN), and cingulate gyrus (CG) of pathologically confirmed PD cases and controls selected using the criteria of Beecham et al. Analysis examined differentially methylated regions (DMRs) between cases and controls for each brain area. RNA sequencing and pathway analysis were also performed for each brain area. RESULTS: Thirty-eight PD cases and 41 controls were included in the analysis. Methylation studies revealed 234 significant DMR in the DMV, 44 in the SN, and 141 in the CG between cases and controls (Sidak p < 0.05). Pathway analysis of these genes showed significant enrichment for the Wnt signaling pathway (FDR < 0.01). CONCLUSIONS: Our data suggest that significant DNA methylation changes exist between cases and controls in PD, especially in the DMV, one of the areas affected earliest in PD. The etiology of these methylation changes is not yet known, but the predominance of methylation changes occurring in the DMV supports the hypothesis that vagus nerve function, perhaps involving the gastrointestinal system, is important in PD pathogenesis. These data also give independent support that genes involved in Wnt signaling are a likely factor in the neurodegenerative processes of PD.