Analyses of polymorphisms of intron 2 of OPRK1 (kappa-opioid receptor gene) in association with opioid and cocaine dependence diagnoses in an African-American population.

RATIONALE: The dynorphin/kappa-opioid receptor (KOR) system (encoded by PDYN and OPRK1 genes respectively) is highly regulated by repeated exposure to drugs of abuse, including mu-opioid agonists and cocaine. These changes in the dynorphin/KOR system can then influence the rewarding effects of these drugs of abuse. Activation of the dynorphin/KOR system is also thought to have a role in the pro-addictive effects of stress. Recent in vitro assays showed that the OPRK1 intron 2 may function as a genomic enhancer in the regulation KOR expression, and contains a glucocorticiod-responsive sequence site. We hypothesize that SNPs in intron 2 of OPRK1 are associated with categorical opioid or cocaine dependence diagnoses, as well as with dimensional aspects of drug use (i.e., magnitude of drug exposure). METHODS: This study includes 577 subjects ≥ 18 years old, with African ancestry (AA) from the USA. They were divided into three groups: 152 control subjects, 142 persons with lifetime opioid dependence diagnosis (OD), and 283 subjects with lifetime cocaine dependence diagnosis (CD). Five SNPs (rs16918909, rs7016778, rs997917, rs6473797, rs10111937) that span 10 Kb nucleotides in intron 2 of OPRK1 were used for the association analyses. Genotyping was performed with the Smokescreen® array or sequencing of PCR-amplified DNA fragments. Association analyses for OD and CD diagnoses and the OPRK1 intron 2 alleles were carried out with Fisher's exact test. The Kreek-McHugh-Schluger-Kellogg (KMSK) scales were used for dimensional measure of maximum exposure to specific drugs, using Mann-Whitney tests. RESULTS: Two SNPs, rs997917 and rs10111937 showed point-wise significant allelic association (p < 0.05) with CD diagnosis, and rs10111937 showed a point-wise significance in association with OD. None of these single SNP associations with categorical diagnoses were significant after correction for multiple testing (pcorr > 0.05). However, significant associations of several genotype patterns (diplotypes) were found with cocaine dependence, but none for opioid dependence. The most significant genotype pattern with cocaine dependence diagnosis occurred for rs6473797 and rs10111937 (pcorr = 0.036, odds ratio = 1.92, FDR < 0.05), and survived correction for multiple testing. Dimensional analyses with KMSK scores show that persons with either rs997917 or rs10111937 variants had greater exposure to cocaine, compared to those with prototype allele (Mann-Whitney tests, point-wise). CONCLUSIONS: This study provides additional support of potential importance of regulatory regions of intron 2 of the OPRK1 gene in development of cocaine and opioid dependence diagnoses, in a population with African-American ancestry.

Association of Serotonin Transporter (SERT) Polymorphisms with Opioid Dependence and Dimensional Aspects of Cocaine Use in a Caucasian Cohort of Opioid Users.

INTRODUCTION: A functional tandem repeat polymorphism in the promoter of the serotonin transporter (SERT) gene (SLC6A4) has been studied for association to neuropsychiatric conditions, including substance use disorders. Short (S) forms of this repeat result in reduced transcription, and presumably greater synaptic levels of serotonin, which are involved in opioid and cocaine-induced reward. Dual exposure to heroin and cocaine is a common pattern of poly-drug use and is associated with considerable morbidity. We hypothesize that SLC6A4 variants are associated with cocaine exposure in subjects with an opioid dependence diagnosis (OD), and also in non-dependent opioid users (NOD). Other single nucleotide polymorphisms (SNPs) of SLC6A4 may also be likewise associated. MATERIALS AND METHODS: This study determined whether variants of the SLC6A4 promoter repeats and two intronic SNPs, rs16965628 and rs2066713, are associated with categorical diagnoses of opioid dependence (DSM-IV criteria) and with dimensional aspects of cocaine use, in a Caucasian cohort (n=591). Three groups of subjects were examined: (1) 276 subjects with opioid dependence diagnosis (OD); (2) 163 subjects who had used opioids for non-medical reasons but never had an opioid dependence diagnosis (NOD); (3) 152 healthy controls (HC). RESULTS: Aside from high exposure to heroin in the OD group, relatively high exposure to cocaine was detected in both OD and NOD groups. The SERT repeat genotype (classified as "long-long" [LL] versus "short-long" plus "short-short" [SL+SS]) was not associated with categorical opioid dependence diagnoses. A nominally significant association was identified with the [SL+SS] genotype of SLC6A4 and cocaine KMSK scores ≥"cutpoint" for a cocaine dependence diagnosis (p=0.026). The [SL+SS] genotype was associated with more rapid cocaine escalation than the LL genotype. No significant associations of rs16965628 and rs2066713 SNPs were found overall. CONCLUSION: The functional SERT promoter tandem repeat genotype may be associated to heavy cocaine exposure and more rapid escalation of cocaine use, in persons with and without opioid dependence diagnosis.

Association of variants of prodynorphin promoter 68-bp repeats in caucasians with opioid dependence diagnosis: Effect on age trajectory of heroin use.

The dynorphin/kappa opioid receptor (Dyn/KOR) system is involved in reward processing and dysphoria/anhedonia. Exposure to mu-opioid receptor agonists such as heroin increases expression of the prodynorphin gene (PDYN) in the brain. In this study in a Caucasian cohort, we examined the association of the functional PDYN 68-bp repeat polymorphism with opioid use disorders. In this case-control study, 554 subjects with Caucasian ancestry (142 healthy controls, 153 opioid-exposed, but never opioid dependent, NOD, and 259 with an opioid dependence diagnosis, OD) were examined for association of the PDYN 68-bp repeats with the diagnosis of opioid dependence (DSM-IV criteria), with a dimensional measure of heroin exposure (KMSK scale), and age trajectory parameters of heroin use (age of heroin first use, and age of onset of heaviest use). The PDYN 68-bp repeat genotype (classified as: "short-short" [SS], "long-long" [LL], and "short-long" [SL], based on the number of repeats) was not associated with categorical opioid dependence diagnoses. However, the LL genotype was associated with later age of first heroin use than the SS + SL genotype (19 versus 18 years; p < 0.01). This was also confirmed by a significant positive correlation between the number of repeats and the age of first use of heroin, in volunteers with OD (Spearman r = 0.16; p = 0.01). This suggests that the functional PDYN 68-bp repeat genotype is associated with the age of first use of heroin in Caucasians diagnosed with opioid dependence.

Chronic Oxycodone Self-administration Altered Reward-related Genes in the Ventral and Dorsal Striatum of C57BL/6J Mice: An RNA-seq Analysis.

Prescription opioid abuse, for example of oxycodone, is a pressing public health issue. This study focuses on how chronic oxycodone self-administration (SA) affects the reward pathways in the mouse brain. In this study, we tested the hypothesis that the expression of reward-related genes in the ventral and dorsal striatum, areas involved in different aspects of opioid addiction models, was altered within 1 h after chronic oxycodone SA, using transcriptome-wide sequencing (RNA-seq). Based on results from earlier human genetic and rodent preclinical studies, we focused on a set of genes that may be associated with the development of addictive diseases and the rewarding effect of drugs of abuse, primarily in the opioid, stress response and classical neurotransmitter systems. We found that 32 transcripts in the ventral striatum, and 7 in the dorsal striatum, were altered significantly in adult mice that had self-administered oxycodone (n = 5) for 14 consecutive days (4 h/day) compared with yoked saline controls (n = 5). The following 5 genes in the ventral striatum showed experiment-wise significant changes: proopiomelanocortin (Pomc) and serotonin 5-HT-2A receptor (Htr2a) were upregulated; serotonin receptor 7 (Htr7), galanin receptor1 (Galr1) and glycine receptor 1 (Glra1) were downregulated. Some genes detected by RNA-seq were confirmed by quantitative polymerase chain reaction (qPCR). Conclusion: A RNA-seq study shows that chronic oxycodone SA alters the expression of several reward-related genes in the dorsal and ventral striatum. These results suggest potential mechanisms underlying neuronal adaptation to chronic oxycodone self-exposure, of relevance to our mechanistic understanding of prescription opioid abuse.

Oxycodone Self-Administration Induces Alterations in Expression of Integrin, Semaphorin and Ephrin Genes in the Mouse Striatum.

Oxycodone is one a commonly used medication for pain, and is also a widely abused prescription opioid, like other short-acting MOPr agonists. Neurochemical and structural adaptations in brain following chronic MOPr-agonist administration are thought to underlie pathogenesis and persistence of opiate addiction. Many axon guidance molecules, such as integrins, semaphorins, and ephrins may contribute to oxycodone-induced neuroadaptations through alterations in axon-target connections and synaptogenesis, that may be implicated in the behaviors associated with opiate addiction. However, little is known about this important area. The aim of this study is to investigate alterations in expression of selected integrin, semaphorin, ephrins, netrin, and slit genes in the nucleus accumbens (NAc) and caudate putamen (CPu) of mice following extended 14-day oxycodone self-administration (SA), using RNAseq. Methods: Total RNA from the NAc and CPu were isolated from adult male C57BL/6J mice within 1 h after the last session of oxycodone in a 14-day self-administration paradigm (4h/day, 0.25 mg/kg/infusion, FR1) or from yoked saline controls. Gene expressions were examined using RNA sequencing (RNA-Seq) technology. RNA-Seq libraries were prepared using Illumina's TruSeq® Stranded Total RNA LT kit. The reads were aligned to the mouse reference genome (version mm10) using STAR. DESeq2 was applied to the counts of protein coding genes to estimate the fold change between the treatment groups. False Discovery Rate (FDR) q < 0.1 were used to select genes that have a significant expression change. For selection of a subset of genes related to axon guidance pathway, REACTOME was used. Results: Among 38 known genes of the integrin, semaphorin, and ephrin gene families, RNA-seq data revealed up-regulation of six genes in the NAc: heterodimer receptor, integrins Itgal, Itgb2, and Itgam, and its ligand semaphorin Sema7a, two semaphorin receptors, plexins Plxnd1 and Plxdc1. There was down-regulation of eight genes in this region: two integrin genes Itga3 and Itgb8, semaphorins Sema3c, Sema4g, Sema6a, Sema6d, semaphorin receptor neuropilin Nrp2, and ephrin receptor Epha3. In the CPu, there were five differentially expressed axon guidance genes: up-regulation of three integrin genes, Itgal, Itgb2, Itga1, and down-regulation of Itga9 and ephrin Efna3 were thus observed. No significant alterations in expression of Netrin-1 or Slit were observed. Conclusion: We provide evidence for alterations in the expression of selective axon guidance genes in adult mouse brain following chronic self-administration of oxycodone. Further examination of oxycodone-induced changes in the expression of these specific axon guidance molecules and integrin genes in relation to behavior may provide new insights into development of addiction to oxycodone.

Gender-specific association of functional prodynorphin 68 bp repeats with cannabis exposure in an African American cohort.

BACKGROUND: Cannabis use disorders (CUDs) cause substantial neuropsychiatric morbidity and comorbidity. There is evidence for gender-based differences in CUDs, for instance, a greater prevalence in males than in females. The main active component of cannabis is delta 9-tetrahydrocannabinol (delta 9-THC), a partial agonist of the cannabinoid type 1 receptor. Preclinical studies show that genetic or pharmacological manipulation of the kappa opioid receptor/dynorphin system modulates the effects of delta 9-THC. METHODS: In this case-control study of adult African Americans (n=476; 206 females, 270 males), we examined the association of the functional prodynorphin 68 bp (PDYN 68 bp) promoter repeats with categorical diagnoses of cannabis dependence (Diagnostic and Statistical Manual of Mental Disorders-IV criteria), as well as with a rapid dimensional measure of maximum lifetime cannabis exposure (the Kreek-McHugh-Schluger-Kellogg cannabis scale). RESULTS: The PDYN 68 bp genotype (examined as short-short [SS], short-long [SL], or long-long [LL], based on the number of repeats) was not significantly associated with categorical cannabis-dependence diagnoses, either in males or in females. However, in males, the PDYN 68 bp SS+SL genotype was associated with both greater odds of any use of cannabis (p<0.05) and earlier age of first cannabis use, compared to the LL genotype (ie, 15 versus 16.5 years of age; p<0.045). Males in the SS+SL group also had greater odds of high lifetime exposure to cannabis, compared to the LL group (p<0.045). Of interest, none of the aforementioned genetic associations were significant in females. CONCLUSION: This study provides the first data on how the PDYN 68 bp genotype is associated with gender-specific patterns of exposure to cannabis. Overall, this study shows that PDYN 68 bp polymorphisms affect behaviors involved in early stages of nonmedical cannabis use and potentially lead to increasing self-exposure. These data may eventually lead to improvements in personalized medicine for the prevention and treatment of highly prevalent CUDs and neuropsychiatric comorbidities.

Dopamine gene variants in opioid addiction: comparison of dependent patients, nondependent users and healthy controls.

AIM: To determine whether specific dopaminergic system gene variants are associated with opioid dependence. PATIENTS & METHODS: Subjects included 153 healthy controls, 163 opioid exposed, but not dependent and 281 opioid dependent. Genotypes of 90 variants in 13 genes were examined. RESULTS: The most significant results were obtained for DA ß-hydroxylase variants, rs2073837 and rs1611131, which were associated with protection from addiction (q = 0.0172, 0.0415, respectively) and the functional TH variant, rs2070762, was associated with more risk (q = 0.0387). The three variants also showed a combined effect that remained significant after correction for multiple testing (pfinal = 0.0039). CONCLUSION: These data offer support that dopaminergic gene variants have a role in opioid dependence and warrant further study.

Alterations of expression of inflammation/immune-related genes in the dorsal and ventral striatum of adult C57BL/6J mice following chronic oxycodone self-administration: a RNA sequencing study.

INTRODUCTION: Non-medical use of prescription opioids such as the mu opioid receptor (MOP-r) agonist oxycodone is a growing problem in the USA and elsewhere. There is limited information about oxycodone's impact on diverse gene systems in the brain. OBJECTIVES: The current study was designed to examine how chronic oxycodone self-administration (SA) affects gene expression in the terminal areas of the nigrostriatal and mesolimbic dopaminergic pathways in mice. METHOD: Adult male C57BL/6J mice underwent a 14-day oxycodone self-administration procedure (4 h/day, 0.25 mg/kg/infusion, FR1) and were euthanized 1 h after the last session. The dorsal and ventral striata were dissected, and total RNAs were extracted. Gene expressions were examined using RNA sequencing. RESULT: We found that oxycodone self-administration exposure led to alterations of expression in numerous genes related to inflammation/immune functions in the dorsal striatum (54 upregulated genes and 1 downregulated gene) and ventral striatum (126 upregulated genes and 15 downregulated genes), with 38 upregulated genes identified in both brain regions. CONCLUSION: This study reveals novel neurobiological mechanisms underlying some of the effects of a commonly abused prescription opioid. We propose that inflammation/immune gene systems may undergo a major change during chronic self-administration of oxycodone.

Variants of opioid system genes are associated with non-dependent opioid use and heroin dependence.

BACKGROUND: Heroin addiction is a chronic, relapsing brain disease. Genetic factors are involved in the development of drug addiction. The aim of this study was to determine whether specific variants in genes of the opioid system are associated with non-dependent opioid use and heroin dependence. METHODS: Genetic information from four subject groups was collected: non-dependent opioid users (NOD) [n=163]; opioid-dependent (OD) patients in methadone maintenance treatment (MMT) [n=143]; opioid-dependent MMT-resistant patients in heroin-assisted treatment (HAT) [n=138]; and healthy controls with no history of opioid use (HC) [n=153]. Eighty-two variants in eight opioid system genes were studied. To establish the role of these genes in (a) non-dependent opioid use, and (b) heroin dependence, the following groups were compared: HC vs. NOD; HC vs. OD (MMT+HAT); and NOD vs. OD (MMT+HAT). RESULTS: Five unique SNPs in four genes showed nominally significant associations with non-dependent opioid use and heroin dependence. The association of the delta opioid receptor (OPRD1) intronic SNP rs2236861 with non-dependent opioid use (HC vs. NOD) remained significant after correction for multiple testing (OR=0.032; pcorrected=0.015). This SNP exhibited a significant gene-gene interaction with prepronociceptin (PNOC) SNP rs2722897 (OR=5.24; pcorrected=0.041) (HC vs. NOD). CONCLUSIONS: This study identifies several new and some previously reported associations of variants with heroin dependence and with non-dependent opioid use, an important and difficult to obtain group not extensively studied previously. Further studies are warranted to confirm and elucidate the potential roles of these variants in the vulnerability to illicit drug use and drug addiction.

Strain and cocaine-induced differential opioid gene expression may predispose Lewis but not Fischer rats to escalate cocaine self-administration.

The aim of the present study was to investigate alterations in gene expression of opioid system components induced by extended access (18 h) cocaine self-administration and to determine the impact of genetic background in the vulnerability to escalate cocaine intake. Comparing two inbred rat strains, we previously reported that Lewis rats progressively escalated cocaine consumption compared to Fischer rats, in a new translational model of intravenous cocaine self-administration, which included 14 sessions of 18-h operant sessions in which rats were allowed to select the cocaine unit dose to self-administer. We compare here Fischer and Lewis rats in the gene expression of endogenous opioid peptides (Pomc, Penk, Pdyn) and cognate receptors (Oprm, Oprk and Oprd) in reward-related brain regions, after exposure to either cocaine self-administration or yoked-saline, in the aforementioned translational paradigm. We performed a correlation analysis between the mRNA level, found in the Dorsal Striatum (DS), Nucleus accumbens (NAcc) shell and core respectively, and individual cocaine intake. Our findings show that the gene expression of all the aforementioned opioid genes exhibit strain-dependent differences in the DS, in absence of cocaine exposure. Also, different strain-specific cocaine-induced mRNA expression of Oprm and Oprk was found in DS. Only few differences were found in the ventral parts of the striatum. Moreover, gene expression level of Pdyn, Penk, Oprk, and Oprm in the DS was significantly correlated with cocaine intake only in Fischer rats. Overall, these data shed light on potential genetic differences which may predispose of subjects to initiate and escalate cocaine consumption.

Genetics of opiate addiction.

Addiction to MOP-r agonists such as heroin (and also addiction to prescription opioids) has reemerged as an epidemic in the twenty first century, causing massive morbidity. Understanding the genetics contributing to susceptibility to this disease is crucial for the identification of novel therapeutic targets, and also for discovery of genetic markers which would indicate relative protection or vulnerability from addiction, and relative responsiveness to pharmacotherapy. This information could thus eventually inform clinical practice. In this review, we focus primarily on association studies of heroin and opiate addiction, and further describe the studies which have been replicated in this field, and are thus more likely to be useful for translational efforts.

Neurocognitive and neuroinflammatory correlates of PDYN and OPRK1 mRNA expression in the anterior cingulate in postmortem brain of HIV-infected subjects.

Chronic inflammation may contribute to neuropsychological impairments in individuals with HIV, and modulation of this inflammatory response by opiate receptor ligands is important in light of the prevalence of drug use in HIV populations. Exogenous MOR and KOR agonists have differential effects on central nervous system (CNS) immunity and, while some data suggest KOR agonists are immunosuppressive, the KOR agonist dynorphin has been shown to stimulate human monocyte chemotaxis. In this study, we examined mRNA levels of endogenous opioid receptors OPRK1 and OPRM1, prodynorphin (PDYN), macrophage scavenger receptor CD163, and microglia/macrophage marker CD68 in the caudate and anterior cingulate of postmortem brains from HIV-positive and HIV-negative subjects. Brain tissues of HIV-infected (n = 24) and control subjects (n = 15) were obtained from the Manhattan HIV Brain Bank. Quantification of the gene mRNA was performed using SYBR Green RT-PCR. CD68 and CD163 were increased in HIV-positive (HIV+) compared to HIV-negative (HIV-) individuals in both brain regions. There were higher OPRK1 (P <0.005), and lower PDYN mRNA (P <0.005) levels in the anterior cingulate of HIV+ compared to HIV- subjects. This difference between the clinical groups was not found in the caudate. There was no difference in the levels of OPRM1 mRNA between HIV+ and HIV- subjects. Using linear regression analysis, we examined the relationship of OPRK1 and PDYN mRNA levels in the HIV+ subjects with seven cognitive domain T scores of a neuropsychological test battery. Within the HIV+ subjects, there was a positive correlation between anterior cingulate PDYN mRNA levels and better T-scores in the motor domain. Within the HIV+ subjects there were also positive correlations of both OPRK1 and PDYN mRNA levels with the anti-inflammatory marker CD163, but not with proinflammatory CD68 levels. In this setting, decreased PDYN mRNA may reflect a homeostatic mechanism to reduce monocyte migration, accompanied by compensatory increases in the cognate receptor (KOR) to dampen pro-inflammatory responses. It is possible that enhanced neuroprotection and better motor performance are associated with higher levels of dynorphin and the recruitment of neuroprotective CD163-positive macrophages. Further studies are needed to test this hypothesis.

Genetic diversity and linkage disequilibrium in the chemokine receptor CCR2-CCR5 region among individuals and populations.

BACKGROUND: Chemokine receptors CCR2 and CCR5 play a key role in immune and inflammatory responses and have been associated with several diseases, including AIDS. In order to comprehend health disparities it is important to understand the nature of genetic variation in specific genes of interest in different populations. Current studies of the CCR2 and CCR5 receptor genes are primarily focused on the CCR5-Δ32, and CCR2-V64I SNPs. METHODS: Sanger sequencing was used to sequence the regions containing 16 SNPs in the adjacent CCR2 and CCR5 genes (including CCR5-Δ32, and CCR2-V64I) in 249 subjects of African, European and Hispanic ancestry. Linkage disequilibrium (LD) and haplotypes were determined using Haploview. RESULTS: The data revealed large differences in allele frequencies of several SNPs and LD patterns among the ethnic groups, including SNPs that were restricted to Africans or Europeans. Seven known CCR5 haplotypes and six novel CCR2 haplotypes were identified. A rare case of an HIV+ subject with the CCR5-Δ32/Δ32 was identified. CONCLUSIONS: These data demonstrate a LD between CCR2 and CCR5 at several loci and provide new information about CCR2 that contributes to our understanding of its population-specific genetic variability. The data indicate that in addition to CCR5-Δ32 and CCR2-V64I, other SNPs and haplotypes may be important genetic determinants of disease and should be investigated.

Polymorphisms of the kappa opioid receptor and prodynorphin genes: HIV risk and HIV natural history.

OBJECTIVE: Studies indicate cross-desensitization between opioid receptors (eg, kappa opioid receptor, OPRK1) and chemokine receptors (eg, CXCR4) involved in HIV infection. Whether gene variants of OPRK1 and its ligand, prodynorphin (PDYN), influence the outcome of HIV therapy was tested. METHODS: Three study points, admission to the Women's Interagency HIV Study, initiation of highly active antiretroviral therapy (HAART), and the most recent visit, were chosen for analysis as crucial events in the clinical history of the HIV patients. Regression analyses of 17 variants of OPRK1 and 11 variants of PDYN with change of viral load (VL) and CD4 count between admission and initiation of HAART and initiation of HAART to the most recent visit to Women's Interagency HIV Study were performed in 598 HIV+ subjects, including African Americans, Hispanics, and Whites. Association with HIV status was done in 1009 subjects. RESULTS: Before HAART, greater VL decline (improvement) in carriers of PDYN IVS3+189C>T and greater increase of CD4 count (improvement) in carriers of OPRK -72C>T were found in African Americans. Also, greater increase of CD4 count in carriers of OPRK1 IVS2+7886A>G and greater decline of CD4 count (deterioration) in carriers of OPRK1 -1205G>A were found in Whites. After HAART, greater decline of VL in carriers of OPRK1 IVS2+2225G>A and greater increase of VL in carriers of OPRK1 IVS2+10658G>T and IVS2+10963A>G were found in Whites. Also, a lesser increase of CD4 count was found in Hispanic carriers of OPRK1 IVS2+2225G>A. CONCLUSIONS: OPRK1 and PDYN polymorphisms may alter severity of HIV infection and response to treatment.

Expression of ephrin receptors and ligands in postmortem brains of HIV-infected subjects with and without cognitive impairment.

Despite the successes of combination antiretroviral therapy, HIV-associated neurocognitive disorders persist in many infected individuals. Earlier studies showed that neurocognitive impairment was associated with glutamate toxicity and synaptodendritic damage. We examined alterations in expression of four ephrin genes that are involved in synapse formation and recruitment of glutamate receptors to synapses, in the caudate and anterior cingulate in postmortem brain of cognitively characterized HIV-infected subjects, along with expression of neuronal and astroglial/macroglial markers. Postmortem tissues of HIV-infected and control subjects were obtained from the Manhattan HIV Brain Bank. HIV-infected subjects underwent neurocognitive assessment prior to death. Quantification of mRNA of genes of chemokine receptors and chemokines (CCR5, CXCR4, CCL2), astroglial/microglial markers (GFAP, CD163, CD68), the neuronal marker SNAP25, ephrin receptors EPHA4 and EPHB2, and ephrin ligands EFNB1 and EFNB2 was performed using SYBR Green RT-PCR. Proinflammatory chemokine and glial/macrophage mRNA levels in both regions were significantly greater in HIV+ than in HIV- subjects. Levels of EPHA4 and EFNB2 mRNA in the caudate, and EPHB2 mRNA in anterior cingulate were significantly lower in HIV+ subjects (p < 0.002, p < 0.02, p < 0.05, respectively). These transcripts also showed correlations with immune status and cognitive function within the HIV-infected group. Decreased levels of EFNB2 mRNA in the caudate correlated with lower CD4 counts (P < 0.05). Cognitive associations were limited to the cingulate, where decreased levels of EPHB2 mRNA were associated with better global cognitive status. Decreased cingulate expression of EPHB2 may represent a compensatory mechanism minimizing excitotoxic injury in the face of chronic inflammation.

κ-opioid receptor/dynorphin system: genetic and pharmacotherapeutic implications for addiction.

Addictions to cocaine or heroin/prescription opioids [short-acting µ-opioid receptor (MOPr) agonists] involve relapsing cycles, with experimentation/escalating use, withdrawal/abstinence, and relapse/re-escalation. κ-Opioid receptors (KOPr; encoded by OPRK1), and their endogenous agonists, the dynorphins (encoded by PDYN), have counter-modulatory effects on reward caused by cocaine or MOPr agonist exposure, and exhibit plasticity in addictive-like states. KOPr/dynorphin activation is implicated in depression/anxiety, often comorbid with addictions. In this opinion article we propose that particular stages of the addiction cycle are differentially affected by KOPr/dynorphin systems. Vulnerability and resilience can be due to pre-existing (e.g., genetic) factors, or epigenetic modifications of the OPRK1 or PDYN genes during the addiction cycle. Pharmacotherapeutic approaches limiting changes in KOPr/dynorphin tone, especially with KOPr partial agonists, may hold potential for the treatment of specific drug addictions and psychiatric comorbidity.

The genetics of the opioid system and specific drug addictions.

Addiction to drugs is a chronic, relapsing brain disease that has major medical, social, and economic complications. It has been established that genetic factors contribute to the vulnerability to develop drug addiction and to the effectiveness of its treatment. Identification of these factors may increase our understanding of the disorders, help in the development of new treatments and advance personalized medicine. In this review, we will describe the genetics of the major genes of the opioid system (opioid receptors and their endogenous ligands) in connection to addiction to opioids, cocaine, alcohol and methamphetamines. Particular emphasis is given to association and functional studies of specific variants. We will provide information on the sample populations and the size of each study, as well as a list of the variants implicated in association with addiction-related phenotypes, and with the effectiveness of pharmacotherapy for addiction.

Tissue-specific DNA methylation of the human prodynorphin gene in post-mortem brain tissues and PBMCs.

OBJECTIVE: Dynorphins, the endogenous ligands for the κ opioid receptor, are implicated in neuropsychiatric disorders through modulation of basal and stimuli-induced dopaminergic, glutamatergic, and serotonergic tones. Expression of the prodynorphin gene (PDYN) is critical for rewarding properties of drugs of abuse and stress-induced responses. Epigenetic factors, such as DNA methylation, play an important role in modulation of gene expression. METHODS: We analyzed DNA methylation patterns of three CpG-rich regions of PDYN, a CpG island, and cluster A in the proximal promoter, and cluster B in coding exon 4, by bisulfite sequencing of DNA from the caudate and anterior cingulate cortex from post-mortem brain of 35 individuals (22 HIV seropositive), and in peripheral blood mononuclear cells from 21 of these individuals. RESULTS: We found remarkably similar patterns of methylation across CpG sites in these tissues. However, there were tissue-specific differences in methylation levels (P=0.000001) of the CpG island: higher levels in peripheral blood mononuclear cells (82%) than in the brain tissues, the caudate (62%), and the anterior cingulate cortex (44%). But there was higher PDYN expression in the caudate than in the anterior cingulate cortex. In contrast, cluster A near the transcription start site is hypomethylated. CONCLUSION: This DNA methylation profile of the PDYN gene is typical for primary responsive genes with regulatory elements for both basal and tissue-specific transcription. Our findings provide a rationale for further studies of the role of other epigenetic factors in the regulation of PDYN expression in individuals with psychiatric and neurological disorders.

Cell-specific effects of variants of the 68-base pair tandem repeat on prodynorphin gene promoter activity.

A polymorphic 68-bp tandem repeat has been identified within the promoter of the human prodynorphin (PDYN) gene. We found that this 68-bp repeat in the PDYN promoter occurs naturally up to five times. We studied the effect of the number of 68-bp repeats, and of a SNP (rs61761346) found within the repeat on PDYN gene promoter activity. Thirteen promoter forms, different naturally occurring combinations of repeats and the internal SNP, were cloned upstream of the luciferase reporter gene, transfected into human SK-N-SH, H69, or HEK293 cells. Cells were then stimulated with TPA or caffeine. We found cell-specific effects of the number of 68-bp repeats on the transcriptional activity of the PDYN promoter. In SK-N-SH and H69 cells, three or four repeats led to lower expression of luciferase than did one or two repeats. The opposite effect was found in HEK293 cells. The SNP also had an effect on PDYN gene expression in both SK-N-SH and H69 cells; promoter forms with the A allele had significantly higher expression than promoter forms with the G allele. These results further our understanding of the complex transcriptional regulation of the PDYN gene promoter.

Genome-wide association study identifies genes that may contribute to risk for developing heroin addiction.

OBJECTIVES: We have used genome-wide association studies to identify variants that are associated with vulnerability to develop heroin addiction. METHODS: DNA from 325 methadone stabilized, former severe heroin addicts and 250 control individuals were pooled by ethnicity (Caucasian and African-American) and analyzed using the Affymetrix GeneChip Mapping 100 K Set. Genome-wide association tests were conducted. RESULTS: The strongest association with vulnerability to develop heroin addiction, with experiment-wise significance (P=0.035), was found in Caucasians with the variant rs10494334, a variant in an unannotated region of the genome (1q23.3). In African Americans, the variant most significantly associated with the heroin addiction vulnerability was rs950302, found in the cytosolic dual specificity phosphatase 27 gene DUSP27 (point-wise P=0.0079). Furthermore, analysis of the top 500 variants with the most significant associations (point-wise P< or =0.0036) in Caucasians showed that three of these variants are clustered in the regulating synaptic membrane exocytosis protein 2 gene RIMS2. Of the top 500 variants in African-Americans (point-wise P< or =0.0238), three variants are in the cardiomyopathy associated 3 gene CMYA3. CONCLUSION: This study identifies new genes and variants that may increase an individual's vulnerability to develop heroin addiction.