Genes and Pathways Co-associated with the Exposure to Multiple Drugs of Abuse,Including Alcohol,Amphetamine/Methamphetamine,Cocaine, Marijuana,Morphine, and/or Nicotine: a Review of Proteomics Analyses

Drug addiction is a chronic neuronal disease. In recent years, proteomics technology has been widely used to assess the protein expression in the brain tissues of both animals and humans exposed to addictive drugs. Through this approach, a large number of proteins potentially involved in the etiology of drug addictions have been identified, which provide a valuable resource to study protein function, biochemical pathways, and networks related to the molecular mechanisms underlying drug dependence. In this article, we summarize the recent application of proteomics to profiling protein expression patterns in animal or human brain tissues after the administration of alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine/heroin/butorphanol, or nicotine. From available reports, we compiled a list of 497 proteins associated with exposure to one or more addictive drugs, with 160 being related to exposure to at least two abused drugs. A number of biochemical pathways and biological processes appear to be enriched among these proteins, including synaptic transmission and signaling pathways related to neuronal functions. The data included in this work provide a summary and extension of the proteomics studies on drug addiction. Furthermore, the proteins and biological processes highlighted here may provide valuable insight into the cellular activities and biological processes in neurons in the development of drug addiction.     

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.     

Gene expression profile from the striatum of amphetamine-treated rats: a cDNA array and in situ hybridization histochemical study

Changes in gene/protein expression markedly outlast the transient changes in behavior evoked by a single dose of a psychostimulant. These changes in gene expression are thought to underlie and/or trigger enduring changes in neuroplasticity that lead to drug addiction. We used cDNA arrays to gain a more complete picture of changes in striatal gene expression 1 and 3 h after an acute injection of amphetamine. Consistent, reliable gene expression changes were detected when criteria of at least a 1.5-fold difference and three replicate hybridizations using independent samples were performed. Using these criteria, the mRNA for three immediate early genes (IEGs), coding for activity-regulated cytoskeletal-associated protein (Arc), nerve growth factor-induced protein A (NGFI-A; early growth response protein 1) and nerve growth factor-induced protein B (NGFI-B), were upregulated 1 and 3 h after amphetamine as previously described. Novel genes, RL/IF-1 (coding for I kappa B alpha chain) and serum/glucocorticoid-regulated serine/threonine protein kinase (SGK) also were increased throughout the striatum, at 1 but not 3 h. Conversely, amphetamine increased the mRNA coding for the secretogranin II precursor (chromogranin C) only at the 3 h time point when a specific decrease in regulator of G-protein signaling 4 (RGS4) mRNA was also observed. Gene changes and unique patterns of expression were verified by in situ hybridization, providing valuable information about changes in gene expression in response to acute amphetamine.     

Investigating the Potential Influence of Cause of Death and Cocaine Levels on the Differential Expression of Genes Associated with Cocaine Abuse

The development of new therapeutic strategies for the treatment of complex brain disorders such as drug addiction is likely to be advanced by a more complete understanding of the underlying molecular pathophysiology. Although the study of postmortem human brain represents a unique resource in this regard, it can be challenging to disentangle the relative contribution of chronic pathological processes versus perimortem events to the observed changes in gene expression. To begin to unravel this issue, we analyzed by quantitative PCR the midbrain expression of numerous candidate genes previously associated with cocaine abuse. Data obtained from chronic cocaine abusers (and matched control subjects) dying of gunshot wounds were compared with a prior study of subjects with deaths directly attributable to cocaine abuse. Most of the genes studied (i.e., tyrosine hydroxylase, dopamine transporter, forkhead box A2, histone variant H3 family 3B, nuclear factor kappa B inhibitor alpha, growth arrest and DNA damage-inducible beta) were found to be differentially expressed in chronic cocaine abusers irrespective of immediate cause of death or perimortem levels of cocaine, suggesting that these may represent core pathophysiological changes arising with chronic drug abuse. On the other hand, chemokine C-C motif ligand 2 and jun proto-oncogene expression were unaffected in cocaine-abusing subjects dying of gunshot wounds, in contrast to the differential expression previously reported in cocaine-related fatalities. The possible influence of cause of death and other factors on the cocaine-responsiveness of these genes is discussed.     

Cortical–striatal gene expression in neonatal hippocampal lesion (NVHL)‐amplified cocaine sensitization

Cortical–striatal circuit dysfunction in mental illness may enhance addiction vulnerability. Neonatal ventral hippocampal lesions (NVHL) model this dual diagnosis causality by producing a schizophrenia syndrome with enhanced responsiveness to addictive drugs. Rat genome‐wide microarrays containing >24 000 probesets were used to examine separate and co‐occurring effects of NVHLs and cocaine sensitization (15 mg/kg/day × 5 days) on gene expression within medial prefrontal cortex (MPFC), nucleus accumbens (NAC), and caudate‐putamen (CAPU). Two weeks after NVHLs robustly amplified cocaine behavioral sensitization, brains were harvested for genes of interest defined as those altered at P < 0.001 by NVHL or cocaine effects or interactions. Among 135 genes so impacted, NVHLs altered twofold more than cocaine, with half of all changes in the NAC. Although no genes were changed in the same direction by both NVHL and cocaine history, the anatomy and directionality of significant changes suggested synergy on the neural circuit level generative of compounded behavioral phenotypes: NVHL predominantly downregulated expression in MPFC and NAC while NVHL and cocaine history mostly upregulated CAPU expression. From 75 named genes altered by NVHL or cocaine, 27 had expression levels that correlated significantly with degree of behavioral sensitization, including 11 downregulated by NVHL in MPFC/NAC, and 10 upregulated by NVHL or cocaine in CAPU. These findings suggest that structural and functional impoverishment of prefrontal‐cortical‐accumbens circuits in mental illness is associated with abnormal striatal plasticity compounding with that in addictive disease. Polygenetic interactions impacting neuronal signaling and morphology within these networks likely contribute to addiction vulnerability in mental illness .     

Differential Regulation of the Period Genes in Striatal Regions following Cocaine Exposure

Several studies have suggested that disruptions in circadian rhythms contribute to the pathophysiology of multiple psychiatric diseases, including drug addiction. In fact, a number of the genes involved in the regulation of circadian rhythms are also involved in modulating the reward value for drugs of abuse, like cocaine. Thus, we wanted to determine the effects of chronic cocaine on the expression of several circadian genes in the Nucleus Accumbens (NAc) and Caudate Putamen (CP), regions of the brain known to be involved in the behavioral responses to drugs of abuse. Moreover, we wanted to explore the mechanism by which these genes are regulated following cocaine exposure. Here we find that after repeated cocaine exposure, expression of the Period (Per) genes and Neuronal PAS Domain Protein 2 (Npas2) are elevated, in a somewhat regionally selective fashion. Moreover, NPAS2 (but not CLOCK (Circadian Locomotor Output Cycles Kaput)) protein binding at Per gene promoters was enhanced following cocaine treatment. Mice lacking a functional Npas2 gene failed to exhibit any induction of Per gene expression after cocaine, suggesting that NPAS2 is necessary for this cocaine-induced regulation. Examination of Per gene and Npas2 expression over twenty-four hours identified changes in diurnal rhythmicity of these genes following chronic cocaine, which were regionally specific. Taken together, these studies point to selective disruptions in Per gene rhythmicity in striatial regions following chronic cocaine treatment, which are mediated primarily by NPAS2.     

Drug-induced Alterations in the Extracellular Signal-regulated Kinase (ERK) Signalling Pathway: Implications for Reinforcement and Reinstatement

Drug addiction, characterized by high rates of relapse, is recognized as a kind of neuroadaptive disorder. Since the extracellular signal-regulated kinase (ERK) pathway is critical to neuroplasticity in the adult brain, understanding the role this pathway plays is important for understanding the molecular mechanism underlying drug addiction and relapse. Here, we review previous literatures that focus on the effects of exposure to cocaine, amphetamine, Δ⁹-tetrahydrocannabinol (THC), nicotine, morphine, and alcohol on ERK signaling in the mesocorticolimbic dopamine system; these alterations of ERK signaling have been thought to contribute to the drug’s rewarding effects and to the long-term maladaptation induced by drug abuse. We then discuss the possible upstreams of the ERK signaling pathway activated by exposure of drugs of abuse and the environmental cues previously paired with drugs. Finally, we argue that since ERK activation is a key molecular process in reinstatement of conditioned place preference and drug self-administration, the pharmacological manipulation of the ERK pathway is a potential treatment strategy for drug addiction. Haifeng Zhai and Yanqin Li contributed equally to this paper.     

Glutamate receptor metabotropic 7 is cis-regulated in the mouse brain and modulates alcohol drinking

Alcoholism is a heritable disease that afflicts about 8% of the adult population. Its development and symptoms, such as craving, loss of control, physical dependence, and tolerance, have been linked to changes in mesolimbic, mesocortical neurotransmitter systems utilizing biogenic amines, GABA, and glutamate. Identification of genes predisposing to alcoholism, or to alcohol-related behaviors in animal models, has been elusive because of variable interactions of multiple genes with relatively small individual effect size and sensitivity of the predisposing genotype to lifestyle and environmental factors. Here, using near-isogenic advanced animal models with reduced genetic background interactions, we integrate gene mapping and gene mRNA expression data in segregating and congenic mice and identify glutamate receptor metabotropic 7 (Grm7) as a cis-regulated gene for alcohol consumption. Traditionally, the mesoaccumbal dopamine reward hypothesis of addiction and the role of the ionotropic glutamate receptors have been emphasized. Our results lend support to an emerging direction of research on the role of metabotropic glutamate receptors in alcoholism and drug addiction. These data suggest for the first time that Grm7 is a risk factor for alcohol drinking and a new target in addiction therapy.