Ruiming Zhu

Taurine attenuates methamphetamine-induced autophagy and apoptosis in PC12 cells through mTOR signaling pathway

Methamphetamine (METH), a commonly abused psychostimulant, has been shown to induce neuronal damage by causing reactive oxygen species (ROS) formation, apoptosis and autophagy. Taurine (2-aminoethanesulfonic acid) is involved in several physiological actions in the brain, including neuroprotection, osmoregulation and neurotransmission. In this study, we investigate the protective effect of taurine against METH-induced neurotoxicity in PC12 cells and the underlying mechanism. The results showed that taurine significantly increased the cell viability inhibited by METH. LC3-II expression was elevated by METH treatment, whereas such increase was obviously attenuated by taurine. Co-treatment of taurine strongly reversed the decline of antioxidase activities induced by METH. Moreover, phosphorylated mammalian target of rapamycin (p-mTOR) was significantly inhibited by METH, whereas complementation of taurine markedly increased the expression of p-mTOR in PC12 cells, rather than phosphorylated Erk. Interestingly, taurine-induced decreasing expression of LC3-II was partially blocked by pretreatment of RAD001, an mTOR inhibitor. These results indicated that taurine inhibits METH-induced autophagic process through activating mTOR rather than Erk signaling. Collectively, our study shows that taurine protects METH-induced PC12 cells damage by attenuating ROS production, apoptosis and autophagy, at least in part, via mTOR signaling pathway.

Transcriptome analysis of long non-coding RNAs of the nucleus accumbens in cocaine-conditioned mice

Cocaine dependence involves in the brains reward circuit as well as nucleus accumbens (NAc), a key region of the mesolimbic dopamine pathway. Many studies have documented altered expression of genes and identified transcription factor networks and epigenetic processes that are fundamental to cocaine addiction. However, all these investigations have focused on mRNA of encoding genes, which may not always reflect the involvement of long non‐coding RNAs (lncRNAs), which has been implied in a broad range of biological processes and complex diseases including brain development and neuropathological process. To explore the potential involvement of lncRNAs in drug addiction, which is viewed as a form of aberrant neuroplasticity, we used a custom‐designed microarray to examine the expression profiles of mRNAs and lncRNAs in brain NAc of cocaine‐conditioned mice and identified 764 mRNAs, and 603 lncRNAs were differentially expressed. Candidate lncRNAs were identified for further genomic context characterization as sense‐overlap, antisense‐overlap, intergenic, bidirection, and ultra‐conserved region encoding lncRNAs. We found that 410 candidate lncRNAs which have been reported to act in cis or trans to their targeted loci, providing 48 pair mRNA‐lncRNAs. These results suggest that the modification of mRNAs expression by cocaine may be associated with the actions of lncRNAs. Taken together, our results show that cocaine can cause the genome‐wide alterations of lncRNAs expressed in NAc, and some of these modified RNA transcripts may to play a role in cocaine‐induced neural plasticity and addiction.

Proteomic analysis of the nucleus accumbens in rhesus monkeys of morphine dependence and withdrawal intervention

It has been known that the reinforcing effects and long-term consequences of morphine are closely associated with nucleus accumbens (NAc) in the brain, a key region of the mesolimbic dopamine pathway. However, the proteins involved in neuroadaptive processes and withdrawal symptom in primates of morphine dependence have not been well explored. In the present study, we performed proteomes in the NAc of rhesus monkeys of morphine dependence and withdrawal intervention with clonidine or methadone. Two-dimensional electrophoresis was used to compare changes in cytosolic protein abundance in the NAc. We found a total of 46 proteins differentially expressed, which were further identified by mass spectrometry analysis. The identified proteins can be classified into 6 classes: metabolism and mitochondrial function, synaptic transmission, cytoskeletal proteins, oxidative stress, signal transduction and protein synthesis and degradation. Importantly, we discovered 14 proteins were significantly but similarly altered after withdrawal therapy with clonidine or methadone, revealing potential pharmacological strategies or targets for the treatment of morphine addiction. Our study provides a comprehensive understanding of the neuropathophysiology associated with morphine addiction and withdrawal therapy in primate, which is helpful for the development of opiate withdrawal pharmacotherapies.

1H NMR-based metabonomic analysis of brain in rats of morphine dependence and withdrawal intervention

Metabolic consequences of morphine dependence and withdrawal intervention have not been well explored. In the present study, the metabolic changes in brain hippocampus, nucleus accumbens (NAc), prefrontal cortex (PFC) and striatum of rats with morphine dependence and withdrawal intervention were explored by using ¹H nuclear magnetic resonance coupled with principal component analysis, partial least squares and orthogonal signal correction analysis. We found that the concentrations of neurotransmitters including glutamate, glutamine and gamma-aminobutyric acid changed differentially in hippocampus, NAc, PFC and striatum after repeated morphine treatment. Significant changes were also found in a number of cerebral metabolites including N-acetyl aspartate (NAA), lactic acid, creatine, myo-inositol and taurine. These findings indicate the profound disturbances of energy metabolism, amino acid metabolism and neurotransmitters caused by chronic morphine treatment. Interestingly, morphine-induced changes in lactic acid, creatine and NAA were clearly reversed by intervention of methadone or clonidine. Our study provides a comprehensive understanding of the metabolic alteration associated with morphine addiction and withdrawal therapy, which may help to develop new pharmacotherapies.

Mechanisms of Metabonomic for a Gateway Drug: Nicotine Priming Enhances Behavioral Response to Cocaine with Modification in Energy Metabolism and Neurotransmitter Level

Nicotine, one of the most commonly used drugs, has become a major concern because tobacco serves as a gateway drug and is linked to illicit drug abuse, such as cocaine and marijuana. However, previous studies mainly focused on certain genes or neurotransmitters which have already been known to participate in drug addiction, lacking endogenous metabolic profiling in a global view. To further explore the mechanism by which nicotine modifies the response to cocaine, we developed two conditioned place preference (CPP) models in mice. In threshold dose model, mice were pretreated with nicotine, followed by cocaine treatment at the dose of 2 mg/kg, a threshold dose of cocaine to induce CPP in mice. In high-dose model, mice were only treated with 20 mg/kg cocaine, which induced a significant CPP. 1H nuclear magnetic resonance based on metabonomics was used to investigate metabolic profiles of the nucleus accumbens (NAc) and striatum. We found that nicotine pretreatment dramatically increased CPP induced by 2 mg/kg cocaine, which was similar to 20 mg/kg cocaine-induced CPP. Interestingly, metabolic profiles showed considerable overlap between these two models. These overlapped metabolites mainly included neurotransmitters as well as the molecules participating in energy homeostasis and cellular metabolism. Our results show that the reinforcing effect of nicotine on behavioral response to cocaine may attribute to the modification of some specific metabolites in NAc and striatum, thus creating a favorable metabolic environment for enhancing conditioned rewarding effect of cocaine. Our findings provide an insight into the effect of cigarette smoking on cocaine dependence and the underlying mechanism.

Arginine Methyltransferase 1 in the Nucleus Accumbens Regulates Behavioral Effects of Cocaine.

Recent evidence suggests that histone modifications play a role in the behavioral effects of cocaine in rodent models. Histone arginine is known to be methylated by protein arginine N-methyltransferases (PRMTs). Evidence shows that PRMT1 contributes to >90% of cellular PRMT activity, which regulates histone H4 arginine 3 asymmetric dimethylation (H4R3me2a). Though histone arginine methylation represents a chemical modification that is relatively stable compared with other histone alterations, it is less well studied in the setting of addiction. Here, we demonstrate that repeated noncontingent cocaine injections increase PRMT1 activity in the nucleus accumbens (NAc) of C57BL/6 mice. We, subsequently, identify a selective inhibitor of PRMT1, SKLB-639, and show that systemic injections of the drug decrease cocaine-induced conditioned place preference to levels observed with genetic knockdown of PRMT1. NAc-specific downregulation of PRMT1 leads to hypomethylation of H4R3me2a, and hypoacetylation of histone H3 lysine 9 and 14. We also found that H4R3me2a is upregulated in NAc after repeated cocaine administration, and that H4R3me2a upregulation in turn controls the expression of Cdk5 and CaMKII. Additionally, the suppression of PRMT1 in NAc with lentiviral-short hairpin PMRT1 decreases levels of CaMKII and Cdk5 in the cocaine-treated group, demonstrating that PRMT1 affects the ability of cocaine to induce CaMKII and Cdk5 in NAc. Notably, increased H4R3me2a by repeated cocaine injections is relatively long-lived, as increased expression was observed for up to 7 d after the last cocaine injection. These results show the role of PRMT1 in the behavioral effects of cocaine. SIGNIFICANCE STATEMENT This work demonstrated that repeated cocaine injections led to an increase of protein arginine N-methyltransferase (PRMT1) in nucleus accumbens (NAc). We then identified a selective inhibitor of PRMT1 (SKLB-639), which inhibited cocaine-induced conditioned place preference (CPP). Additionally, genetic downregulation of PRMT1 in NAc also attenuated cocaine-caused CPP and locomotion activity, which was associated with decreased expression of histone H4 arginine 3 asymmetric demethylation (H4R3me2a) and hypoacetylation of histone H3 lysine 9 and 14 (acH3K9/K14). This study also showed that H4R3me2a controlled transcriptions of Cdk5 and CaMKII, and that PRMT1 negatively affected the ability of cocaine to induce CaMKII and Cdk5 in NAc. Notably, increased H4R3me2a by repeated cocaine injection was relatively long-lived as increased expression was observed up to 7 d after withdrawal from cocaine. Together, this study suggests that PRMT1 inhibition may serve as a potential therapeutic strategy for cocaine addiction.UNLABELLED Recent evidence suggests that histone modifications play a role in the behavioral effects of cocaine in rodent models. Histone arginine is known to be methylated by protein arginine N-methyltransferases (PRMTs). Evidence shows that PRMT1 contributes to >90% of cellular PRMT activity, which regulates histone H4 arginine 3 asymmetric dimethylation (H4R3me2a). Though histone arginine methylation represents a chemical modification that is relatively stable compared with other histone alterations, it is less well studied in the setting of addiction. Here, we demonstrate that repeated noncontingent cocaine injections increase PRMT1 activity in the nucleus accumbens (NAc) of C57BL/6 mice. We, subsequently, identify a selective inhibitor of PRMT1, SKLB-639, and show that systemic injections of the drug decrease cocaine-induced conditioned place preference to levels observed with genetic knockdown of PRMT1. NAc-specific downregulation of PRMT1 leads to hypomethylation of H4R3me2a, and hypoacetylation of histone H3 lysine 9 and 14. We also found that H4R3me2a is upregulated in NAc after repeated cocaine administration, and that H4R3me2a upregulation in turn controls the expression of Cdk5 and CaMKII. Additionally, the suppression of PRMT1 in NAc with lentiviral-short hairpin PMRT1 decreases levels of CaMKII and Cdk5 in the cocaine-treated group, demonstrating that PRMT1 affects the ability of cocaine to induce CaMKII and Cdk5 in NAc. Notably, increased H4R3me2a by repeated cocaine injections is relatively long-lived, as increased expression was observed for up to 7 d after the last cocaine injection. These results show the role of PRMT1 in the behavioral effects of cocaine. SIGNIFICANCE STATEMENT This work demonstrated that repeated cocaine injections led to an increase of protein arginine N-methyltransferase (PRMT1) in nucleus accumbens (NAc). We then identified a selective inhibitor of PRMT1 (SKLB-639), which inhibited cocaine-induced conditioned place preference (CPP). Additionally, genetic downregulation of PRMT1 in NAc also attenuated cocaine-caused CPP and locomotion activity, which was associated with decreased expression of histone H4 arginine 3 asymmetric demethylation (H4R3me2a) and hypoacetylation of histone H3 lysine 9 and 14 (acH3K9/K14). This study also showed that H4R3me2a controlled transcriptions of Cdk5 and CaMKII, and that PRMT1 negatively affected the ability of cocaine to induce CaMKII and Cdk5 in NAc. Notably, increased H4R3me2a by repeated cocaine injection was relatively long-lived as increased expression was observed up to 7 d after withdrawal from cocaine. Together, this study suggests that PRMT1 inhibition may serve as a potential therapeutic strategy for cocaine addiction.

Taurine protects methamphetamine-induced developmental angiogenesis defect through antioxidant mechanism.

Investigations have characterized addictive drug-induced developmental cardiovascular malformation in human, non-human primate and rodent. However, the underlying mechanism of malformation caused by drugs during pregnancy is still largely unknown, and preventive and therapeutic measures have been lacking. Using 1H NMR spectroscopy, we profiled the metabolites from human embryo endothelial cells exposed to methamphetamine (METH) and quantified a total of 226 peaks. We identified 11 metabolites modified robustly and found that taurine markedly increased. We then validated the hypothesis that this dramatic increase in taurine could attribute to its effect in inhibiting METH-induced developmental angiogenesis defect. Taurine supplement showed a more significant potential than other metabolites in protecting against METH-induced injury in endothelial cells. Taurine strongly attenuated METH-induced inhibition of proliferation and migration in endothelial cells. Furthermore, death rate and vessel abnormality of zebrafish embryos treated with METH were greatly reversed by taurine. In addition, taurine supplement caused a rapid decrease in reactive oxygen species generation and strongly attenuated the excitable arise of antioxidase activities in the beginning of METH exposure prophase. Dysregulations of NF-κB, p-ERK as well as Bax, which reflect apoptosis, cell cycle arrest and oxidative stress in vascular endothelium, were blocked by taurine. Our results provide the first evidence that taurine prevents METH-caused developmental angiogenesis defect through antioxidant mechanism. Taurine could serve as a potential therapeutic or preventive intervention of developmental vascular malformation for the pregnant women with drug use.

1H NMR-based metabonomics in brain nucleus accumbens and striatum following repeated cocaine treatment in rats

Studies have shown a few cerebral metabolites modified by cocaine in brain regions; however, endogenous metabolic profiling has been lacking. Ex vivo (1)H NMR (hydrogen-1 nuclear magnetic resonance) spectroscopy-based metabonomic approach coupled with partial least squares was applied to investigate the changes of cerebral metabolites in nucleus accumbens (NAc) and striatum of rats subjected to cocaine treatment. Our results showed that both single and repeated cocaine treatment can induce significant changes in a couple of cerebral metabolites. The increase of neurotransmitters glutamate and gamma-amino butyric acid (GABA) were observed in NAc and striatum from the rats repeatedly treated with cocaine. Creatine and taurine increased in NAc whereas taurine increased and creatine decreased in striatum after repeated cocaine treatment. Elevation of N-acetylaspartate in NAc and striatum and decrease of lactate in striatum were observed, which may reflect the mitochondria dysregulation caused by cocaine; moreover, alterations of choline, phosphocholine and glycerol in NAc and striatum could be related to membrane disruption. Moreover, groups of rats with and without conditioned place preference (CPP) apparatus are presenting difference in metabolites. Collectively, our results provide the first evidence of metabonomic profiling of NAc and striatum in response to cocaine, exhibiting a regionally-specific alteration patterns. We find that repeated cocaine administration leads to significant metabolite alterations, which are involved in neurotransmitter disturbance, oxidative stress, mitochondria dysregulation and membrane disruption in brain.

Prenatal cocaine exposure impairs cognitive function of progeny via insulin growth factor II epigenetic regulation.

Studies have showed that prenatal cocaine exposure (PCOC) can impair cognitive function and social behavior of the offspring; however, the mechanism underlying such effect is poorly understood. Insulin-like growth factor II (Igf-II), an imprinted gene, has a critical role in memory consolidation and enhancement. We hypothesized that epigenetic regulation of hippocampal Igf-II may attribute to the cognitive deficits of PCOC offspring. We used Morris water maze and open-field task to test the cognitive function in PCOC offspring. The epigenetic alteration involved in hippocampal Igf-II expression deficit in PCOC offspring was studied by determining Igf-II methylation status, DNA methyltransferases (DNMT) expressions and L-methionine level. Moreover, IGF-II rescue experiments were performed and the downstream signalings were investigated in PCOC offspring. In behavioral tests, we observed impaired spatial learning and memory and increased anxiety in PCOC offspring; moreover, hippocampal IGF-II mRNA and protein expressions were significantly decreased. Hippocampal methylation of cytosine-phospho-guanine (CpG) dinucleotides in differentially methylated region (DMR) 2 of Igf-II was elevated in PCOC offspring, which may be driven by the upregulation of L-methionine and DNA methyltransferase (DNMT) 1. Importantly, intra-hippocampal injection of recombinant IGF-II reactivated the repressed calcium calmodulin kinase II α (CaMKIIα) and reversed cognitive deficits in PCOC offspring. Collectively, our findings suggest that cocaine exposure during pregnancy impairs cognitive function of offspring through epigenetic modification of Igf-II gene. Enhancing IGF-II signaling may represent a novel therapeutical strategy for cocaine-induced cognitive impairment.

1H-nuclear magnetic resonance-based metabonomic analysis of brain in rhesus monkeys with morphine treatment and withdrawal intervention

Comprehensive cerebral metabolites involved in morphine dependence have not been well explored. To gain a better understanding of morphine dependence and withdrawal therapy in a model highly related to humans, metabolic changes in brain hippocampus and prefrontal cortex (PFC) of rhesus monkeys were measured by 1H‐nuclear magnetic resonance spectroscopy, coupled with partial least squares and orthogonal signal correction analysis. The results showed that concentrations of myoinositol (M‐Ins) and taurine were significantly reduced, whereas lactic acid was increased in hippocampus and PFC of morphine‐dependent monkeys. Phosphocholine and creatine increased in PFC but decreased in hippocampus after chronic treatment of morphine. Moreover, N‐acetyl aspartate (NAA), γ‐aminobutyric acid, glutamate, glutathione, methionine, and homocysteic acid also changed in these brain regions. These results suggest that chronic morphine exposure causes profound disturbances of neurotransmitters, membrane, and energy metabolism in the brain. Notably, morphine‐induced dysregulations in NAA, creatine, lactic acid, taurine, M‐Ins, and phosphocholine were clearly reversed after intervention with methadone or clonidine. Our study highlights the potential of metabolic profiling to enhance our understanding of metabolite alteration and neurobiological actions associated with morphine addiction and withdrawal therapy in primates.