Adrian M. Gomez

Mutation of Tyrosine 470 of Human Dopamine Transporter is Critical for HIV-1 Tat-Induced Inhibition of Dopamine Transport and Transporter Conformational Transitions

HIV-1 Tat protein plays a crucial role in perturbations of the dopamine (DA) system. Our previous studies have demonstrated that Tat decreases DA uptake, and allosterically modulates DA transporter (DAT) function. In the present study, we have found that Tat interacts directly with DAT, leading to inhibition of DAT function. Through computational modeling and simulations, a potential recognition binding site of human DAT (hDAT) for Tat was predicted. Mutation of tyrosine470 (Y470H) attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of this residue for Tat binding to hDAT. Y470H reduced the maximal velocity of [3H]DA uptake without changes in the Km and IC50 values for DA inhibition of DA uptake but increased DA uptake potency for cocaine and GBR12909, suggesting that this residue does not overlap with the binding sites in hDAT for substrate but is critical for these inhibitors. Furthermore, Y470H also led to transporter conformational transitions by affecting zinc modulation of DA uptake and WIN35,428 binding as well as enhancing basal DA efflux. Collectively, these findings demonstrate Tyr470 as a functional recognition residue in hDAT for Tat-induced inhibition of DA transport and transporter conformational transitions. The consequence of mutation at this residue is to block the functional binding of Tat to hDAT without affecting physiological DA transport.

Genetically expressed HIV-1 viral proteins attenuate nicotine-induced behavioral sensitization and alter mesocorticolimbic ERK and CREB signaling in rats

The prevalence of tobacco smoking in HIV-1 positive individuals is 3-fold greater than that in the HIV-1 negative population; however, whether HIV-1 viral proteins and nicotine together produce molecular changes in mesolimbic structures that mediate psychomotor behavior has not been studied. This study determined whether HIV-1 viral proteins changed nicotine-induced behavioral sensitization in HIV-1 transgenic (HIV-1Tg) rats. Further, we examined cAMP response element binding protein (CREB) and extracellular regulated kinase (ERK1/2) signaling in the prefrontal cortex (PFC), nucleus accumbens (NAc) and ventral tegmental area (VTA). HIV-1Tg rats exhibited a transient decrease of activity during habituation, but showed attenuated nicotine (0.35mg/kg, s.c.)-induced behavioral sensitization compared to Fisher 344 (F344) rats. The basal levels of phosphorylated CREB and ERK2 were lower in the PFC of HIV-1Tg rats, but not in the NAc and VTA, relative to the controls. In the nicotine-treated groups, the levels of phosphorylated CREB and ERK2 in the PFC were increased in HIV-1Tg rats, but decreased in F344 animals. Moreover, repeated nicotine administration reduced phosphorylated ERK2 in the VTA of HIV-1Tg rats and in the NAc of F344 rats, but had no effect on phosphorylated CREB, indicating a region-specific change of intracellular signaling. These results demonstrate that HIV-1 viral proteins produce differences in basal and nicotine-induced alterations in CREB and ERK signaling that may contribute to the alteration in psychomotor sensitization. Thus, HIV-1 positive smokers are possibly more vulnerable to alterations in CREB and ERK signaling and this has implications for motivated behavior, including tobacco smoking, in HIV-1 positive individuals who self-administer nicotine.

Effect of environmental enrichment on methylphenidate-induced locomotion and dopamine transporter dynamics

Rats raised in an enriched condition (EC) are less sensitive to the locomotor effects of stimulant drugs than rats raised in an impoverished condition (IC). Methylphenidate (MPD), a primary pharmacotherapy for attention-deficit/hyperactivity disorder, has abuse potential. This study determined whether environmental enrichment differentially altered the effects of MPD on locomotor activity and dopamine (DA) transporter (DAT) function. Acute and repeated MPD (3 or 10 mg/kg, s.c.) increased locomotion in EC, IC and social condition (SC) rats; however, EC rats showed a blunted response to repeated MPD (3 mg/kg). The maximal velocity (V(max)) of [(3)H]DA uptake in the presence of the combination of phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator and okadaic acid, a protein phosphatase inhibitor was decreased in EC and IC rats by 68% and 40%, respectively, indicating that DAT in prefrontal cortex (PFC) is more sensitive to PKC-mediated down-regulation in EC rats. Acute MPD (10 mg/kg) administration decreased the V(max) of [(3)H]DA uptake in PFC and striatum in EC rats, but not in IC rats. Furthermore, [(3)H]WIN 35,428 binding density was decreased in PFC of EC and IC rats, and in striatum of EC rats given repeated MPD (10 mg/kg). These results demonstrate that environmental enrichment modulates DAT dynamics in PFC. However, since the change in DAT function was observed only following the high dose of MPH (10 mg/kg), the attenuated locomotor response to repeated MPD (3 mg/kg) in EC rats is not likely due to a specific DAT alteration in the brain regions examined.

Environmental Enrichment Alters Nicotine-Mediated Locomotor Sensitization and Phosphorylation of DARPP-32 and CREB in Rat Prefrontal Cortex

Exposure within an environmental enrichment paradigm results in neurobiological adaptations and decreases the baseline of locomotor activity. The current study determined activation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32) and CREB (cAMP response element binding protein), and locomotor activity in rats raised in enriched (EC), impoverished (IC), and standard (SC) conditions following repeated administration of nicotine or saline. In the saline-control group, the basal phosphorylation state of DARPP-32 at Threonine-34 site (pDARPP-32 Thr34) in the prefrontal cortex (PFC) was lower in EC compared to IC and SC rats, which was positively correlated with their respective baseline activities. While nicotine (0.35 mg/kg, freebase) produced locomotor sensitization across all housing conditions when the nicotine-mediated locomotor activity was expressed as a percent change from their respective saline control, EC rats displayed greater sensitization to nicotine than IC and SC rats. Consistent with the behavioral findings, repeated nicotine injection increased pDARPP-32 Thr34 in PFC of EC and IC rats and in nucleus accumbens of EC rats; however, the magnitude of change from saline control in nicotine-induced enhancement of pDARPP-32 Thr34 in PFC was strikingly increased in EC rats relative to IC rats. Moreover, EC rats had lower basal phosphorylation levels of CREB at serine 133 in PFC and nucleus accumbens compared to IC and SC rats, whereas the nicotine-induced increase in phosphorylated CREB-Ser133 was more pronounced in PFC of EC rats relative to IC and SC rats. Collectively, these findings suggest innovative insights into advancing our understanding of the molecular mechanisms of enrichment-induced changes in the motivational effects of nicotine, and aiding in the identification of new therapeutic strategies for tobacco smokers.

Intra-ventral tegmental area HIV-1 Tat1–86 attenuates nicotine-mediated locomotor sensitization and alters mesocorticolimbic ERK and CREB signaling in rats

Cigarette smoking prevalence in the HIV-positive individuals is profoundly higher than that in the HIV-negative individuals. We have demonstrated that HIV-1 transgenic rats exhibit attenuated nicotine-mediated locomotor activity, altered cAMP response element binding protein (CREB) and extracellular regulated kinase (ERK1/2) signaling in the mesocorticolimbic regions. This study investigated the role of HIV-1 transactivator of transcription (Tat) protein in the alterations of nicotine-mediated behavior and the signaling pathway observed in the HIV-1 transgenic rats. Rats received bilateral microinjection of recombinant Tat1–86 (25 μg/side) or vehicle directed at ventral tegmental area (VTA) followed by locomotor testing in response to 13 daily intravenous injections of nicotine (0.05 mg/kg, freebase, once/day) or saline. Further, we examined the phosphorylated levels of CREB (pCREB) and ERK1/2 (pERK1/2) in the prefrontal cortex (PFC), nucleus accumbens (NAc) and VTA. Tat diminished baseline activity in saline control rats, and attenuated nicotine-induced behavioral sensitization. Following repeated saline injection, the basal levels of pERK1 in the NAc and VTA and pERK2 in VTA were lower in the vehicle control group, relative to the Tat group. After repeated nicotine injection, pERK1 in NAc and VTA and pERK2 in VTA were increased in the vehicle group, but not in the Tat group. Moreover, repeated nicotine injections decreased pCREB in the PFC and VTA in the Tat group but not in the vehicle group. Thus, these findings indicate that the direct injection of Tat at the VTA may mediate CREB and ERK activity in response to nicotine-induced locomotor activity.

Effects of environmental enrichment on ERK1/2 phosphorylation in the rat prefrontal cortex following nicotine-induced sensitization or nicotine self-administration

Rats raised in an enriched condition (EC) exhibit alterations in the neurobiological and behavioral response to nicotine compared with rats reared in an impoverished condition (IC) or a standard condition (SC). The current study determined whether environmental enrichment differentially regulates extracellular signal‐regulated kinase1/2 (ERK1/2) activity in the prefrontal cortex in rats following nicotine sensitization or nicotine self‐administration. Under the saline control condition, EC rats displayed diminished baseline activity and greater sensitization to repeated administration of nicotine compared with IC and SC rats. After repeated saline injections, the basal levels of phosphorylated ERK1/2 (pERK1/2) were higher in EC compared with IC and SC rats, which was negatively correlated with their respective baseline activities. Repeated nicotine (0.35 mg/kg) injections induced pERK1/2 to similar levels in SC and IC rats; however, the induction of pERK1/2 in EC rats by nicotine was not significantly different from saline controls, owing to their high baseline. In the self‐administration paradigm, EC rats self‐administered less nicotine (0.03 mg/kg/infusion) relative to IC or SC rats on a fixed ratio‐1 schedule of reinforcement. Accordingly, no differences in pERK1/2 were found between EC and IC rats self‐administering saline, whereas nicotine self‐administration resulted in an increase in pERK1/2 in IC rats but not in EC rats. Furthermore, the levels of pERK1/2 in EC and IC rats were positively correlated with their respective total number of nicotine infusions. Thus, these findings suggest that environmental enrichment alters the basal and nicotine‐mediated pERK1/2, which may contribute to enrichment‐induced behavioral alterations in response to nicotine.

Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine

Background: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine. Methods: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity. Results: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and phosphorylated cAMP-response element-binding protein in the medial prefrontal cortex of impoverished condition but not enriched condition rats. Conclusion: These findings suggest that environmental enrichment, via upregulation of prefrontal microRNA-221 expression, suppresses the nicotine-induced activation of extracellular signal-regulated kinase and cAMP-response element-binding protein, which provides a potential mechanism underlying enhanced locomotor sensitivity to nicotine.