HDAC superfamily promoters acetylation is differentially regulated by modafinil and methamphetamine in the mouse medial prefrontal cortex.

Dysregulation of histone deacetylases (HDAC) has been proposed as a potential contributor to aberrant transcriptional profiles that can lead to changes in cognitive functions. It is known that METH negatively impacts the prefrontal cortex (PFC) leading to cognitive decline and addiction whereas modafinil enhances cognition and has a low abuse liability. We investigated if modafinil (90 mg/kg) and methamphetmine (METH) (1 mg/kg) may differentially influence the acetylation status of histones 3 and 4 (H3ac and H4ac) at proximal promoters of class I, II, III, and IV HDACs. We found that METH produced broader acetylation effects in comparison with modafinil in the medial PFC. For single dose, METH affected H4ac by increasing its acetylation at class I Hdac1 and class IIb Hdac10, decreasing it at class IIa Hdac4 and Hdac5. Modafinil increased H3ac and decreased H4ac of Hdac7. For mRNA, single-dose METH increased Hdac4 and modafinil increased Hdac7 expression. For repeated treatments (4 d after daily injections over 7 d), we found specific effects only for METH. We found that METH increased H4ac in class IIa Hdac4 and Hdac5 and decreased H3/H4ac at class I Hdac1, Hdac2, and Hdac8. At the mRNA level, repeated METH increased Hdac4 and decreased Hdac2. Class III and IV HDACs were only responsive to repeated treatments, where METH affected the H3/H4ac status of Sirt2, Sirt3, Sirt7, and Hdac11. Our results suggest that HDAC targets linked to the effects of modafinil and METH may be related to the cognitive-enhancing vs cognitive-impairing effects of these psychostimulants.

Cocaine alters the mouse testicular epigenome with direct impact on histone acetylation and DNA methylation marks.

RESEARCH QUESTION: Recent evidence suggests that cocaine administration in animal models can trigger non-genetic inheritance of addiction traits from father to offspring, affecting development and behaviour. Is chronic cocaine intake involved in alterations of epigenetic homeostasis in the testis? DESIGN: Epigenetic marks and mediators in testis and isolated germ cells of adult mice treated with cocaine (10 mg/kg) or vehicle (sterile saline solution) were evaluated in an intermittent binge protocol: three intraperitoneal injections, 1 h apart, one day on/off for 13 days, collecting tissue 24 h after the last binge administration (day 14). RESULTS: It was shown that chronic cocaine intake in mice disrupts testicular epigenetic homeostasis, increasing global methylated cytosine levels in DNA from germ cells and sperm. Cocaine also increased testicular and germ cell acetylated histone 3 and 4 and decreased expression of histone deacetylases HDAC1/2. Immunolocalization studies showed that HDAC1/2 and acetylated histone 3 and 4 proteins localize to meiotic germ cells. Analysis of mRNA expression in isolated germ cells shows decreased levels of Hdac1/2/8, Dnmt3b and Tet1 and increased levels of Dnmt3a gene expression after cocaine treatment. CONCLUSIONS: Cocaine intake is associated with testicular toxicity and significant reproductive function impairment. The results presented here broaden the basic knowledge of the impact of addictive stimulants on testicular pathophysiology, fertility and male reproductive health and imply that altered epigenetic homeostasis by cocaine may have potential consequences on future generations.

Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase.

This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj_PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj_PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj_PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn(2+), and [Formula: see text] , being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn(2+). Therefore in Lmj_PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj_PEPCK.

Acute Regulation of the Arousal-Enhancing Drugs Caffeine and Modafinil on Class IIa HDACs In Vivo and In Vitro: Focus on HDAC7.

Psychostimulant drugs, such as modafinil and caffeine, induce transcriptional alterations through the dysregulation of epigenetic mechanisms. We have previously demonstrated that acute modafinil administration is accompanied by multiple changes in the expression of histone deacetylases (HDACs) within the mouse medial prefrontal cortex (mPFC). Herein, we compared alterations in class IIa HDACs in the mouse mPFC and dorsal striatum (DS) after a single exposure to each psychostimulant. We treated male C57BL/6 mice with modafinil (90 mg/kg, i.p.), caffeine (10 mg/kg, i.p.), or vehicle and evaluated locomotor activity. Following, we examined hdac4, hdac5, and hdac7 mRNA expression using qRT-PCR and HDAC7, pHDAC7, and pHDACs4/5/7 using Western blot. Last, we explored generalized effects in N2a cell line using modafinil (100 µM and 1 mM) or caffeine (80 µM and 800 µM). Our results indicate that modafinil had greater effects on locomotor activity compared with caffeine. qRT-PCR experiments revealed that modafinil decreased hdac5 and hdac7 mRNA expression in the DS, while caffeine had no effects. In the mPFC, modafinil increased hdac7 mRNA expression, with no effects observed for caffeine. Western blot revealed that within the DS, modafinil induced increases in HDAC7, pHDAC7, and pHDACs4/5/7 protein expression, while, in the mPFC, caffeine induced decreases in HDAC7, pHDAC7, and pHDACs4/5/7 protein levels. In vitro studies revealed that modafinil increased hdac4, hdac5, and hdac7 mRNA levels in N2a, while caffeine only increased hdac5 at a higher dose. These findings support the notion that modafinil and caffeine exert distinct regulation of class IIa HDAC family members and that these transcriptional and translational consequences are region-specific.

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of ß3-Adrenergic Receptor.

G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although ß3-adrenergic receptor (ß3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce ß3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of ß3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human ß3AR presented a short-term desensitization of cAMP response stimulated by the ß3AR agonist, BRL37344, and not by forskolin. We found that ß3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased ß3AR desensitization. Consistently, stimulation of ß3AR increased interaction between GRK2 and Gαs subunit. Furthermore, in rat cardiomyocytes endogenously expressing ß3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the ß3AR in heart function and disease.

Participation of Gαi-Adenylate Cyclase and ERK1/2 in Mas Receptor Signaling Pathways.

The MasR receptor (MasR) is an orphan G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang-(1-7) protective axis of renin-angiotensin system. This receptor has been suggested to participate in several physiological processes including cardio- and reno-protection and regulation of the central nervous system function. Although the knowledge of the signaling mechanisms associated with MasR is essential for therapeutic purposes, these are still poorly understood. Accordingly, in the current study we aimed to characterize the signaling pathways triggered by the MasR. To do that, we measured cAMP and Ca2+ levels in both naïve and MasR transfected cells in basal conditions and upon incubation with putative MasR ligands. Besides, we evaluated activation of ERK1/2 by Ang-(1-7) in MasR transfected cells. Results indicated the existence of a high degree of MasR constitutive activity toward cAMP modulation. This effect was not mediated by the PDZ-binding motif of the MasR but by receptor coupling to Gαi-adenylyl cyclase signaling pathway. Incubation of MasR transfected cells with Ang-(1-7) or the synthetic ligand AVE 0991 amplified MasR negative modulation of cAMP levels. On the other hand, we provided evidence for lack of MasR-associated modulation of Ca2+ levels by Ang-(1-7). Finally, it was determined that the MasR attenuated Ang-(1-7)-induced ERK1/2 phosphorylation mediated by AT1R. We provided further characterization of MasR signaling mechanisms regarding its constitutive activity and response to putative ligands. This information could prove useful to better describe MasR physiological role and development of therapeutic agents that could modulate its action.

The effects of single-dose injections of modafinil and methamphetamine on epigenetic and functional markers in the mouse medial prefrontal cortex: potential role of dopamine receptors.

METH use causes neuroadaptations that negatively impact the prefrontal cortex (PFC) leading to addiction and associated cognitive decline in animals and humans. In contrast, modafinil enhances cognition by increasing PFC function. Accumulated evidence indicates that psychostimulant drugs, including modafinil and METH, regulate gene expression via epigenetic modifications. In this study, we measured the effects of single-dose injections of modafinil and METH on the protein levels of acetylated histone H3 (H3ac) and H4ac, deacetylases HDAC1 and HDAC2, and of the NMDA subunit GluN1 in the medial PFC (mPFC) of mice euthanized 1 h after drug administration. To test if dopamine (DA) receptors (DRs) participate in the biochemical effects of the two drugs, we injected the D1Rs antagonist, SCH23390, or the D2Rs antagonist, raclopride, 30 min before administration of METH and modafinil. We evaluated each drug effect on glutamate synaptic transmission in D1R-expressing layer V pyramidal neurons. We also measured the enrichment of H3ac and H4ac at the promoters of several genes including DA, NE, orexin, histamine, and glutamate receptors, and their mRNA expression, since they are responsive to chronic modafinil and METH treatment. Acute modafinil and METH injections caused similar effects on total histone acetylation, increasing H3ac and decreasing H4ac, and they also increased HDAC1, HDAC2 and GluN1 protein levels in the mouse mPFC. In addition, the effects of the drugs were prevented by pre-treatment with D1Rs and D2Rs antagonists. Specifically, the changes in H4ac, HDAC2, and GluN1 were responsive to SCH23390, whereas those of H3ac and GluN1 were responsive to raclopride. Whole-cell patch clamp in transgenic BAC-Drd1a-tdTomato mice showed that METH, but not modafinil, induced paired-pulse facilitation of EPSCs, suggesting reduced presynaptic probability of glutamate release onto layer V pyramidal neurons. Analysis of histone 3/4 enrichment at specific promoters revealed: i) distinct effects of the drugs on histone 3 acetylation, with modafinil increasing H3ac at Drd1 and Adra1b promoters, but METH increasing H3ac at Adra1a; ii) distinct effects on histone 4 acetylation enrichment, with modafinil increasing H4ac at the Drd2 promoter and decreasing it at Hrh1, but METH increasing H4ac at Drd1; iii) comparable effects of both psychostimulants, increasing H3ac at Drd2, Hcrtr1, and Hrh1 promoters, decreasing H3ac at Hrh3, increasing H4ac at Hcrtr1, and decreasing H4ac at Hcrtr2, Hrh3, and Grin1 promoters. Interestingly, only METH altered mRNA levels of genes with altered histone acetylation status, inducing increased expression of Drd1a, Adra1a, Hcrtr1, and Hrh1, and decreasing Grin1. Our study suggests that although acute METH and modafinil can both increase DA neurotransmission in the mPFC, there are similar and contrasting epigenetic and transcriptional consequences that may account for their divergent clinical effects.

Involvement of histamine H1 and H2 receptor inverse agonists in receptor's crossregulation.

Histamine [2-(4-Imidazolyl)-ethylamine] modulates different biological processes, through histamine H1 and H2 receptors, and their respective blockers are widely used in treating allergic and gastric acid-related disorders. Histamine H1 and H2 receptor crossdesensitization and cointernalization induced by its agonists have been previously described. In this study, we show how this crosstalk determines the response to histamine H1 and H2 receptor inverse agonists and how histamine H1 and H2 receptor inverse agonists interfere with the other receptor's response to agonists. By desensitization assays we demonstrate that histamine H1 and H2 receptor inverse agonists induce a crossregulation between both receptors. In this sense, the histamine H1 receptor inverse agonists desensitize the cAMP response to amthamine, a histamine H2 receptor agonist. In turn, histamine H2 receptor inverse agonists interfere with histamine H1 receptor signaling. We also determine that the crossdesensitization induced by histamine H1 or H2 receptor agonists alters the histamine inverse agonists receptor response: activation of histamine H1 receptor affects cAMP response induced by histamine H2 receptor inverse agonists, whereas histamine H2 receptor agonist induces a negative regulation on the anti-inflammatory response of histamine H1 receptor inverse agonists. Binding studies revealed that histamine H1 and H2 receptors cointernalize after stimulus with histamine receptor inverse agonists. In addition, the inhibition of the internalization process prevents receptor crossregulation. Our study provides new insights in the mechanisms of action of histamine H1 and H2 receptors that explain the effect of histamine H1 and H2 receptor inverse agonists and opens up new venues for novel therapeutic applications.

Molecular and functional characterization of two malic enzymes from Leishmania parasites.

Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites.

Repeated methamphetamine and modafinil induce differential cognitive effects and specific histone acetylation and DNA methylation profiles in the mouse medial prefrontal cortex.

Methamphetamine (METH) and modafinil are psychostimulants with different long-term cognitive profiles: METH is addictive and leads to cognitive decline, whereas modafinil has little abuse liability and is a cognitive enhancer. Increasing evidence implicates epigenetic mechanisms of gene regulation behind the lasting changes that drugs of abuse and other psychotropic compounds induce in the brain, like the control of gene expression by histones 3 and 4 tails acetylation (H3ac and H4ac) and DNA cytosine methylation (5-mC). Mice were treated with a seven-day repeated METH, modafinil or vehicle protocol and evaluated in the novel object recognition (NOR) test or sacrificed 4days after last injection for molecular assays. We evaluated total H3ac, H4ac and 5-mC levels in the medial prefrontal cortex (mPFC), H3ac and H4ac promotor enrichment (ChIP) and mRNA expression (RT-PCR) of neurotransmitter systems involved in arousal, wakefulness and cognitive control, like dopaminergic (Drd1 and Drd2), α-adrenergic (Adra1a and Adra1b), orexinergic (Hcrtr1 and Hcrtr2), histaminergic (Hrh1 and Hrh3) and glutamatergic (AMPA Gria1 and NMDA Grin1) receptors. Repeated METH and modafinil treatment elicited different cognitive outcomes in the NOR test, where modafinil-treated mice performed as controls and METH-treated mice showed impaired recognition memory. METH-treated mice also showed i) decreased levels of total H3ac and H4ac, and increased levels of 5-mC, ii) decreased H3ac enrichment at promoters of Drd2, Hcrtr1/2, Hrh1 and Grin1, and increased H4ac enrichment at Drd1, Hrh1 and Grin1, iii) increased mRNA of Drd1a, Grin1 and Gria1. Modafinil-treated mice shared none of these effects and showed increased H3ac enrichment and mRNA expression at Adra1b. Modafinil and METH showed similar effects linked to decreased H3ac in Hrh3, increased H4ac in Hcrtr1, and decreased mRNA expression of Hcrtr2. The specific METH-induced epigenetic and transcriptional changes described here may be related to the long-term cognitive decline effects of the drug and its detrimental effects on mPFC function. The lack of similar epigenetic effects of chronic modafinil administration supports this notion.

Cocaine and Caffeine Effects on the Conditioned Place Preference Test: Concomitant Changes on Early Genes within the Mouse Prefrontal Cortex and Nucleus Accumbens.

Caffeine is the world's most popular psychostimulant and is frequently used as an active adulterant in many illicit drugs including cocaine. Previous studies have shown that caffeine can potentiate the stimulant effects of cocaine and cocaine-induced drug seeking behavior. However, little is known about the effects of this drug combination on reward-related learning, a key process in the maintenance of addiction and vulnerability to relapse. The goal of the present study was thus to determine caffeine and cocaine combined effects on the Conditioned Place Preference (CPP) test and to determine potential differential mRNA expression in the Nucleus Accumbens (NAc) and medial prefrontal cortex (mPFC) of immediate-early genes (IEGs) as well as dopamine and adenosine receptor subunits. Mice were treated with caffeine (5 mg/kg, CAF), cocaine (10 mg/kg, COC), or their combination (caffeine 5 mg/kg + cocaine 10 mg/kg, CAF-COC) and trained in the CPP test or treated with repeated injections inside the home cage. NAc and mPFC tissues were dissected immediately after the CPP test, after a single conditioning session or following psychostimulant injection in the home cage for mRNA expression analysis. CAF-COC induced a marked change of preference to the drug conditioned side of the CPP and a significant increase in locomotion compared to COC. Gene expression analysis after CPP test revealed specific up-regulation in the CAF-COC group of Drd1a, cFos, and FosB in the NAc, and cFos, Egr1, and Npas4 in the mPFC. Importantly, none of these changes were observed when animals received same treatments in their home cage. With a single conditioning session, we found similar effects in both CAF and CAF-COC groups: increased Drd1a and decreased cFos in the NAc, and increased expression of Drd1a and Drd2, in the mPFC. Interestingly, we found that cFos and Npas4 gene expression were increased only in the mPFC of the CAF-COC. Our study provides evidence that caffeine acting as an adulterant could potentiate reward-associated memories elicited by cocaine. This is associated with specific changes in IEGs expression that were observed almost exclusively in mice that received the combination of both psychostimulants in the context of CPP memory encoding and retrieval. Our results highlight the potential relevance of caffeine in the maintenance of cocaine addiction which might be mediated by modifying neural plasticity mechanisms that strengthen learning of the association between drug and environment.