LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing.

Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

Catestatin, an endogenous chromogranin A-derived peptide, inhibits in vitro growth of Plasmodium falciparum.

Catestatin, an endogenous peptide derived from bovine chromogranin A, and its active domain cateslytin display powerful antimicrobial activities. We have tested the activities of catestatin and other related peptides on the growth of Plasmodium falciparum in vitro. Catestatin inhibits growth of the chloroquine-sensitive strain of P. falciparum 3D7, exhibiting 88% inhibition at 20 microM. A similar partial inhibition of parasite growth was observed for the chloroquine-resistant strain, 7G8 (64%,) and the multidrug-resistant strain, W2 (62%). In the presence of parasite-specific lactate dehydrogenase, a specific protein-protein interaction between catestatin and plasmepsin II precursor was demonstrated. In addition, catestatin partially inhibited the parasite-specific proteases plasmepsin in vitro. A specific interaction between catestatin and plasmepsins II and IV from P. falciparum and plasmepsin IV from the three remaining species of Plasmodium known to infect man was observed, suggesting a catestatin-induced reduction in availability of nutrients for protein synthesis in the parasite.

A single acute pharmacological dose of γ-hydroxybutyrate modifies multiple gene expression patterns in rat hippocampus and frontal cortex.

γ-Hydroxybutyrate (GHB) is a natural brain neuromodulator that has its own enzymatic machinery for synthesis and degradation, release, and transport systems and several receptors that belong to the G protein-coupled receptor (GPCR) family. Targeting of this system with exogenous GHB is used in therapy to induce sleep and anesthesia and to reduce alcohol withdrawal syndrome. GHB is also popular as a recreational drug for its anxiolytic and mild euphoric effects. However, in both cases, GHB must be administered at high doses in order to maintain GHB concentrations in brain of ∼800-1,000 µM. These high concentrations are thought to be necessary for interactions with low-affinity sites on GABA(B) receptor, but the molecular targets and cellular mechanisms modulated by GHB remain poorly characterized. Therefore, to provide new insights into the elucidation of GHB mechanisms of action and open new tracks for future investigations, we explored changes of GHB-induced transcriptomes in rat hippocampus and prefrontal cortex by using DNA microarray studies. We demonstrate that a single acute anesthetic dose of 1 g/kg GHB alters a large number of genes, 121 in hippocampus and 53 in prefrontal cortex; 16 genes were modified simultaneously in both brain regions. In terms of molecular functions, the majority of modified genes coded for proteins or nucleotide binding sites. In terms of Gene Ontology (GO) functional categories, the largest groups were involved in metabolic processing for hippocampal genes and in biological regulation for prefrontal cortex genes. The majority of genes modified in both structures were implicated in cell communication processes. Western blot and immunohistochemical studies carried out on eight selected proteins confirmed the microarray findings.

Inhibition of histone deacetylases in rats self-administering cocaine regulates lissencephaly gene-1 and reelin gene expression, as revealed by microarray technique.

Injection of the histone deacetylase inhibitor trichostatin A (TsA) to rats has been shown to decrease their motivation to self-administer cocaine. In the present study, we investigated alterations in gene expression patterns in the anterior cingulate cortex and nucleus accumbens of rats self-administering cocaine and treated with TsA. Using oligonucleotide microarrays, we identified 722 probe sets in the cortex and 136 probe sets in the nucleus accumbens that were differentially expressed between vehicle and TsA-treated rats that self-administered cocaine. Microarray data were validated by real-time PCR for seven genes. Using immunohistochemistry, we further investigated the expression of Lis1 and reelin genes, because (i) they were similarly regulated by TsA at the mRNA level; (ii) they belong to the same signal transduction pathway; (iii) mutations within both genes cause lissencephaly. Cocaine self-injection was sufficient to activate the two genes at both the mRNA and protein levels. TsA treatment was found to up-regulate both Lis1 and reelin protein expression in the cortex and to down-regulate it in the nucleus accumbens of rats self-administering cocaine. The data suggest that the two proteins contribute to establish neurobiological mechanisms underlying brain plasticity whereby TsA lowers the motivation for cocaine.

CDKL5 is a brain MeCP2 target gene regulated by DNA methylation.

Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long-term plasticity triggered by pharmacological agents.

Abnormal nociception and opiate sensitivity of STOP null mice exhibiting elevated levels of the endogenous alkaloid morphine.

BACKGROUND: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission. RESULTS: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice. CONCLUSIONS: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

[Molecular basis of HIV-1 latency - part I: physiology of HIV-1 latency]. / Un virus tapi dans l'ombre : les bases moléculaires de la latence du VIH-1 - Partie I : la physiologie de la latence du VIH-1.

The introduction of the highly active antiretroviral therapy (HAART) in 1996 has greatly extended survival and raised hopes for the eradication of HIV-1. Unfortunately, the optimism declined by revealing the existence of latent HIV-1 reservoirs in cells targeted by the virus. The long-lived HIV-1 reservoirs constitute a major obstacle to the eradication of HIV-1. Understanding the molecular mechanisms of virus latency is essential for efficient therapeutic intervention against the virus.

[Molecular basis of HIV-1 latency - Part II: HIV-1 reactivation and therapeutic implications]. / Un virus tapi dans l'ombre : les bases moléculaires de la latence du VIH-1 - Partie II : la réactivation de la latence du VIH-1 et ses implications thérapeutiques.

The latent HIV-1 reservoirs established early during infection present a major obstacle for virus eradication. Complete eradication of the virus from infected patients may require a purge of the reservoirs. Since the development of a HIV-1 vaccine is not achieved, and therefore remains a major challenge for the immunologists, future direction towards an effective curative therapy for HIV-1 infection will rely on the development of original therapeutic strategies which take into account latency, chronic replication and accessibility to tissue-sanctuary.

Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats.

Regulation of gene expression is known to contribute to the long-term adaptations taking place in response to drugs of abuse. Recent studies highlighted the regulation of gene transcription in neurons by chromatin remodeling, a process in which posttranslational modifications of histones play a major role. To test the involvement of epigenetic regulation on drug-reinforcing properties, we submitted rats to the cocaine operant self-administration paradigm. Using the fixed ratio 1 schedule, we found that the histone deacetylase (HDAC) inhibitors trichostatin A and phenylbutyrate dose-dependently reduced cocaine self-administration. Under the progressive ratio schedule, both trichostatin A and depudecin significantly reduced the breaking point, indicating that HDAC inhibition attenuated the motivation of rats for cocaine. Conversely, HDAC inhibition did not decrease self-administration for the natural reinforcer sucrose. This observation was correlated with measurements of HDAC activity in the frontal cortex, which was inhibited in response to cocaine, but not to sucrose self-administration. Control experiments showed that the decrease in the motivation for the drug was not attributable to a general motivational dysfunction because trichostatin A had no adverse effect on locomotion during the habituation session or on cocaine-induced hyperlocomotion. It was not attributable to anhedonia because the inhibitor had no effect on the sucrose preference test. In contrast, trichostatin A completely blocked the cocaine-induced behavioral sensitization. Together, the data show that epigenetic regulation of gene transcription in adult brain is able to influence a motivated behavior and suggest that HDAC inhibition may counteract the neural sensitization leading to drug dependence.

Characterization of human and bovine phosphatidylethanolamine-binding protein (PEBP/RKIP) interactions with morphine and morphine-glucuronides determined by noncovalent mass spectrometry.

BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP/RKIP), initially found to bind phosphatidylethanolamine (PE), has been shown to be associated with morphine derivatives. Our recent study on bovine primary chromaffin cells showed that inside secretory granules, PEBP is noncovalently associated to endogenous morphine-6-glucuronide (M6G), a highly analgesic morphine metabolite. During stress, M6G-PEBP complexes may be released into circulation to target peripheral opioid receptors. We now report the investigation of PEBP binding properties towards morphine and morphine analogs. MATERIAL/METHODS: Noncovalent electrospray ionization mass spectrometry (ESI-MS) was used to investigate bovine and human PEBP binding properties towards morphine and morphine-glucuronides. RESULTS: We describe for the first time that: (i) PEBP directly interacts with morphine glucuronides (M3G and M6G) but not with morphine, (ii) that the presence of a glucuronide group either on the 3rd or the 6th morphine's carbon does not affect these interactions, (iii) that M6G binds PEBP in a similar manner as the reference ligand PE and (iv) that PEBP displays a similar affinity for PE, M6G and M3G. CONCLUSIONS: Our results suggest that PEBP might protect M6G following its secretion into blood, leading to a longer half life. This study highlights the potentialities of ESI-MS to validate / invalidate the formation of protein: ligand noncovalent complexes when low affinity binders (i.e., compounds with affinities lower than 10(3) M(-1)) are concerned.

Xanthurenic acid distribution, transport, accumulation and release in the rat brain.

Tryptophan metabolism through the kynurenine pathway leads to several neuroactive compounds, including kynurenic and picolinic acids. Xanthurenic acid (Xa) has been generally considered as a substance with no physiological role but possessing toxic and apoptotic properties. In the present work, we present several findings which support a physiological role for endogenous Xa in synaptic signalling in brain. This substance is present in micromolar amounts in most regions of the rat brain with a heterogeneous distribution. An active vesicular synaptic process inhibited by bafilomycin and nigericin accumulates xanthurenate into pre-synaptic terminals. A neuronal transport, partially dependant on adenosine 5'-triphosphate (ATP), sodium and chloride ions exists in NCB-20 neurons which could participate in the clearance of extracellular xanthurenate. Both transports (neuronal and vesicular) are greatly enhanced by the presence of micromolar amounts of zinc ions. Finally, electrical in vivo stimulation of A10-induced Xa release in the extracellular spaces of the rat prefrontal cortex. This phenomenon is reproduced by veratrine, K+ ions and blocked by EGTA and tetrodotoxin. These results strongly argue for a role for Xa in neurotransmission/neuromodulation in the rat brain, thus providing the existence of specific Xa receptors.

Prognostic value of chromogranin A at admission in critically ill patients: a cohort study in a medical intensive care unit.

BACKGROUND: Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. The acute response to stress causes the release of catecholamines from the adrenal medulla accompanied by chromogranin A (CGA). To date, no study has evaluated the prognostic value of CGA in critically ill intensive care unit patients. METHODS: We conducted a prospective study of intensive care unit patients by measuring serum procalcitonin (PCT), C-reactive protein (CRP), and CGA at the time of admission. Univariate and multivariate analyses were performed to evaluate the ability of these biomarkers to predict mortality. RESULTS: In 120 consecutive patients, we found positive correlations between CGA and the following: CRP (r(2) = 0.216; P = 0.02), PCT (r(2) = 0.396; P < 0.001), Simplified Acute Physiologic Score II (SAPS II) (r(2) = 0.438; P < 0.001), and the Logistic Organ Dysfunction System (LODS) score (r(2) = 0.374; P < 0.001). Nonsurvivors had significantly higher CGA and PCT concentrations than survivors [median (interquartile range): 293.0 microg/L (163.5-699.5 microg/L) vs 86.0 microg/L (53.8-175.3 microg/L) for CGA, and 6.78 microg/L (2.39-22.92 microg/L) vs 0.54 microg/L (0.16-6.28 microg/L) for PCT; P < 0.001 for both comparisons]. In a multivariable linear regression analysis, creatinine (P < 0.001), age (P < 0.001), and SAPS II (P = 0.002) were the only significant independent variables predicting CGA concentration (r(2) = 0.352). A multivariate Cox regression analysis identified 3 independent factors predicting death: log-normalized CGA concentration [hazard ratio (HR), 7.248; 95% confidence interval (CI), 3.004-17.487], SAPS II (HR, 1.046; 95% CI, 1.026-1.067), and cardiogenic shock (HR, 3.920; 95% CI, 1.731-8.880). CONCLUSIONS: CGA is a strong and independent indicator of prognosis in critically ill nonsurgical patients.

Cloning and functional characterization of a gamma-hydroxybutyrate receptor identified in the human brain.

Two parent clones of a gamma-hydroxybutyrate (GHB) receptor, C12K32 and GHBh1, were isolated from a human frontal cortex cDNA library. The two clones differ by a deleted cytosine in C12K32. CHO cells transfected with either C12K32 or GHBh1 responded positively to submicromolar GHB stimulation. However, unlike C12K32, GHBh1 desensitizes rapidly on application of low concentrations of GHB. GHB receptor properties were then studied on C12K32 expressed in CHO cells. C12K32 bound GHB with a Kd of 114 nM and has no affinity for GABA or glutamate. GHB and NCS-382 displaced [3H]GHB with an IC50 of 53 +/- 8 and 120 +/- 18 nM, respectively. In patch-clamp experiments, GHB induced a dose-dependent response with an EC50 of 130 nM. This response was antagonized by NCS-382, was not reproduced by GABA, and was sensitive to the addition of GTP-gamma-S in the recording pipette. CHO cells transfected with C12K32 exhibited GTPgamma-35S binding with an EC50 of 462 nM for GHB and an IC50 of 2.9 microM for NCS-382. The present data led to the conclusion that both C12K32 and GHBh1 are two closely related isoforms of a human GHB receptor, GHBh1, that is described in the databank as the GPCR 172A.

COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells.

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415-5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1alpha, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region.

Transmembrane peptides as inhibitors of ErbB receptor signaling.

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.

Immunohistochemical localization of a GHB receptor-like protein isolated from rat brain.

Gamma-hydroxybutyrate (GHB) is a substance derived from the metabolism of GABA and is heterogeneously distributed in various regions of the brain. This compound possesses a neuromodulatory role on several types of synapses, particularly those using GABA as a neurotransmitter. At physiological concentrations, this effect of GHB is mediated via specific receptors that induce neuronal hyperpolarization and bind radioactive GHB with a specific distribution, ontogenesis, kinetics, and pharmacology. A membrane protein that possesses six to seven transmembrane domains and which binds and is activated by micromolar amounts of GHB was recently cloned from rat brain hippocampus. In order to study the regional and cellular distribution of this receptor in rat brain, we selected several specific peptides belonging to the extracellular domains of the receptor to be used as specific immunogens to raise polyclonal antibodies in the rabbit. Among the antisera obtained, one of them gave particularly good results in terms of specificity and reactivity at high dilution. Immunohistochemical analyses, both at the confocal and electron microscopic level, showed receptor protein distribution closely resembling the distribution of GHB high-affinity binding sites, except for cerebellum, where GHB receptor(s) of lower affinity exist(s). In all regions studied the GHB receptor-like protein labeling appears to be distributed specifically in neurons and not in glial cells. At the cellular level the antibody specifically labels dendrites, and no immunoreactivity was detected in presynaptic endings or in axons. Accordingly, electron microscopy reveals strong labeling of postsynaptic densities and of neuronal cytosol.

Characterization of natural vasostatin-containing peptides in rat heart.

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1-76) and CGA(1-113), respectively] have been reported to affect vessel motility and exert concentration-dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin-containing peptides in rat heart. Rat heart extracts were purified by RP-HPLC, and the resulting fractions analyzed for the presence of CGA N-terminal fragments using dot-blot analysis. CGA-immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N-terminal CGA-derived peptides [CGA(4-113), CGA(1-124), CGA(1-135) and CGA(1-199)] containing the vasostatin sequence were characterized. The following post-translational modifications of these fragments were identified: phosphorylation at Ser96, O-glycosylation (trisaccharide, NAcGal-Gal-NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA-derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.

Induction by cocaine of the serotonergic 5-HT3 receptor in rat cerebellum.

The expression of the 5-HT(3) receptor, a member of the serotonin receptor family, was examined in rat cerebellum of saline- or cocaine-treated rats. Both immunohistochemistry and Western blot analysis were used. We found that the expression of this serotonin receptor subtype was increased in the cerebellum of rats injected either acutely or repeatedly (1 injection/day for 10 days) with cocaine. The stimulation was more pronounced after a single injection than after a series of 10 injections. Surprisingly, the expression of the 5-HT(3) receptor was mainly localized in Bergmann glial cells, as assessed from the co-localization of the receptor with the glial cell marker glial fibrillary acidic protein (GFAP). Our data emphasize the importance of the 5-HT(3) receptor induction in the cerebellum as part of the neuroadaptations taking place in rat brain in response to the psychostimulant cocaine.

Activation of the cGMP pathway in dopaminergic structures reduces cocaine-induced EGR-1 expression and locomotor activity.

Nitric oxide (NO) and the C-type natriuretic peptide (CNP) exert their action on brain via the cGMP signaling pathway. NO, by activating soluble guanylyl cyclase, and CNP, by stimulating membrane-bound guanylyl cyclase, cause intracellular increases of cGMP, activating cGMP-dependent protein kinases (PKGs). We show here that injection of CNP into the rat ventral tegmental area strongly reduced cocaine-induced egr-1 expression in the nucleus accumbens in a dose-dependent manner. The effect of CNP was reversed by the previous injection of a selective PKG inhibitor, KT5823. Activation of PKG by 8-bromo-cGMP reduced, like CNP, cocaine-induced gene transcription in dopaminergic structures. To confirm the involvement of PKG, this was overexpressed in either the mesencephalon or the caudate-putamen. Using the polyethyleneimine delivery system, an active protein was expressed by injecting a plasmid vector containing the human PKG-Ialpha cDNA. PKG was overexpressed in dopaminergic and GABAergic neurons when the plasmid was injected in the ventral tegmental area, whereas overexpression was observed in medium spiny GABAergic neurons and in both cholinergic and GABAergic interneurons when the PKG vector was injected into the caudate-putamen. Activation of the overexpressed PKG reduced cocaine-induced egr-1 expression in dopaminergic structures and affected behavior (i.e., locomotor activity). These effects were again reversed by previous injection of the selective PKG inhibitor. The current data suggest that NO and the neuropeptide CNP are potential regulators of cocaine-related effects on behavior.