Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFß-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (ß) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 ß-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.

Histone deacetylase inhibition decreases preference without affecting aversion for nicotine.

Epigenetic mechanisms have recently been shown to be involved in the long-term effects of drugs of abuse. A well described epigenetic mechanism modulating transcriptional activity consists in the binding to DNA of methyl-CpG binding proteins, such as MeCP2, recruiting histone deacetylases (HDACs). Nicotine causes long-term changes in the brain, but little is known concerning the mechanisms involved in nicotine-preference. Using a nicotine-conditioned place preference protocol, we demonstrate here that the histone deacetylase inhibitor phenylbutyrate was able to dramatically reduce the preference for nicotine, without altering the aversive properties of the drug. We measured immunohistochemically the acetylation of lysine-9 of histone H3, and the expression of phosphorylated cAMP-response element-binding protein, HDAC2 and methyl-CpG-binding protein 2 in the striatum and prefrontal cortex of rats displaying nicotine-preference or aversion and treated with phenylbutyrate. We show that, at the dose administered, the inhibitor was effective in inhibiting HDAC activity. The data suggest that phosphorylated cAMP-response element-binding protein participates in the establishment of conditioned place preference, but not in the reduction of nicotine-preference in response to phenylbutyrate. Moreover, striatal expression of HDAC2 in response to phenylbutyrate mirrored the behavioral effects of the inhibitor, suggesting that HDAC2 is involved in promoting synaptic plasticity underlying the preference for nicotine.

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.

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.

Author Correction: Loss of the adaptor protein ShcA in endothelial cells protects against monocyte macrophage adhesion, LDL-oxydation, and atherosclerotic lesion formation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Loss of the adaptor protein ShcA in endothelial cells protects against monocyte macrophage adhesion, LDL-oxydation, and atherosclerotic lesion formation.

ShcA is an adaptor protein that binds to the cytoplasmic tail of receptor tyrosine kinases and of the Low Density Lipoprotein-related receptor 1 (LRP1), a trans-membrane receptor that protects against atherosclerosis. Here, we examined the role of endothelial ShcA in atherosclerotic lesion formation. We found that atherosclerosis progression was markedly attenuated in mice deleted for ShcA in endothelial cells, that macrophage content was reduced at the sites of lesions, and that adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) were severely reduced. Our data indicate that transcriptional regulation of ShcA by the zinc-finger E-box-binding homeobox 1 (ZEB1) and the Hippo pathway effector YAP, promotes ICAM-1 expression independently of p-NF-κB, the primary driver of adhesion molecules expressions. In addition, ShcA suppresses endothelial Akt and nitric oxide synthase (eNOS) expressions. Thus, through down regulation of eNOS and ZEB1-mediated ICAM-1 up regulation, endothelial ShcA promotes monocyte-macrophage adhesion and atherosclerotic lesion formation. Reducing ShcA expression in endothelial cells may represent an obvious therapeutic approach to prevent atherosclerosis.

Cocaine self-administration alters the expression of chromatin-remodelling proteins; modulation by histone deacetylase inhibition.

Injection of the histone deacetylases inhibitor trichostatin A to rats has been shown to decrease the reinforcing properties of cocaine. In the present study, we investigated alterations in gene expression patterns in the anterior cingulate cortex, caudate-putamen and nucleus accumbens of rats self-administering cocaine and treated with trichostatin A. As recent studies highlighted the importance of chromatin remodelling in the regulation of gene transcription in neurons, we studied the expression of Mecp2 and of several histone deacetylases. Cocaine self-administration was accompanied by an increased synthesis of Mecp2, HDAC2 and HDAC11 and by a decreased nuclear localization of HDAC5 and of the phospho-form of HDAC5, suggesting a nuclear export of this protein in response to the drug. The latter mechanism was further addressed by the demonstration of an enhanced expression of MEF2C transcription factor. Among the genes we examined, treatment with trichostatin A before each cocaine self-administration session was found to mostly affect Mecp2 and HDAC11 expression. A correlation was found between the modification of Mecp2 and MEF2C gene expression and the reinforcing property of cocaine. The two factors known to regulate gene transcription are likely to play a role in the neurobiological mechanism underlying a decrease in the reinforcing properties of cocaine.

The inhibition of histone deacetylases reduces the reinstatement of cocaine-seeking behavior in rats.

Drug addiction is a chronic brain disease characterized by a persistent risk of relapse, even after a long period of abstinence. A current hypothesis states that relapse results from lasting neuroadaptations that are induced in response to repeated drug administration. The adaptations require gene expression, some of which being under the control of stable epigenetic regulations. We have previously demonstrated that pretreatment with histone deacetylase (HDAC) inhibitors reduces the cocaine reinforcing properties as well as the motivation of rats for cocaine. We show here that the same HDAC inhibitors, trichostatin A and phenylbutyrate, significantly reduced the cocaine-seeking behavior induced by the combination of a cocaine injection together with the exposure to a light cue previously associated with cocaine taking. Reinstatement of drug-seeking behavior was carried out after a 3-week withdrawal period, which came after ten daily sessions of cocaine intravenous self-administration. Our results suggest that pharmacological treatment aimed at modulating epigenetic regulation, and particularly treatment that would inhibit HDAC activity, could reduce the risk of relapse, a major drawback in the treatment of drug addiction.

The HDAC Inhibitor Phenylbutyrate Reverses Effects of Neonatal Ventral Hippocampal Lesion in Rats.

Recent evidence suggests that epigenetic mechanisms play a role in psychiatric diseases. In this study, we considered rats with neonatal ventral hippocampal lesions (NVHL) that are currently used for modeling neurodevelopmental aspects of schizophrenia. Contribution of epigenetic regulation to the effects of the lesion was investigated, using a histone deacetylase (HDAC) inhibitor. Lesioned or sham-operated rats were treated with the general HDAC inhibitor phenylbutyrate, which was injected daily from the day after surgery until adulthood. Changes in the volume of the lesion were monitored by magnetic resonance imaging (MRI). Anxiety was analyzed in the Plus Maze Test. Hypersensitivity of the dopaminergic system was evaluated by measuring the locomotor response to apomorphine. An associative conditioning test rewarded with food was used to evaluate learning abilities. The volume of the lesions expanded long after surgery, independently of the treatment, as assessed by MRI. Removal of the ventral hippocampus reduced anxiety, and this remained unchanged when animals were treated with phenylbutyrate. In contrast, NVHL rats' hypersensitivity to apomorphine and deterioration of the associative learning were reduced by the treatment. Global HDAC activity, which was increased in the prefrontal cortex of lesioned non-treated rats, was found to be reversed by HDAC inhibition. The study provides evidence that chromatin remodeling may be useful for limiting behavioral consequences due to lesioning of the ventral hippocampus at an early age. This represents a novel approach for treating disorders resulting from insults occurring during brain development.