Dominique Aunis

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.

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 antimicrobial peptides derived from chromogranin/secretogranin family, new actors of innate immunity

Chromogranins/secretogranins are members of the granin family present in secretory vesicles of nervous, endocrine and immune cells. In chromaffin cells, activation of nicotinic cholinergic receptors induces the release, with catecholamines, of bioactive peptides resulting from a natural processing. During the past decade, our laboratory has characterized new antimicrobial chromogranin-derived peptides in the secretions of stimulated bovine chromaffin cells. They act at the micromolar range against bacteria, fungi, yeasts, and are non-toxic for the mammalian cells. They are recovered in several biological fluids involved in defence mechanisms (human serum, neutrophil secretions and saliva). These new antimicrobial peptides demonstrate the major role of the adrenal medulla in innate immunity. In this review we focus on the antimicrobial peptides derived from human and bovine chromogranin A (CGA), chromogranin B (CGB) and secretogranin II (SGII) emphasizing their direct action against pathogens and their effects on immune cells.

Immunohistochemical Expression of Chromogranins A and B, Prohormone Convertases 2 and 3, and Amidating Enzyme in Carcinoid Tumors and Pancreatic Endocrine Tumors

Although chromogranin A (CgA) is widely distributed in neuroendocrine tumors, the distribution of chromogranin B (CgB) has not been elucidated. Hormones produced by tumors are sometimes prohormones and not necessarily bioactive hormones. Prohormones have to be processed into bioactive peptides by prohormone convertases (PCs), and some of them have to be amidated by peptidylglycine α-amidating monooxygenase (PGM). Whether PCs and PGM are present or not in tumors may explain why some tumors are functioning and some are nonfunctioning. We investigated 45 carcinoids and 16 pancreatic endocrine tumors. Of the carcinoids, CgA was expressed in most of the tumors, except for the rectal and ovarian carcinoids, which expressed CgB strongly. The expressions of PC2, PC3, and PGM were 31%, 100%, and 87%, respectively. In the pancreatic tumors, CgA was expressed in all tumors, whereas CgB was not expressed in any tumor. The expressions of PC2, PC3, and PGM were 63%, 88%, and 63%, respectively. PC3 was expressed in all of the functioning tumors but not in two of the four nonfunctioning tumors. PC2 and PGM were not expressed in three of the four nonfunctioning tumors. In conclusion, expression of CgA and CgB was different depending on the tumor location. High frequency of PCs and PGM may explain why even nonfunctioning tumors produce some inconspicuous peptides.

Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

Background Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. Methodology The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. μ opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. Principal Findings We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca2+. LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca2+. LPS treatment increased μ opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. Conclusions Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.

BOVINE ADRENAL MEDULLARY DOPAMINE‐β‐HYDROXYLASE: STUDIES ON INTERACTION WITH CONCANAVALIN A

Bovine adrenal medullary dopamine‐β‐hydroxylase binds with concanavalin A and forms an enzymically active precipitate. The formation of the insoluble complex is pH‐dependent and can be inhibited by α‐methyl‐D‐mannoside, D‐mannose and D‐glucose. The insoluble complex can be dissociated into two species with α‐methyl‐D‐mannoside. From the results, it appears that the interaction between dopamine‐β‐hydroxylase and concanavalin A is due to the carbohydrate moiety of dopamine‐β‐hydroxylase. This property was used to purify the enzyme from a soluble lysate of chromaffin granules. Of all the proteins contained in the soluble lysate, dopamine‐β‐hydroxylase was the only one to be retained on a column of concanavalin A covalently bound to Sepharose 4B. The preparation of pure dopamine‐β‐hydroxylase exhibits a very high specific activity of 320 μmol of octopamine formed per 30 min per mg of protein.

Gangliosides and phospholipids of the membranes from bovine adrenal medullary chromaffin granules.

The lipid and ganglioside compositions of membranes of chromaffin granules isolated from bovine adrenal medulla have been investigated. The detailed lipid analysis revealed the presence of high levels of lysophosphatidylcholine, in agreement with previous studies, but also of sphingomyelin and plasmalogens. From these membranes, gangliosides have been extracted and separated by thin-layer chromatography and analysed. 95% of the total recovered gangliosides were hematosides (GM3), which migrated as three major species. Sugar analyses have been performed, as well as the fatty acid compositions. The three hematoside gangliosides appeared to differ on the basis of their fatty acid composition. Compared with the brain, chromaffin granule membranes showed a simple ganglioside composition, thus offering a good model for the study of the metabolism and the role of gangliosides. The simple ganglioside composition of chromaffin granule membranes has allowed us to state that there are 60 mol phospholipid and 30 mol cholesterol per mol ganglioside.

Bovine adrenal medullary chromogranin A: Studies on the structure and further evidence for identity with dopamine-β-hydroxylase subunit

1. Chromogranin A was purified by the use of polyacrylamide gel electrophoresis. The amino acid composition of chromogranin A appeared to be nearly identical to that reported by other investigators and, moreover, was confirmed to be similar to that of dopamine beta-hydroxylase. 2. Dansyl-end group analysis revealed the presence of leucine as the only amino-terminal residue and quantitative estimations showed the presence of two leucine residues per molecule of 77 000 molecular weight. 3. Tryptic and CNBr patterns were obtained. Data are in good agreement with the concept of two nearly identical polypeptide chains per chromogranin A molecule of mol. wt 77 000. Patterns were compared with those obtained in parallel dopamine beta-hydroxylase and support the idea that chromogranin A and the dopamine beta-hydroxylase subunit are identical. Digestion with leucine amino peptidase gave further additional evidence for this suggestion. 4. Chromogranin A appeared to be free of carbohydrates. No cross-reaction was detected between chromogranin A and rabbit antibody against bovine adrenal dopamine beta-hydroxylase.

Inhibition of adrenal dopamine-β-hydroxylase by 6-hydroxy dopamine

Abstract Incubation of 6-hydroxydopamine with highly purified bovine adrenal dopamine-β-hydroxylase resulted in a complete inhibition of enzyme activity. When reciprocal rates were plotted vs reciprocal tyramine concentration or reciprocal ascorbate concentration, the inhibition appeared to be of the non-competitive type. The interaction between the inhibitor and the enzyme molecule is strong and irreversible. The addition of catalase protected all systems only when catalase was added during the preincubation time. The data show that the inhibitor is not 6-hydroxydopamine but hydrogen peroxides generated from 6-hydroxydopamine.

Osmotic fragility of chromaffin granules prepared under isoosmotic or hyperosmotic conditions and localization of acetylcholinesterase

In chromaffin cells of the adrenal medulla, catecholamines are stored in secretory granules. Different methods have been described to purify chromaffin granules. In the present study, storage granules were prepared using isoosmotic self-generating Percoll gradients or hyperosmotic sucrose gradients, and a comparison of their physical properties in response to osmotic changes was made. Catecholamines, dopamine beta-hydroxylase activity and protein were detected both in the external medium and in the granule fraction according to the medium osmolality. Suspension turbidity was used as a measure of organelle integrity. Acetylcholinesterase activity was found to be associated with both isoosmotically and hyperosomotically prepared granules. The total acetylcholinesterase activity was determined after adding Triton X-100 to the assay medium. When adrenal medullary tissue was homogenized in buffers containing echothiopate, an inhibitor of acetylcholinesterase, only 15-20% of enzyme activity was inhibited, excluding the possibility that main granule acetylcholinesterase could be due to contamination by plasma membrane fragments, endoplasmic reticulum and Golgi membranes. When granules were suspended in hypoosmotic buffers, a soluble acetylcholinesterase form was released into the external medium, while an insoluble acetylcholinesterase form was still found associated with the membrane fraction. Soluble acetylcholinesterase was found to be released differently than soluble dopamine beta-hydroxylase, indicating that acetylcholinesterase may be associated with a more osmotically resistant granule population.