Véronique Kemmel

Pharmacological doses of gamma-hydroxybutyrate (GHB) potentiate histone acetylation in the rat brain by histone deacetylase inhibition.

Several small chain fatty acids, including butyrate, valproate, phenylbutyrate and its derivatives, inhibit several HDAC activities in the brain at a several hundred micromolar concentration. Gamma-hydroxy-butyrate (GHB), a natural compound found in the brain originating from the metabolism of GABA, is structurally related to these fatty acids. The average physiological tissue concentration of GHB in the brain is below 50 microM, but when GHB is administered or absorbed for therapeutic or recreative purposes, its concentration reaches several hundred micromolars. In the present scenario, we demonstrate that pharmacological concentrations of GHB significantly induce brain histone H3 acetylation with a heterogeneous distribution in the brain and reduce in vitro HDAC activity. The degree of HDAC inhibition was also different according to the region of the brain considered. Taking into account the multiple physiological and functional roles attributed to the modification of histone acetylation and its consequences at the level of gene expression, we propose that part of the therapeutic or toxic effects of high concentrations of GHB in the brain after therapeutic administration of the drug could be partly due to GHB-induced epigenetic factors. In addition, we hypothesize that GHB, being naturally synthesized in the cytosolic compartment of certain neurons, could penetrate into the nuclei and may reach sufficient levels that could significantly modulate histone acetylation and may participate in the epigenetic modification of gene expression.

Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca2+ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.

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.

Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death

Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.

Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons.

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations <50 microM. Above this dose, GHB effects were inactivated. In addition, at GHB concentrations exceeding 50 microM, GTP-gammaS binding was also reduced, confirming the desensitization of GHB receptor(s). Taken together, these results support the existence in NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes.

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.

Skeletal muscle ischemia-reperfusion injury and cyclosporine A in the aging rat.

Old patients exhibit muscle impairments and increased perioperative risk during vascular surgery procedures. Although aging generally impairs protective mechanisms, data are lacking concerning skeletal muscle in elderly. We tested whether cyclosporine A (CsA), which protects skeletal muscle from ischemia–reperfusion (IR) in young rats, might reduce skeletal muscle mitochondrial dysfunction and oxidative stress in aging rats submitted to hindlimb IR. Wistar rats aged 71–73 weeks were randomized to IR (3 h unilateral tourniquet application and 2 h reperfusion) or IR + CsA (10 mg/kg cyclosporine IV before reperfusion). Maximal oxidative capacity (VMax), acceptor control ratio (ACR), and relative contribution of the mitochondrial respiratory chain complexes II, III, IV (VSucc), and IV (VTMPD/Asc), together with calcium retention capacity (CRC) a marker of apoptosis, and tissue reactive oxygen species (ROS) production were determined in gastrocnemius muscles from both hindlimbs. Compared to the nonischemic hindlimb, IR significantly reduced mitochondrial coupling, VMax (from 7.34 ± 1.50 to 2.87 ± 1.22 μMO2/min/g; P < 0.05; −70%), and VSucc (from 6.14 ± 1.07 to 3.82 ± 0.83 μMO2/min/g; P < 0.05; −42%) but not VTMPD/Asc. IR also decreased the CRC from 15.58 ± 3.85 to 6.19 ± 0.86 μMCa2+/min/g; P < 0.05; −42%). These alterations were not corrected by CsA (−77%, −49%, and −32% after IR for VMax, VSucc, and CRC, respectively). Further, CsA significantly increased ROS production in both hindlimbs (P < 0.05; +73%). In old rats, hindlimb IR impairs skeletal muscle mitochondrial function and increases oxidative stress. Cyclosporine A did not show protective effects.

Evidence for effective structure-based neuromodulatory effects of new analogues of neurosteroid allopregnanolone

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic‐pituitary‐adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP‐based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell‐proliferation‐promoting effects, hamper the development of selective/targeted therapies. For example, although AP‐induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP‐evoked cell‐proliferation is contraindicated for steroid‐sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12‐oxo‐epi‐AP), BR297 (O‐allyl‐epi‐AP), BR351 (O‐allyl‐AP) and BR338 (12‐oxo‐AP). First, because AP is well‐known as allosteric modulator of GABAA receptors (GABAA‐R), we used the electrophysiological patch‐clamp technique to determine the structure‐activity relationship of our ANS on GABAA‐activated current in NCB20 cells expressing functional GABAA‐R. We found that the addition of 12‐oxo‐group did not significantly change the respective positive or negative allosteric effects of 3α‐AP or 3β‐(epi)‐AP analogues. Importantly, substitution of the 3α‐hydroxyl‐group by 3α‐O‐allyl highly modified the ANS activities. Unlike AP, BR351 induced a long‐lasting desensitisation/inhibition of GABAA‐R. Interestingly, replacement of the 3β‐hydroxyl by 3β‐O‐allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH‐SY5Y neuronal cell viability/proliferation using MTT‐reduction assays. Different dose‐response curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell‐proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress‐induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH‐SY5Y cells against oxidative stress‐induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell‐proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid‐based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

Neurophysiological responses to unpleasant stimuli (acute electrical stimulations and emotional pictures) are increased in patients with schizophrenia

Patients with schizophrenia have often been described as insensitive to nociceptive signals, but objective evidence is sparse. We address this question by combining subjective behavioral and objective neurochemical and neurophysiological measures. The present study involved 21 stabilized and mildly symptomatic patients with schizophrenia and 21 control subjects. We applied electrical stimulations below the pain threshold and assessed sensations of pain and unpleasantness with rating scales, and Somatosensory Evoked Potentials (SEPs/EEG). We also measured attention, two neurochemical stress indices (ACTH/cortisol), and subjective VEPs/EEG responses to visual emotional stimuli. Our results revealed that, subjectively, patients’ evaluations do not differ from controls. However, the amplitude of EEG evoked potentials was greater in patients than controls as early as 50 ms after electrical stimulations and beyond one second after visual processing of emotional pictures. Such responses could not be linked to the stress induced by the stimulations, since stress hormone levels were stable. Nor was there a difference between patients and controls in respect of attention performance and tactile sensitivity. Taken together, all indices measured in patients in our study were either heightened or equivalent relative to healthy volunteers.

Protective effect of 4-Phenylbutyrate against proteolipid protein mutation-induced endoplasmic reticulum stress and oligodendroglial cell death

ABSTRACT Proteolipid protein (PLP) mutation causes oligodendrocyte degeneration and myelin disorders including Pelizaeus‐Merzbacher Disease (PMD). As the pathophysiological mechanisms involved in PMD are poorly known, the development of therapies remains difficult. To elucidate the pathogenic pathways, an immortalized oligodendroglial cell line (158JP) expressing PLP mutation has been generated. Previous investigations revealed that 158JP oligodendrocytes exhibit several abnormalities including aberrant PLP insertion into the plasma membrane, cAMP, plasmalogen and cell cycle deficits. However, further clarifications of abnormal PLP‐induced oligodendrocyte degeneration are required in order to identify relevant mechanisms to target for efficient protection against oligodendrocyte death. Because PLP overexpression may lead to its accumulation inside the endoplasmic reticulum (ER) and cause ER‐stress, we explored whether ER‐stress may pivotally determine 158JP cell survival/death. Viability assays, RT‐qPCR, western blot and flow cytometry were combined to compare cell survival, ER‐stress and apoptotic markers in 158JP and control (158N) oligodendrocytes. We observed a significant decreased viability/survival of 158JP compared to 158N cells. Consistently, ER‐stress markers (BiP, caspase‐12) increased in 158JP (+30%) compared to the controls. mRNA and protein ratios of apoptotic modulators (Bax/Bcl2) are higher in 158JP oligodendrocytes which are also more vulnerable than 158N cells to tunicamycin‐induced ER‐stress. Interestingly, 4‐Phenylbutyrate (ER‐stress inhibitor), which decreased ER‐stress and apoptotic markers in 158JP cells, significantly increased their survival. Our results, which show a direct link between the viability and endogenous levels of ER‐stress and apoptotic markers in 158JP cells, also suggest that 4‐Phenylbutyrate‐based strategy may contribute to develop effective strategies against oligodendrocyte dysfunctions/death and myelin disorders. Highlights158JP oligodendrocytes harboring PLP mutation are less viable than normal 158N cells.Basal levels of ER stress and apoptosis markers are higher in 158JP than 158N cells.158JP cells are more vulnerable to tunicamycin‐evoked ER‐stress than 158N cells.ER stress pivotally determines PLP mutation‐induced 158JP oligodendrocyte death.4‐PBA which reduces ER‐stress and apoptotic levels protects 158JP cells against death.