Biomedical subjects

Joël Bockaert

Publications and source records attributed to Joël Bockaert.

At least 19 recordsLinked to original sources

PubMed2006

Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability.

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation.

Serotonin (5-hydroxytryptamine, 5-HT) is, without doubt, the neurotransmitter for which the number of receptors is the highest. Fifteen genes encoding functional 5-HT receptors have been cloned in mammalian brain. 5-HT(3) receptors are ionotropic receptors, whereas all the others are metabotropic G-protein-coupled receptors (GPCRs). 5-HT receptor diversity is further increased by post-genomic modifications, such as alternative splicing (up to 10 splice variants for the 5-HT(4) receptor) or by mRNA editing in the case of 5-HT(2C) receptors. The cellular and behavioral implications of 5-HT(2C) receptor editing are of great physiological importance. Signaling of 5-HT receptors involves a great variety of pathways, but only some of these have been demonstrated in neurons. The classical view of neurotransmitter receptors localized within the synaptic cleft cannot be applied to 5-HT receptors, which are mostly (but not exclusively) localized at extra-synaptic locations either pre- or post-synaptically. 5-HT receptors are engaged in pre- or post-synaptic complexes composed of many GPCR-interacting proteins. The functions of these proteins are starting to be revealed. These proteins have been implicated in targeting, trafficking to or from the membrane, desensitization, and fine-tuning of signaling.

Alternative Splicing↗

PubMed2006

The proteomic analysis of mouse choroid plexus secretome reveals a high protein secretion capacity of choroidal epithelial cells.

Choroid plexuses (CP) are involved in multiple functions related to their unique architecture and localization at the interface between the blood and cerebrospinal fluid compartments. These include the release by choroidal epithelial cells (CEC) of biologically active molecules, such as polypeptides, which are distributed globally to the brain. Here, we have used a proteomic approach to get an unbiased overview of the proteins that are secreted by primary cultures enriched in epithelial cells from mice CP. We identified a total of 43 proteins secreted through the classical vesicular pathway in CEC -conditioned medium. They include transport proteins, collagen subunits and other cell matrix proteins, proteases, protease inhibitors and neurotrophic factors. Treating CEC cultures with lipopolysaccharide, increased the secretion of four protein species and induced the release of two additional proteins. Our study also reveals a higher protein secretion capacity of CECs compared with other CP cells or cultured astrocytes. In conclusion, this study provides for the first time the characterization of the major proteins that are secreted by CECs. These proteins may play a critical role in neuronal growth, differentiation and function as well as in brain pathologies.

Animals↗

PubMed2006

Adaptive changes in serotonin neurons of the raphe nuclei in 5-HT(4) receptor knock-out mouse.

Decreased serotonin (5-HT) transmission is thought to underlie several mental diseases, including depression and feeding disorders. However, whether deficits in genes encoding G protein-coupled receptors may down-regulate the activity of 5-HT neurons is unknown currently. Based on recent evidence that stress-induced anorexia may involve 5-HT(4)receptors (5-HT(4)R), we measured various aspects of 5-HT function in 5-HT(4)R knock-out (KO) mice. When compared to dorsal raphe nucleus (DRN) 5-HT neurons from wild-type mice, those from 5-HT(4)R KO mice exhibited reduced spontaneous electrical activity. This reduced activity was associated with diminished tissue levels of 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA). Cumulative, systemic doses of the 5-HT uptake blocker citalopram, that reduced 5-HT cell firing by 30% in wild-type animals, completely inhibited 5-HT neuron firing in the KO mice. This effect was reversed by administration of the 5-HT(1A) receptor (5-HT(1A)R) antagonist, WAY100635, in mice of both genotypes. Other changes in DRN of the KO mice included increases in the levels of 5-HT plasma membrane transporter sites and mRNA, as well as a decrease in the density of 5-HT(1A)R sites without any change in 5-HT(1A) mRNA content. With the exception of increased 5-HT turnover index in the hypothalamus and nucleus accumbens and a decreased density of 5-HT(1A)R sites in the dorsal hippocampus (CA1) and septum, no major changes were detected in 5-HT territories of projection, suggesting region-specific adaptive changes. The mechanisms whereby 5-HT(4)R mediate a tonic positive influence on the firing activity of DRN 5-HT neurons and 5-HT content remain to be determined.

Animals↗

PubMed2006

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels.

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

Amino Acid Sequence↗

PubMed2005

Characterization and localization of cocaine- and amphetamine-regulated transcript (CART) binding sites.

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the brain and various endocrine tissues. CART is implicated in many physiological functions including food intake, drug reward, stress and nociception. No CART receptor has been identified yet. We fused CART(55-102) to the green fluorescent protein (GFP) and found that the ligand suppresses significantly food intake after intracerebroventricular (i.c.v.) injection in mice. Using this ligand, we show specific CART binding sites on HepG2 cells and hypothalamic dissociated cells. In brain sections, CART displaceable binding sites were observed on cell bodies mainly localized in hypothalamic periventricular areas.

Animals↗

PubMed2005

Constitutive secretion of protease nexin-1 by glial cells and its regulation by G-protein-coupled receptors.

Extracellular serine proteases and their inhibitors (serpins) play a key role for synaptic plasticity in the developing and adult CNS. Serpins also counteract the extravasated proteases during brain injury. We studied the mechanisms by which one of the most important serpins, serpinE2 or protease nexin-1 (PN-1), is secreted by glial cells and how its secretion is regulated by extracellular signals. Using time-lapse videomicroscopy and biochemical methods, we demonstrate that PN-1 is constitutively secreted through small vesicles animated by a discontinuous movement using microtubules as tracks. The F-actin network underneath the plasma membrane acting as a barrier hindered PN-1 vesicle exocytosis. Vasointestinal/pituitary adenylate cyclase peptides and the G-protein activator mastoparan increased PN-1 secretion by disrupting the F-actin barrier. The receptor-mediated regulation of PN-1 constitutive secretion may be an important mechanism adapting extracellular proteolytic activity to synaptic activity.

Actins↗

PubMed2005

Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements.

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.

Alternative Splicing↗

PubMed2005

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes.

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Animals↗

PubMed2005

Frontocortical 5-HT4 receptors exert positive feedback on serotonergic activity: viral transfections, subacute and chronic treatments with 5-HT4 agonists.

BACKGROUND: We recently identified a facilitory control exerted by serotonin4 (5-HT4) receptors on the in vivo firing activity of dorsal raphe nucleus (DRN) serotonergic (5-HT) neurons. However, these findings were based on acute administrations of 5-HT4 receptor agonists and antagonists, which were active only in a subpopulation of 5-HT neurons. We had no evidence that this influence was significant when considering the entire DRN, nor if it was persistent after chronic treatments. In addition, the poor distribution of 5-HT4 receptors within the DRN raised the question of the neuroanatomical bases underlying this control. METHODS AND RESULTS: Here we show that the subacute intraperitoneal (IP) injection of the 5-HT4 receptor agonists prucalopride (2.5 mg/kg) and RS 67333 (1.5 mg/kg) 30 minutes before the beginning of recordings augment the mean firing rate of DRN neurons by 40% and 66%, respectively. These increases remain stable when the compounds are administered continuously during 3 and 21 days; the effects of the 3-day treatment are blocked by the 5-HT4 receptor antagonist GR 125487 (1000 microg/kg, intravenous [i.v.]). In addition, stereotaxic microinjections of herpes simplex viruses, transformed to overexpress 5-HT4 receptors, increase DRN 5-HT neuronal mean activity when performed in the medial prefrontal cortex (mPFC) but not in the striatum or in the hippocampus. CONCLUSIONS: This finding suggests the existence of a 5-HT(4)-dependent activation of DRN that may involve the mPFC, unveiling the 5-HT4 receptor as a putative player in the physiopathology of several disorders related to central 5-HT dysfunction.

Aniline Compounds↗

PubMed2005

Shank expression is sufficient to induce functional dendritic spine synapses in aspiny neurons.

Shank proteins assemble glutamate receptors with their intracellular signaling apparatus and cytoskeleton at the postsynaptic density. Whether Shank plays a role in spinogenesis and synaptogenesis remained unclear. Here, we report that knock-down of Shank3/prolinerich synapse-associated protein-2 by RNA interference reduces spine density in hippocampal neurons. Moreover, transgene expression of Shank 3 is sufficient to induce functional dendritic spines in aspiny cerebellar neurons. Transfected Shank protein recruits functional glutamate receptors, increases the number and size of synaptic contacts, and increases amplitude, frequency, and the AMPA component of miniature EPSCs, similar to what is observed during synapse developmental maturation. Mutation/deletion approaches indicate that these effects require interactions of Shank3 with the glutamate receptor complex. Consistent with this observation, chronic treatment with glutamate receptor antagonists alters maturation of the Shank3-induced spines. These results strongly suggest that Shank proteins and the associated glutamate receptors participate in a concerted manner to form spines and functional synapses.

Animals↗

PubMed2005

Proteomic detection of prostate-specific antigen using a serum fractionation procedure: potential implication for new low-abundance cancer biomarkers detection.

One of the major obstacles in proteomic analysis of biological fluids is the presence of highly abundant proteins such as albumin and immunoglobulins, which can interfere with the resolution and sensitivity of the proteome profiling techniques used. In this paper, we describe an anion exchange fractionation procedure for serum using denaturating conditions allowing protein-protein interaction disruption before analysis by surface-enhanced laser desorption/ionization and by two-dimensional electrophoresis. This method simplifies the serum proteome into subproteomes and markedly increases resolution and sensitivity without any loss of minor proteins. To confirm the applicability of this method, fractionated serum of a patient with prostate cancer was analyzed for the presence of the prostate-specific antigen (PSA) which is a low-abundance tumor marker protein. The results demonstrate that PSA can be detected by two-dimensional electrophoresis only in serum following fractionation. Hence, this procedure may facilitate the identification of other, so far unknown, tumor markers in patient sera.

Animals↗

PubMed2005

Increased number of neural progenitors in human temporal lobe epilepsy.

An increased neurogenesis is reported in animal models of mesial temporal lobe epilepsy (MTLE) but the fate of newborn cells is unknown. Here, we attempted to demonstrate neurogenesis in adult epileptic tissue obtained after hippocampectomy. MTLE hippocampi showed increased expression of division markers and of Musashi-1, a marker of neural progenitors, compared to control hippocampi. Large quantities of Musashi-1+ cells were obvious in the subgranular layer and the subventricular zone, both known neurogenic areas, and in the fissura hippocampi. Musashi-1 was expressed by small cells that were mainly vimentin+ or nestin+, rarely Dcx+ or PSA-NCAM+ and negative for markers of mature neurons or astrocytes. Some of them are present in the granular layer, the hilus, and CA1 area resembling the ectopic positions described in rodents. These findings demonstrate that neural progenitors proliferate in chronic epilepsy and suggest that the fissura hippocampi behaves like another neurogenic area.

Adolescent↗

PubMed2005

3,4-N-methlenedioxymethamphetamine-induced hypophagia is maintained in 5-HT1B receptor knockout mice, but suppressed by the 5-HT2C receptor antagonist RS102221.

3,4-Methylenedioxy-N-methamphetamine (MDMA or 'ecstasy') is a psychoactive substance, first described as an appetite suppressant in humans, inducing side effects and even death. MDMA increases serotonin (5-HT) levels, and 5-HT inhibits food intake, but the 5-HT receptors involved in MDMA-induced changes in feeding behavior are unknown. We examined whether a systemic MDMA injection would reduce the physiological drive to eat in starved mice and tested if the inactivation of 5-HT1B or 5-HT2C receptors could restore this response. Our results indicate that in starved mice, MDMA (10 mg/kg) provoked an initial hypophagia for 1 h (-77%) followed by a period of hyperphagia (studied between 1 and 3 h). This biphasic feeding behavior due to MDMA treatment was maintained in 5-HT1B receptor-null mice or in animals treated with the 5-HT1B/1D receptor antagonist GR127935 (3 or 10 mg/kg). In contrast, MDMA-induced hypophagia (for the first 1 h period) was suppressed when combined with the 5-HT2C receptor antagonist RS102221 (2 mg/kg). However, RS102221 did not alter MDMA-induced hyperphagia (for the 1-3 h period) but did exert a stimulant effect, when administered alone, during that period. We have previously shown that MDMA or 5-HT1A/1B receptor agonist RU24969 fails to stimulate locomotor activity in 5-HT1B receptor-null mice. Our present data indicate that the 5-HT2C receptor antagonist RS102221 suppresses MDMA-induced hyperlocomotion. These findings provide the first evidence that the inactivation of 5-HT2C receptors may reduce hypophagia and motor response to MDMA, while a genetic deficit or pharmacological inactivation of 5-HT1B receptors was insufficient to alter the feeding response to MDMA.

Animals↗

PubMed2005

Dynamic reorganization of the astrocyte actin cytoskeleton elicited by cAMP and PACAP: a role for phosphatidylInositol 3-kinase inhibition.

Cyclic AMP (cAMP)-raising agents induce astrocytes grown in vitro to adopt a stellate morphology resembling their in vivo appearance, through the depolymerization of actomyosin stress fibres. The signalling pathways responsible for cAMP-induced astrocyte stellation have thus far remained largely elusive. We showed in this study that the neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) mimicked the effect of forskolin, a direct activator of adenylate cyclase, on the actin cytoskeleton of primary rat astrocytes. The depolymerization of stress fibres induced by PACAP or forskolin was prevented by the expression of a constitutively active mutant of RhoA, but not by a protein kinase A (PKA) blocker, indicating that cAMP-raising agents act upstream of RhoA, in a PKA-independent manner. In addition, PACAP and forskolin inhibited basal Akt phosphorylation, and basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3-kinase (PI 3-K) activities. Incubation with a PI 3-K blocker resulted in the depolymerization of stress fibres. This effect was blocked by the expression of a constitutively active mutant of RhoA, indicating that PI 3-K inhibition acted upstream of RhoA. Together, these data demonstrate for the first time that depolymerization of stress fibres, and the resulting astrocyte stellation, induced by stimulation of cAMP production involves the inhibition of the PI 3-K-RhoA pathway.

Actins↗

PubMed2004

The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology.

In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased ( approximately 10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.

Action Potentials↗

PubMed2004

New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting.

The 5-hydroxytryptamine type 4 receptor (5-HT4R) is involved in learning, feeding, respiratory control and gastrointestinal transit. This receptor is one of the G-protein-coupled receptors for which alternative mRNA splicing generates the most variants that differ in their C-terminal extremities. Some 5-HT4R variants (a, e and f) express canonical PDZ ligands at their C-termini. Here, we have examined whether some mouse 5-HT4R variants associate with specific sets of proteins, using a proteomic approach based on peptide-affinity chromatography, two-dimensional electrophoresis and mass spectrometry. We have identified ten proteins that interact specifically with the 5-HT4(a)R and three that only associate with the 5-HT4(e)R. Most of them are PDZ proteins. Among the proteins that associated specifically with the 5-HT4(a)R variant, NHERF greatly modified its subcellular localization. Moreover, NHERF recruited the 5-HT4(a)R to microvilli, where it localized with activated ezrin, consistent with the role of 5-HT4(a)R in cytoskeleton remodelling. The 5-HT4(a)R also interacted with both the constitutive and inducible (upon methamphetamine treatment) forms of the recently cloned sorting nexin 27 (SNX27a and b, respectively). We found that SNX27a redirected part of 5-HT4(a)R to early endosomes. The interaction of the 5-HT4R splice variants with distinct sets of PDZ proteins might specify their cellular localization as well as their signal transduction properties.

Alternative Splicing↗

PubMed2004

Shaggy, the homolog of glycogen synthase kinase 3, controls neuromuscular junction growth in Drosophila.

A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3 alpha and beta, two serine-threonine kinases, was concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, we show that Sgg controlled the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, was required for Shaggy function on synaptic growth.

Animals↗