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Marc Fontaine

Publications and source records attributed to Marc Fontaine.

13 recordsLinked to original sources
PubMed2006

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes.

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([(125)I]rUII) revealed the presence of high- and low-affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII(4-11) and [3-iodo-Tyr9]hUII(4-11) were also very potent in displacing [(125)I]rUII from its binding sites, whereas the non-cyclic analogue [Ser5,10]hUII(4-11) and somatostatin-14 could only displace [(125)I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [(125)I-Tyr0,D-Trp8]somatostatin-14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [(3)H]inositol incorporation and increased intracellular Ca(2+) concentration in a dose-dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [(125)I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high- and low-affinity UII binding sites coupled to G proteins, the high-affinity receptor exhibiting the same pharmacological and functional characteristics as UT.

Animals↗
PubMed2006

Interleukin-1beta and anaphylatoxins exert a synergistic effect on NGF expression by astrocytes.

C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological activities through interaction with two G protein-coupled receptors named C3aR and C5aR, respectively. In the brain, these receptors are expressed on glial cells, and some recent data have suggested that anaphylatoxins could mediate neuroprotection. In this study, we used RT-PCR and ribonuclease protection assays (RPA) to investigate the role of anaphylatoxins on neurotrophin expression by the human glioblastoma cell line T98G and by rat astrocytes. Our data show that for both cell types, anaphylatoxins upregulate expression of NGF mRNA. This response depended on a G protein-coupled pathway since pre-treatment of cells with pertussis toxin (PTX) completely blocked NGF mRNA increases. This effect was anaphylatoxin-specific since pre-incubation with anti-C3a or anti-C5aR antibodies abolished the effects of C3a and C5a, respectively. The regulation of NGF mRNA by anaphylatoxins was not accompanied by translation into protein expression, but there was a significant synergic effect of anaphylatoxins/IL-1b costimulation. Our demonstration of involvement of anaphylatoxins in the NGF release process by astrocytes suggests that C3a and C5a could modulate neuronal survival in the CNS.

Journal Article↗
PubMed2006

Large-scale expression study of human mesial temporal lobe epilepsy: evidence for dysregulation of the neurotransmission and complement systems in the entorhinal cortex.

Human mesial temporal lobe epilepsies (MTLE) are the most frequent form of partial epilepsies and display frequent pharmacoresistance. The molecular alterations underlying human MTLE remain poorly understood. A two-step transcriptional analysis consisting in cDNA microarray experiments followed by quantitative RT-PCR validations was performed. Because the entorhinal cortex (EC) plays an important role in the pathophysiology of the MTLE and usually discloses no detectable or little cell loss, resected EC and each corresponding lateral temporal neocortex (LTC) of MTLE patients were used as the source of disease-associated and control RNAs, respectively. Six genes encoding (i) a serotonin receptor (HTR2A) and a neuropeptide Y receptor type 1 (NPY1R), (ii) a protein (FHL2) associating with the KCNE1 (minK) potassium channel subunit and with presenilin-2 and (iii) three immune system-related proteins (C3, HLA-DR-gamma and CD99), were found consistently downregulated or upregulated in the EC of MTLE patients as compared with non-epileptic autopsy controls. Quantitative western blot analyses confirmed decreased expression of NPY1R in all eight MTLE patients tested. Immunohistochemistry experiments revealed the existence of a perivascular infiltration of C3 positive leucocytes and/or detected membrane attack complexes on a subset of neurons, within the EC of nine out of eleven MTLE patients. To summarize, a large-scale microarray expression study on the EC of MTLE patients led to the identification of six candidate genes for human MTLE pathophysiology. Altered expression of NPY1R and C3 was also demonstrated at the protein level. Overall, our data indicate that local dysregulation of the neurotransmission and complement systems in the EC is a frequent event in human MTLE.

Adult↗
PubMed2006

PACAP and ceramides exert opposite effects on migration, neurite outgrowth, and cytoskeleton remodeling.

During brain development, cells that fail to reach their final destination or to establish proper connections are eliminated. It has been shown that the proinflammatory cytokine second messenger ceramides and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) play pivotal roles in the histogenesis of the cerebellum. However, little is known regarding the effects of these two factors on cerebellar granule cell migration. We have found that PACAP prevents the effects of C2-ceramide on granule cell motility and neurite outgrowth. These actions are attributable to opposite effects on actin distribution, tubulin polymerization, and Tau phosphorylation. These data suggest that PACAP and factors inducing ceramide production may control granule cell migration during cerebellar development.

Apoptosis↗
PubMed2005

Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling.

During neuronal migration, cells that do not reach their normal destination or fail to establish proper connections are eliminated through an apoptotic process. Recent studies have shown that the proinflammatory cytokine tumor necrosis factor alpha (and its second messengers ceramides) and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) play a pivotal role in the histogenesis of the cerebellar cortex. However, the effects of ceramides and PACAP on migration of cerebellar granule cells have never been investigated. Time-lapse videomicroscopy recording showed that C2-ceramide, a cell-permeable ceramide analog, and PACAP induced opposite effects on cell motility and neurite outgrowth. C2-ceramide markedly stimulated cell movements during the first hours of treatment and inhibited neuritogenesis, whereas PACAP reduced cell migration and promoted neurite outgrowth. These actions of C2-ceramide on cell motility and neurite outgrowth were accompanied by a disorganization of the actin filament network, depolarization of tubulin, and alteration of the microtubule-associated protein Tau. In contrast, PACAP strengthened the polarization of actin at the emergence cone, increased Tau phosphorylation, and abolished C2-ceramide-evoked alterations of the cytoskeletal architecture. The caspase-inhibitor Z-VAD-FMK, like PACAP, suppressed the "dance of the death" provoked by C2-ceramide. Finally, Z-VAD-FMK and the PP2A inhibitor okadaic acid both prevented the impairment of Tau phosphorylation induced by C2-ceramide. Taken together, these data indicate that the reverse actions of C2-ceramide and PACAP on cerebellar granule cell motility and neurite outgrowth are attributable to their opposite effects on actin distribution, tubulin polymerization, and Tau phosphorylation.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Expression of a functional C5a receptor in regenerating hepatocytes and its involvement in a proliferative signaling pathway in rat.

Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.

Animals↗
PubMed2004

Characterization of C3a and C5a receptors in rat cerebellar granule neurons during maturation. Neuroprotective effect of C5a against apoptotic cell death.

There is now clear evidence that the Complement anaphylatoxin C3a and C5a receptors (C3aR and C5aR) are expressed in glial cells, notably in astrocytes and microglia. In contrast, very few data are available concerning the possible expression of these receptors in neurons. Here, we show that transient expression of C3aR and C5aR occurs in cerebellar granule neurons in vivo with a maximal density in 12-day-old rat, suggesting a role of these receptors during development of the cerebellum. Expression of C3aR and C5aR mRNAs and proteins was also observed in vitro in cultured cerebellar granule cells. Quantification of the mRNAs by real-time reverse transcription-PCR showed a peak of expression at day 2 in vitro (DIV 2); the C3aR and C5aR proteins were detected by Western blot analysis at DIV 4 and by flow cytometry and immunocytochemistry in differentiating neurons with a maximum density at DIV 4-9. Apoptosis of granule cells plays a crucial role for the harmonious development of the cerebellar cortex. We found that, in cultured granule neurons in which apoptosis was induced by serum deprivation and low potassium concentration, a C5aR agonist promoted cell survival and inhibited caspase-3 activation and DNA fragmentation. The neuroprotective effect of the C5aR agonist was associated with a marked inhibition of caspase-9 activity and partial restoration of mitochondrial integrity. Our results provide the first evidence that C3aR and C5aR are both expressed in cerebellar granule cells during development and that C5a, but not C3a, is a potent inhibitor of apoptotic cell death in cultured granule neurons.

Aging↗
PubMed2004

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells.

The sphingomyelin-derived messenger ceramides provoke neuronal apoptosis through caspase-3 activation, while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase-3 activity. However, the mechanisms leading to the opposite regulation of caspase-3 by C2-ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2-ceramide-induced inhibition of mitochondrial potential and C2-ceramide-evoked cytochrome c release. C2-ceramide stimulated Bax expression, but had no effect on Bcl-2, while PACAP abrogated the action of C2-ceramide on Bax and stimulated Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked, respectively, by the JNK inhibitor L-JNKI1 and the MEK inhibitor U0126. L-JNKI1 prevented the alteration of mitochondria induced by C2-ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2-ceramide on mitochondrial potential. Moreover, L-JNKI1 inhibited the stimulatory effect of C2-ceramide on caspase-9 and -3 and prevented C2-ceramide-induced cell death. U0126 blocked PACAP-induced Bcl-2 expression, abrogated the inhibitory effect of PACAP on ceramide-induced caspase-9 activity, and promoted granule cell death. The present study reveals that C2-ceramide and PACAP exert opposite effects on Bax and Bcl-2 through, respectively, JNK- and ERK-dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro- and anti-apoptotic effects of C2-ceramide and PACAP.

Animals↗
PubMed2003

Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells.

The sphingolipid metabolites, ceramides, are critical mediators of the cellular stress response and play an important role in the control of programmed cell death. In particular, ceramides have been shown to induce apoptosis of cerebellar granule cells. We show that pituitary adenylate cyclase-activating polypeptide (PACAP) prevents C2-ceramide-induced apoptosis. The neuroprotective effect of PACAP was dose-dependent and blocked by its antagonist, PACAP6-38, whereas the PACAP-related peptide VIP was inactive. The effect of PACAP on cell survival was mimicked by dibutyryl-cAMP (dbcAMP) and forskolin and prevented by the MEK inhibitor U0126, indicating that both the adenylyl-cyclase and MAP-kinase pathways contribute to the neuroprotective action of the peptide. C2-ceramide and PACAP induced opposite effects on phosphorylated forms of ERK and JNK without affecting the total amounts of ERK and JNK, suggesting that a balance between these two MAP-kinases is critical for the cell survival/death decision. The effect of PACAP on ERK phosphorylation was blocked by U0126, but was not affected by H89 or chelerythrine indicating that PACAP activates ERK through a PKA- and PKC-independent mechanism. C2-ceramide induced a time-dependent activation of caspase-3, enhanced the amount of cleaved caspase-3 and stimulated the DNA fragmentation process, while PACAP strongly inhibited the C2-ceramide-induced activation of caspase-3, reduced the expression of cleaved caspase-3 and blocked DNA fragmentation. Taken together, the present results show that C2-ceramide induces apoptosis of cerebellar granule cells through a mechanism involving activation of caspase-3. Our data also demonstrate that PACAP is a potent inhibitor of C2-ceramide-induced apoptosis.

Adenylyl Cyclases↗
PubMed2003

Complement component anaphylatoxins upregulate chemokine expression by human astrocytes.

The complement (C) system, a major component of the innate immune system, has been described as a factor implicated in some brain disorders. C activation leads to the release of anaphylatoxins, two proinflammatory polypeptides acting through specific receptors that have been detected on brain cells. Here, we examined the effect of anaphylatoxins on chemokine expression by human astrocytes. We showed that anaphylatoxins significantly increase chemokine mRNA expression. However, anaphylatoxin-induced chemokine secretion (interleukin-8) was observed only in the presence of interleukin-1beta. Thus, anaphylatoxins could initiate a chemokine cascade and, at least in part, be involved in pathogenesis of the brain.

Anaphylatoxins↗
PubMed2003

Optimization of an animal model of experimental autoimmune encephalomyelitis achieved with a multiple MOG(35-55)peptide in C57BL6/J strain of mice.

The severity of the experimental autoimmune encephalomyelitis (EAE) induced by peptide myelin oligodendrocyte glycoprotein(35-55)(pMOG(35-55)) is thought to be predominantly influenced by the major histocompatibility complex (MHC), so that C57BL6/J mice, on H2(b) strain, were only mildly sick. However, it remains unclear as to how non-MHC gene regions affect EAE. To determine whether the immunization protocol could have an influence on clinical signs, C57BL6/J mice were immunized with a multiple antigen peptide (MAP) containing eight pMOG(35-55)branches synthesized directly onto a lysine core, myelin oligodendrocyte glycoprotein (35-55)-multiple antigen peptide (MOG(35-55)-MAP), in complete Freund's adjuvant (CFA). In most of the mice, clinical onset (marked weakness) occurred approximately at day 15. All mice injected with MOG(35-55)-MAP had more severe symptoms than those injected with pMOG(35-55), which developed no leg paralysis. All MOG(35-55)-MAP-immunized mice developed EAE symptoms, but 50% had primary-progressive EAE, while the other 50% had relapsing-remitting disease. Leukocyte infiltrations, associated with increased glial fibrillary acidic protein (GFAP) expression by reactive astrocytes, were observed around the lateral ventricles and blood vessels in the brain. Significant positive correlations were established between anti-MOG(35-55)antibody levels and clinical scores or GFAP positivity in the spinal cord. The heterogeneity of EAE progression, observed in these genetically identical individuals, suggests that the environment rather than the genetics plays a role. This observation is highly pertinent as it corresponds to what is seen in clinical MS.

Animals↗
PubMed2002

Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death.

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.

Animals↗