No description available.

Oxidized low-density lipoproteins (OxLDL) mediate many of the pathological events associated with atherosclerosis. Oxysterols, the major cytotoxic component of OxLDL, induce apoptosis in macrophages by a calcium flux mediated activation of cytosolic phospholipase A2 resulting in the release of arachidonic acid (AA). Inhibition of AA metabolism has been shown to protect macrophages from oxysterol-induced apoptosis. The current study explores the steps in the oxysterol-induced apoptosis signaling pathway in murine macrophages subsequent to the liberation of AA. To elucidate this mechanism, two oxysterols, 7-ketocholesterol and 25-hydroxycholesterol (25-OHC), were used to induce apoptosis in murine macrophage cell lines (P388D1, and Raw 264.7) and mouse peritoneal macrophages (MPMs). Pharmacological inhibition of eicosanoid synthesis or genetic knockout of important eicosanoid biosynthetic genes had no significant effect on the induction of apoptosis by oxysterols in macrophages. The induction of apoptosis in macrophage cell lines and MPMs by oxysterols and OxLDL was suppressed by Sandoz 58-035, an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT). Furthermore, in comparison to wild-type MPMs, ACAT-1 deficient MPMs were found to be resistant to apoptosis induced by oxysterols or OxLDL. Macrophages treated with 7KC accumulated ACAT-derived cholesteryl and 7-ketocholesteryl esters. An inhibitor of cholesterol trafficking, U18666A, specifically prevented the accumulation of cholesteryl esters, but not 7-ketocholesteryl esters nor the induction of apoptosis. An inhibitor of cPLA2 prevented the accumulation of 7-ketocholesteryl esters. This inhibition was reversed by supplementing oleic acid or AA; however, only AA supplementation restored the induction of apoptosis. These results suggest that oxysterols not only initiate the apoptosis pathway by activating cPLA2, but also participate in the downstream signaling pathway when esterified by ACAT to form arachidonyl oxysterols. We also demonstrate that macrophages lacking the cannabinoid type-2 (CB2) receptor have reduced levels of apoptosis when treated with oxysterols or OxLDL in comparison to wild-type macrophages and that a CB2 specific antagonist blocks oxysterol-induced apoptosis in macrophages suggesting that the CB2 receptor is involved in this pathway, perhaps by interacting with the arachidonyl oxysterols.

No description available.

Cannabinoids have been shown to alter synaptic plasticity but the mechanism by which this occurs at hippocampal CA3-CA1 synapses in vivo is not yet known. Utilizing in vivo electrophysiological recordings of field excitatory postsynaptic potentials (fEPSP) on anesthetized rats and mice as well as three lines of conditional knockout mouse models, the objective was to show a two-part mechanistic breakdown of cannabinoid-evoked CA3-CA1 long-term depression (LTD) in its induction as well as early and later-phase expression stages. It was determined that this cannabinoid-induced in vivo LTD requires cannabinoid type-1 receptors (CB1Rs) on astrocytes, but not CB1Rs on glutamatergic or GABAergic neuronal axons/terminals. Pharmacological testing determined that cannabinoid-induced in vivo LTD also requires activation of NMDA receptors (NMDAR) and subsequent postsynaptic endocytosis of AMPA receptors (AMPAR). There exists a clear role for NR2B-containing NMDARs in a persistent, transitory form, potentially related to prolonged or delayed glutamate release (possibly as a result of the astrocytic network). A key determination of the expression phase is the involvement of new protein synthesis (using translation and transcription inhibitors) – further evidence of the long-term action of the synaptic plasticity from a single cannabinoid dose.

Au cours du développement, les axones des cellules ganglionnaires de la rétine (CGRs) voyagent sur de longues distances pour établir des connexions avec leurs cellules cibles. La navigation des cônes de croissance est guidée par différentes molécules chimiotropiques présentes dans leur environnement. Les endocannabinoïdes (eCB) sont d’importants neuromodulateurs qui régulent de manière rétrograde la fonction de nombreuses synapses du cerveau. Ils agissent principalement par le biais de leurs récepteurs liés à une protéine Gi/o CB1 (CB1R) et CB2 (CB2R). La présence des eCBs durant le stade fœtal et la période postnatale suggère leur implication dans des événements régulant le développement du système nerveux. Cette thèse confirme l’expression des récepteurs aux cannabinoïdes CB1 et CB2 ainsi que l’enzyme dégradant les eCBs lors du développement embryonnaire et perinatal des CGRs et de la voie rétinothalamique in vivo. La manipulation pharmacologique de l’activité de CB1R et CB2R réorganise la morphologie du cône de croissance des CGRs et des neurones corticaux in vitro. De plus, la stimulation locale avec un agoniste de CB1R ou de CB2R modifie le comportement du cône de croissance entraînant sa répulsion. CB1R et CB2R modulent par le biais de la voie de signalisation AMPc/PKA, la mobilisation de DCC à la membrane plasmique. Par ailleurs, les résultats de cette recherche démontrent également l’implication de CB1R et CB2R dans la ségrégation des projections ipsi- et controlatérales et le développement de la voie rétinothalamique. / Following differentiation, retinal ganglion cell (RGC) axons, tipped at their distal end by the growth cone (GC), navigate through relatively long distances in a highly directed manner in order to establish functional synapses with thalamic and superior colliculus (SC) neurons. This is achieved with the help of extracellular guidance molecules which steer RGC axon growth by regulating GC morphology by means of attractive and/or repulsive mechanisms. In the adult brain, endocannabinoids (eCBs) exert an important neuromodulatory function by acting as retrograde messengers to regulate the function of many synapses. Endocannabinoids act mainly via their Gi/o protein coupled receptors CB1 (CB1R) and CB2 (CB2R). Due to their presence at the fetal and early postnatal periods, it has been proposed that eCBs and their receptors might be involved in several developmental events, such as cell proliferation and migration, axon guidance and synaptogenesis. We observed that during early postnatal development, components of the eCB system are expressed along the visual pathway (the optic chiasm, the lateral geniculate nucleus and the SC). To assess the implication of the eCB system, in vitro, embryonic retinal explant and primary neuron cultures were treated with pharmacological agonists and inverse agonists of CB1R and CB2R. These experiments demonstrated that these cannabinoid receptors modify the GC’s morphology. Most importantly, CB1R and CB2R act through the cAMP/PKA pathway to modulate the presence of DCC at the plasma membrane. In vivo, CB1R and CB2R play a major role and the absence of either one of them induces a decrease in eye-specific segregation of retinal projections. These results show an implication of CB1R and CB2R during RGC growth and retinothalamic development.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

La schizophrénie est une maladie complexe et a une prévalence approximative de 1% dans la population générale. Au sein des paradigmes neurochimiques, la théorie étiologique de la dopamine est celle qui prévaut alors que sont de plus en plus impliqués d’autres circuits de neurotransmission comme celui du glutamate. En clinique, les patients atteints de schizophrénie ont une grande propension à consommer des substances, particulièrement du cannabis. Nous avons cherché à étayer l’hypothèse d’un désordre du système cannabinoïde endogène, un important neuromodulateur. Ce mémoire propose d’abord dans un premier article une revue exhaustive de la littérature explorant le système endocannabinoïde et ses implications dans la schizophrénie. Puis, nous exposons dans un second article les résultats d’une recherche clinique sur les endocannabinoïdes plasmatiques dans trois groupes de sujets avec schizophrénie et/ou toxicomanie, pendant 12 semaines. Nous avons observé un effet miroir de deux ligands endocannabinoïdes, l’anandamide et l’oleylethanolamide, qui étaient élevés chez les patients avec double diagnostic et abaissés chez les toxicomanes, au début de l’étude. Au terme de l’étude, l’élévation des endocannabinoïdes s’est maintenue et nous avons supposé un marqueur de vulnérabilité psychotique dans un contexte de consommation. Finalement, nous avons analysé les résultats en les intégrant aux connaissances moléculaires et pharmacologiques ainsi qu’aux théories neurochimiques et inflammatoires déjà développées dans la schizophrénie. Nous avons aussi tenu compte des principales comorbidités observées en clinique: la toxicomanie et les troubles métaboliques. Cela nous a permis de proposer un modèle cannabinoïde de la schizophrénie et conséquemment des perspectives de recherche et de traitement. / Schizophrenia is a complex disease that has 1% worldwide prevalence. Dopamine etiological theory leads neurochemical paradigms although glutamate hypothesis is gaining in importance among several neurotransmission circuits involved. Schizophrenia patients are more prone to substance use disorders, particularly to cannabis dependence, than the general population. Therefore, we have aimed to explain the hypothesis of a deregulation in the endogenous cannabinoid system, a very important neurodulator.  First, this thesis proposes in the first article an exhaustive literature review on the endocannabinoid system and its implications in schizophrenia. Then, we present results from our clinical research on plasmatic endocannabinoids in three groups of subjects with schizophrenia and/or substance use disorders, during twelve weeks. We have observed a mirror effect involving two endocannabinoid ligands, anandamide and oleylethanolamide, which were elevated in patients with dual diagnosis and reduced in patients with only substance use disorders. At the end of the study, it seems that endocannabinoid elevation was maintained and we supposed a vulnerability to psychosis in a substance use disorder context.   Finally, we analyzed our results by integrating explanations from molecular biology and neuropharmacology and also from neurochemical and inflammatory theories already well-known in schizophrenia. We also considered the main comorbidities observed in clinic such as substance use and metabolic disorders. Then, we proposed an endogenous cannabinoid model of schizophrenia. Ultimately, this thesis suggested research perspectives and potential treatments.

The potential antitumor activity of mixed CB1/CB2 cannabinoid receptor agonists, such as the aminoalkylindole WIN55,212-2 (WIN2), has been extensively studied, but little information is available as to their potential interaction with conventional cancer therapies, such as ionizing radiation (IR). In the present work, we investigated the effects of WIN2 on the antiproliferative effects of radiation in human (MCF-7 and MDA-MB-231) and murine (4T1) breast cancer cells, as well as an immortalized human breast epithelial cell line (MCF-10A). WIN2 or radiation alone inhibited breast tumor growth, while the combination of WIN2 and radiation was more effective than either agent alone in breast cancer cells. WIN2 showed lower potency in MCF-10A cells than MCF-7 cells, but was still able to augment the effects of radiation at higher doses. The stereoisomer of WIN2, WIN55,212-3 (WIN3) failed to inhibit growth or potentiate the growth-inhibitory effects of radiation, indicating stereospecificity in all cell lines tested. The combination of WIN2 and IR was examined in vivo but the results were inconclusive. Interestingly, while other aminoalkylindoles, pravadoline and JWH-015, enhanced the antiproliferative effects of radiation, this was not the case for other synthetic cannabinoids (i.e., nabilone, CP55,940 and methanandamide) or phytocannabinoids (i.e., ∆9-tetrahydrocannabinol and cannabidiol). The antiproliferative actions of WIN2 were not ameliorated by CB1, CB2, TRPV1, or PPAR receptor antagonists, suggesting the possibility of a novel site of action. Studies utilizing sphingosine-1-phosphate (S1P) agonists and estradiol suggest that WIN2 interferes with S1P signaling in cell proliferation, but agonist stimulated [³⁵S]GTPγS binding assays show that this antagonism is not occurring at the level of S1P receptors. In addition, WIN2 did not alter radiation-induced DNA damage or the rate of DNA repair based on γH2AX staining. Treatment with WIN2 and radiation promoted both autophagy and senescence, but not apoptosis or necrosis. Time course studies combined with senescence and cell death data suggest that radiation-induced senescence, while WIN2 induced classical growth arrest and the WIN2/IR combination produced parallel mechanisms of both senescent growth arrest and classical growth arrest. Taken together, these findings raise the possibility that aminoalkylindole compounds targeting a novel site of action represents a potential strategy to augment the effectiveness of radiation treatment in breast cancer.

Protein structure predication is a field of computational molecular modeling with an enormous potential for improvement. Side-chain geometry prediction is a critical component of this process that is crucial for computational protein structure predication as well as crystallographers in refining experimentally determined protein crystal structures. The cornerstone of side-chain geometry prediction are side-chain rotamer libraries, usually obtained through exhaustive statistical analysis of existing protein structures. Little is known, however, about the driving forces leading to the preference or suitability of one rotamer over another. Construction of 3D hydropathic interaction maps for nearly 30,000 tyrosines extracted from the PDB reveals their environments, in terms of hydrophobic and polar (collectively “hydropathic”) interactions. Using a unique 3D similarity metric, these environments were clustered with k-means. In the ϕ, ψ region (–200° < ϕ < –155°; –205° < ψ < –160°) representing 631 tyrosines, clustering reduced the set to 14 unique hydropathic environments, with most diversity arising from favorable hydrophobic interactions. Polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic OH and a handful of unique environments surrounding the backbone. The memberships of all but one of the 14 environments are dominated by a single χ1/χ2 rotamer. Each tyrosine residue attempts to fulfill its hydropathic valence. Structural water molecules are thus used in a variety of roles throughout protein structure. A second project involves elucidating the 3D structure of CRIP1a, a cannabinoid 1 receptor (CB1R) binding protein that could provide information for designing small molecules targeting the CRIP1a-CB1R interaction. The CRIP1a protein was produced in high purity. Crystallization experiments failed, both with and without the last 9 or 12 amino acid peptide of the CB1R C-terminus. Attempts were made to use NMR for structure determination; however, the protein precipitated out during data acquisition. A model was thus built computationally to which the CB1R C-terminus peptide was docked. HINT was used in selecting optimum models and analyzing interactions involved in the CRIP1a-CB1R complex. The final model demonstrated key putative interactions between CRIP1a and CB1R while also predicting highly flexible areas of the CRIP1a possibly contributing to the difficulties faced during crystallization.

The endogenous cannabinoids (endocannabinoids) anandamide (AEA) and 2-arachidonylglycerol (2-AG) exert their effects predominantly through cannabinoid CB1 and CB2 receptors, but these actions are short-lived because of rapid hydrolysis by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective inhibition of either enzyme elevates CNS levels of the appropriate endocannabinoid and produces analgesic effects with fewer psychomimetic side effects than Δ9-tetrahydrocannabinol (THC), the primary active constituent of marijuana. While cannabinoid receptor agonists and FAAH inhibitors reliably produce anti-inflammatory and anti-hyperalgesic effects in the carrageenan test and other inflammatory pain models, much less is known about the consequences of inhibiting MAGL in these assays. Here, we tested whether the selective MAGL inhibitor JZL184 would reduce nociceptive behavior in the carrageenan test. JZL184 significantly attenuated carrageenan-induced paw edema and mechanical allodynia, whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Interestingly, the anti-allodynic effects of the combination of low dose of JZL184 (4mg/kg) and high dose of the selective and long-acting FAAH inhibitor PF-3845 (10 mg/kg) was augmented compared with each drug alone. On the contrary, the combination treatment did not reduce edema more than either JZL184 or PR-3845 given alone. These results suggest that low doses of MAGL inhibitors alone or in combination with FAAH inhibitors, reduce inflammatory nociception through the activation of both CB1 and CB2 receptors with no evidence of tolerance following repeated administration.