Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

A neurodegeneração é o resultado da destruição progressiva e irreversível dos neurônios no sistema nervoso central, apresentando causas desconhecidas e mecanismos patológicos não totalmente elucidados. Fatores como a idade, o aumento da formação de radicais livres e/ou estresse oxidativo, defeito no metabolismo energético, a inflamação e acúmulo de elementos neurotóxicos e de proteínas malformadas no lúmen do retículo endoplasmático (RE) contribuem para o desenvolvimento dos processos neurodegenerativos. O sistema canabinoide tem sido proposto como neuroprotetor em diversos modelos de neurodegeneração como hipóxia aguda e epilepsia, isquemia cerebral, lesão cerebral e modelos de estresse oxidativo. Assim, este trabalho teve como objetivo investigar o papel do sistema canabinoide em uma linhagem de neuroblastoma (Neuro 2a) submetida a condições de estresse oxidativo (H2O2), inflamação (LPS) e estresse do RE (tunicamicina), avaliando parâmetros de viabilidade celular e vias de sinalização envolvidas. Nossos resultados mostram que o agonista canabinoide ACEA foi capaz de proteger as células da morte celular causada pela inflamação e pelo estresse de retículo endoplasmático, mas não pelo estresse oxidativo. Esse efeito neuroprotetor exercido pelo ACEA parece pelo menos em parte ocorrer via receptor CB1 no modelo de inflamação e ser independente deste receptor no modelo de estresse de RE. Os efeitos neuroprotetores observados envolveram a modulação dos níveis de proteínas pré-apoptóticas, CHOP e Caspase 12, e da proteína relacionada à sobrevivência celular ERK 1/2. Nossos dados sugerem um papel neuroprotetor do sistema canabinoide em mecanismos relacionados aos processos neurodegenerativos e propõem a manipulação desse sistema como possível alvo terapêutico. / Neurodegeneration is the result of progressive and irreversible destruction of neurons in the central nervous system, with unknown causes and pathological mechanisms not fully elucidated. Factors such as age, increased formation of free radicals and/or oxidative stress, defects in energetic metabolism, inflammation and accumulation of neurotoxic factors and misfolded proteins in the lumen of the endoplasmic reticulum (ER) contribute to the development of neurodegenerative processes. The cannabinoid system has been proposed as neuroprotector in several models of neurodegeneration such as acute hypoxia and epilepsy, cerebral ischaemia, brain injury and oxidative stress models. This work aimed to investigate the role of the cannabinoid system in a neuroblastoma line (Neuro 2a) submitted to oxidative stress (H2O2), inflammation (LPS) and ER stress (tunicamycin) conditions, assessing cell viability parameters and signaling pathways involved. Our results show that the ACEA cannabinoid agonist was able to protect cells from cell death caused by inflammation and ER stress, but not from oxidative stress. This neuroprotective effect exerted by ACEA appears to occur at least in part via the CB1 receptor in inflammation model and it seems to be independent of this receptor in the ER stress model. The neuroprotective effects observed involved the modulation of the levels of pre-apoptotic proteins CHOP and Caspase 12 and the cell survival related protein ERK 1/2. Our data suggest a neuroprotective role of the cannabinoid system in mechanisms related to neurodegenerative processes and propose it as possible therapeutic target.

As doenças neurodegenerativas são desordens progressivas que afetam determinadas populações neuronais do sistema nervoso central, levando a morte neuronal. Vários fatores contribuem para o desenvolvimento das doenças neurodegenerativas, dentre eles o aumento da formação de radicais livres e/ou estresse oxidativo. O sistema canabinóide vem sendo sugerido como um importante sistema neuroprotetor em diversos modelos de neurodegeneração. Os objetivos do presente estudo foram avaliar a participação do sistema canabinóide em um modelo de morte celular in vitro induzida pelo peróxido de hidrogênio. O modelo celular utilizado foi a linhagem Neuro 2A. Realizamos os tratamentos conjugados para verificar a possível função neuroprotetora do sistema canabinóide. Obtivemos resultados de neuroproteção da Metanandamida revertendo o quadro de morte celular induzido pelo peróxido de hidrogênio. Acreditamos que esta neuroproteção seja via CB1 que inibe os canais de cálcio dependentes de voltagem talvez contribuindo para a redução da progressão da excitotoxicidade. / Neurodegenerative diseases are progressive disorder affecting specific neuronal populations in the central nervous system, leading to neuronal death. Several factors contribute to the development of neurodegenerative diseases, including increased free radical formation and / or oxidative stress. The cannabinoid system has been suggested as an important system neuroprotective in several models of neurodegeneration. The objectives of this study were to evaluate the involvement of the cannabinoid system in a model of in vitro cell death induced by hydrogen peroxide. The model used was the cell line Neuro 2A. Performed the treatments combined to verify the possible neuroprotective effects of the cannabinoid system. We obtained results of neuroprotection Methanandamide reversing the framework of cell death induced by hydrogen peroxide. We believe that this is via neuroprotection CB1 that inhibits calcium channels of voltage-dependent perhaps contributing to the reduction of the progression of excitotoxicity.

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.

New psychoactive substances are becoming increasingly common in many parts of the world, and some of them are marketed as “legal highs” and are produced to circumvent the drug legislation, and they come in many unregulated forms. The aim of this research was to characterize the metabolites of a new psychoactive substance and hence provide the fundamental data needed for further research of toxicity and future drug testing. The synthetic cannabinoid 4-fluoro-CUMYL-5-fluoro-PICA was incubated in cryopreserved hepatocytes for 1, 3 and 5 hour and then the formed metabolites was analyzed with an LC-QTOF-MS method, data analysis was performed by using the software MassHunter Qualitative Analysis. For 4-fluoro-CUMYL-5-fluoro-PICA a total of ten metabolites were identified, with three hydroxylations, two oxidative defluorinations to carboxylation, three oxidative defluorination and two fluoropentyl dealkylation. The metabolite with the highest intensity was oxidative defluorination.

Objective: To determine if cannabinoid receptor 2 (CB2) plays a role in atherosclerosis, we investigated the effects of systemic CB2 gene deletion on hyperlipidemia-induced atherogenesis in low density lipoprotein receptor-deficient (Ldlr-/-) mice. Methods and results: Ldlr-/- and CB2/Ldlr double knockout (CB2-/-Ldlr-/-) mice were fed an atherogenic diet for 8 and 12 weeks. Morphometric analysis revealed no significant difference between the atherosclerotic lesion area in the proximal aortas of Ldlr-/- and CB2-/-Ldlr-/- mice after 8 or 12 weeks on the atherogenic diet. The macrophage and smooth muscle cell (SMC) content, as revealed by immunohistochemical staining, did not differ significantly between Ldlr-/- and CB2-/-Ldlr-/- lesions after 8 weeks. However, after 12 weeks, CB2-/-Ldlr-/- lesions displayed greater macrophage content (86.6±4.1 versus 75.2±7.5%, P<0.05) and SMC content (11.1±5.1 versus 4.2±2.4%, P<0.05) compared to controls. Lesional apoptosis, as determined by in situ TUNEL analysis, was reduced ∼50% in CB2-/-Ldlr-/- lesions after 12 weeks. CB2-/-Ldlr-/- lesions displayed significantly reduced collagen content and increased elastin fiber fragmentation after 12 weeks, which was associated with an ∼57% increase in matrix metalloproteinase 9 (MMP) levels. In vitro, CB2-/- macrophages secreted ∼1.8-fold more MMP9 activity than CB2+/+ macrophages. Conclusions: CB2 receptor deficiency affects atherogenesis in Ldlr-null mice by increasing lesional macrophage and SMC content, reducing lesional apoptosis and altering extracellular matrix components, in part, by upregulating MMP9. These results suggest that pharmacological manipulation of CB2 receptors might exert multiple and complex effects on atherogenesis and plaque stability.

Newly designed pyrrolo[2,1-c][1,4]benzodiazepines tricyclic skeleton has shown potential clusters of cannabinoid receptors CB1/CB2 selective ligands. CB2 plays a critical role in microglial-derived neuroinflammation, where it modulates cell proliferation, migration, and differentiation into M1 or M2 phenotypes. Beginning with computer-based docking studies accounting the recently discovered X-ray crystal structure of CB2, we designed a series of PBD analogs as potential ligands of CB2 and tested their binding affinities. Interestingly, computational studies and theoretical binding affinities of several selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs, have revealed the presence of potential selectivity in binding attraction toward CB1 and CB2. Reported here is the discovery of the first representatives of this series of selective binding to CB2. Preliminary data showed that this class of molecules display potential binding efficacy toward the cannabinoid receptors tested. Intriguingly, initial cannabinoid binding assay showed a selective binding affinity of 4g and 4h showed Ki of 0.49 and 4.7 μM toward CB2 receptors while no binding was observed to CB1. The designed leads have shown remarkable stability pattern at the physiological pH magnifying their therapeutic values. We hypothesize that the PBD tricyclic structure offers the molecule an appropriate three-dimensional conformation to fit snugly within the active site of CB2 receptors, giving them superiority over the reported CB2 agonists/inverse agonists. Our findings suggested that the attachment of heterocyclic ring through the condensation of diazepine hydrazone and S- or N-heterocyclic aldehydes enhances the selectivity of CB2 over CB1. [Figure not available: see fulltext.].

No description available.

Several studies have reported that administration of cannabinoid receptor agonists in inflammatory/autoimmune and CNS injury models resulted in significant attenuation of clinical disease. The beneficial effects correlated with the observed reduction of inflammatory mediators and peripheral immune cell infiltration into the site of inflammation. Previous studies from our laboratories demonstrated that administration of cannabinoid type 2 receptor agonist attenuated disease score and improved recovery in two murine models of neuroinflammation; spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. The goal of the current investigation was to evaluate the mechanisms through which administration of selective cannabinoid-2 receptor (CB2R) agonists modify inflammatory responses and help to improve function in SCI and EAE. In SCI, an acute neuroinflammatory disorder, administration of CB2R agonist at 1 h and 24 h following contusion injury to the cord resulted in improved recovery of motor function and bladder function (the ability to spontaneously void) compared to control animals. Evaluation of inflammatory mediators at 48h demonstrated a dramatic reduction in the expression of the chemokines CXCL9, 10, 11 and cytokines IL-23 and its receptor in CB2R agonist-treated cords. There was also a reduction in the expression of toll-like receptors (TLR1, TLR4, TLR6, and TLR7), which correlated with a decreased number of immunoreactive microglia. Interestingly, at seven days post injury, CB2R agonist-treated injured cords showed a significant reduction in both hematopoietic and myeloid cell infiltration. In EAE, a chronic neuroinflammatory disorder, our laboratories demonstrated previously that administration of a CB2R agonist led to lower disease scores and improved recovery. In this study, we observed reduced numbers of infiltrating hematopoietic and myeloid cells into the spinal cord and brain of CB2 agonist-treated mice. This reduction was observed at the peak of disease (day 17) and the effect was maintained at the chronic stage of disease (day 30). Evaluation of molecules associated with cell migration showed decreased levels of the adhesion molecule VCAM-1 and matrix metalloproteinases MMP-2 and 9 at peak of EAE in treated mice. The decrease in VCAM-1 correlates with our previous observation of decreased leukocyte rolling and adhesion to brain microvasculature. However, the reduction in MMP-2/9 expression suggests that CB2R agonists may also affect leukocyte transmigration into the perivascular space and further infiltration into the CNS parenchyma. This process requires both chemokine cues and the gelatinases MMP2/9. Animals deficient in these MMPs show leukocyte accumulation in the perivascular space and are resistant to EAE. There are no reports in the literature on possible CB2R agonist effects on gelatinases in myeloid cells. Although both MMP-2 and -9 are produced by antigen-presenting cells and act on similar substrates, MMP-9 appears to play a crucial role in EAE. Therefore, we decided to examine the effects of CB2 signaling on MMP-9 expression in myeloid cells, focusing on myeloid bone marrow-derived dendritic cells (BMDC). Activation of bone marrow-derived macrophages, dendritic cells, and primary microglia with the cytokine cocktail TNFα, IL-1ß, IL-6, containing PGE2, which mimicked an inflammatory milieu, resulted in expression of high levels of MMP-9. Treatment with CB2R agonists reduced MMP-9 in all three cell types. Since migration of DC to various sites is required for their activation and for the initiation of adaptive immune responses, we evaluated the effects of CB2R agonists on migration. The reduced levels of MMP-9 correlated with reduced migration of DC to the draining lymph nodes in vivo, as well as reduced migration vitro in the matrigel migration assay. The effect on MMP-9 expression was mediated through CB2R, resulting in reduction in cAMP levels, subsequent decrease in ERK activation, and reduced binding of c-Fos and c-Jun to the AP-1 site in the MMP-9 promoter. We postulate that, by dampening production of MMP-9 and subsequent MMP-9-dependent DC migration, cannabinoids contribute to resolve acute inflammation and to reestablish homeostasis. Selective CB2R agonists might be valuable future therapeutic agents for the treatment of chronic inflammatory conditions by targeting activated immune cells including DC. / Physiology

Activation of the CB1 receptor is modulated by aspartate residue D2.63176 in transmembrane helix (TMH) II. Interestingly, D2.63 does not affect the affinity for ligand binding at the CB1 receptor. Studies in class A GPCRs have suggested an ionic interaction between residues of TMHII and VII. In this report, modeling studies identified residue K373, in the extracellular (EC)-3 loop, in charged interactions with D2.63. We investigated this possibility by performing reciprocal mutations and biochemical studies. D2.63176A, K373A, D2.63176A-K373A, and the reciprocal mutant with the interacting residues juxtaposed, D2.63176K-K373D were characterized using radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. None of the mutations resulted in a significant change in the binding affinity of CP55,940 or SR141716A. Computational results indicate that the D2.63176-K373 ionic interaction strongly influences the conformation(s) of the EC-3 loop, providing a structure-based rationale for the importance of the EC-3 loop to signal transduction in CB1. Specifically, the putative ionic interaction results in the EC-3 loop pulling over the top (extracellular side) of the receptor; this EC-3 loop conformation may serve protective and mechanistic roles. These results suggest that the ionic interaction between D2.63176 and K373 is crucial for CB1 signal transduction. This work may help to aide drug design efforts for the effective treatment of different diseases. The cannabinoid receptors of osteoblasts may represent a target for the treatment of bone disorders such as osteoporosis. Our research demonstrates that cannabinoids can affect important signaling molecules in osteoblasts. In MC3T3-E1 osteoblastic cells, the CB1 antagonist, AM251, has been reported to induce increases in Runx2 mRNA, mineralized bone nodule formation, and activation of signaling molecules such as ERK and AKT (Wu et al., 2011). Studies from our lab characterizing mice in which both CB1 and CB2 receptors were inactivated by homologous recombination have demonstrated increased bone mass coupled with enhanced osteoblast differentiation of bone marrow stromal cells in culture (manuscript in preparation). We explored the effect of antagonizing CB1 and CB2 cannabinoid receptors in osteoblastic cells to gain insights into molecular pathways that may help to explain the effects of the endocannabinoid system (ECS) in bone development. Our data was generated by running time course experiments with MC3T3-E1 cells under the influence of SR141716A, SR144528 or both in combination. The cells were harvested with a lysis buffer at specific time points and analyzed by western blot analysis. Quantification of protein activation was calculated using LiCor imaging equipment and software. Within 15 minutes, treatment with the CB1 receptor antagonist SR141716A resulted in several fold increases in pERK, pSMAD158, and pAKT. SR144528, a CB2 receptor antagonist, caused increases in pERK and pSMAD158, but not pAKT. When both antagonists were applied together, pERK and pSMAD158 levels increased, while pAKT signaling was diminished compared to SR141716A alone. The finding that cannabinoid receptor antagonists alter the activity of the SMAD158 complex is a novel finding, which suggests that cannabinoids can influence bone morphogenic signaling pathways, and therefore play a significant role in osteoblast differentiation and function. / Cell Biology