La leucémie lymphoïde chronique (LLC) est caractérisée par une accumulation anormale de lymphocytes B matures. Les thérapies actuelles reposent sur l'utilisation d'inhibiteurs ciblant les kinases impliquées dans la voie du récepteur des cellules B (BCR), mais elles sont limitées par le niveau élevé de résistance à l’apoptose des cellules leucémiques. En effet, celles-ci échouent à éradiquer les cellules résistantes à l'apoptose, il est donc essentiel d'identifier d'autres voies de survie comme nouvelles cibles pour les thérapies anticancéreuses. La surexpression des récepteurs de surface couplés aux protéines G (RCPGs) entraîne une transformation cellulaire et joue ainsi un rôle essentiel dans les tumeurs malignes. Dans cette étude, nous montrons que le récepteur de la neurotensine de type 2 (NTSR2), un récepteur couplé aux protéines G, est un acteur essentiel dans les mécanismes de résistance à l'apoptose dans les cellules leucémiques. Le récepteur NTSR2 est surexprimé et constitutivement actif dans les cellules leucémiques, son activation dépend de son interaction avec le récepteur TrkB (Tropomyosin-related kinase B) et du recrutement des protéines Giα à la place de son interaction avec son ligand naturel, la neurotensine (NTS). L'interaction NTSR2-TrkB agit comme un oncogène conditionnel nécessitant le BDNF (Brain-Derived Neurotrophic Factor), le ligand de TrkB, qui est fortement exprimé dans les cellules B leucémiques, contrairement à son ligand naturel la NTS. L'interaction NTSR2-TrkB active les voies de signalisation de survie, y compris les voies de Src et Akt, ainsi que l'expression des protéines anti-apoptotiques Bcl-2 (B-cell lymphoma-2) et Bcl-xL (B-cell lymphoma-extra large). Néanmoins le récepteur TrkB seul ne protège pas les cellules B leucémiques d'une diminution drastique de la viabilité par apoptose lorsque NTSR2 est inactivé. L’ensemble de ces résultats suggèrent que l'interaction NTSR2-TrKB et l'activation soutenue des voies de signalisation dépendante de cette interaction constituent un mécanisme essentiel d’échappement à l'apoptose des cellules B leucémiques. Le ciblage du récepteur NTSR2 représente une stratégie prometteuse pour le traitement de cette pathologie. / Chronic lymphocytic leukemia (CLL) is characterized by the abnormal accumulation of mature B lymphocytes. Current therapies for CLL rely on using kinase inhibitors targeting B-cell receptor (BCR) pathways, but they are limited by the high level of apoptosis-resistant B-CLL cells, which results in a high frequency of patient relapse. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors (GPCRs) drives cell transformation, and thus plays a critical role in malignancies. In this study, we show that neurotensin receptor 2 (NTSR2), a G-protein-coupled receptor, is an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed and constitutively active in B-CLL cells, and its activation depended on its interaction with the tropomyosin-related kinase B receptor (TrkB) and the recruitment of Gi proteins, instead of its interaction with its natural ligand, neurotensin (NTS). The NTSR2-TrkB interaction acted as a conditional oncogenic driver requiring the TrkB ligand BDNF (Brain-Derived Neurotrophic Factor), which is highly expressed in B-CLL cells, unlike its natural ligand NTS. The NTSR2-TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-xL (B-cell lymphoma-extra large). TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death when NTSR2 was down-regulated. Taken together, the results suggest that the NTSR2-TrKB interaction and the sustained activation of signaling pathways reliant on this interaction constitute an essential driving force for apoptosis evasion of B-CLL cells. Targeting NTSR2 could represent a promising strategy for treating B-CLL malignancy.

Meduloblastoma é o tumor maligno intracranial mais comum em crianças. A desregulação da sinalização BDNF/TrkB tem sido associada a aumento da proliferação, invasão e resistência a quimioterapia, em diversos tipos de câncer, incluindo tumores de sistema nervoso. No entanto, seus efeitos biológicos e relevância clínica em meduloblastoma não estão compreendidos. Neste estudo foram analisados os efeitos do inibidor seletivo de TrkB, ANA-12, na viabilidade, sobrevivência e ciclo celular de linhagens de meduloblastoma humano. Este estudo demonstrou que o bloqueio seletivo de TrkB reduziu significativamente a viabilidade e sobrevivência de linhagens celulares representativas de diferentes subgrupos moleculares de meduloblastoma. Estes resultados fornecem uma base racional para investigar a inibição de TrkB como uma nova e potencial estratégia para o tratamento de meduloblastoma. / Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Deregulation of BDNF/TrkB signaling has been associated with increased proliferative capabilities, invasiveness and chemo-resistance in several types of cancer. However, the relevance of this pathway in MB remains unknown. Here, we show that the selective TrkB inhibitor ANA-12 markedly reduced the viability and survival of human cell lines representative of different MB molecular subgroups. These findings provide a rationale to further investigate TrkB inhibition as a potential novel strategy for MB treatment.

Os efeitos benéficos da administração crônica de cafeína sobre a memória têm sido observados em diferentes condições e modelos animais, mas os mecanismos subjacentes aos seus efeitos permanecem desconhecidos. Este estudo buscou investigar se a administração crônica de cafeína pode melhorar o desempenho em uma tarefa de memória em ratos adultos e de meia-idade. Além disso, os efeitos da cafeína sobre o imunoconteúdo do fator neurotrófico derivado encéfalo(BDNF) foi analisado para estabelecer uma conexão entre os achados comportamentais e BDNF, uma das neurotrofinas estritamente envolvida na memória e processos de aprendizagem. Além disso, analisou-se o imunoconteúdo do receptor tirosina cinase (Trk B), o precursor do BDNF (proBDNF) e o fator de transcrição CREB. Ratos Wistar adultos (2 meses) e de meia-idade (12 meses de idade) receberam água ou cafeína (1 mg / mL) na água de beber durante 30 dias. Ambos os grupos foram submetidos às tarefas de avaliação da atividade locomotora e esquiva inibitória. Ratos de meia-idade apresentaram uma diminuição da atividade locomotora em relação aos adultos e o tratamento com cafeína não modificou esse parâmetro em nenhuma das idades. Na tarefa de esquiva inibitória, a memória de curta e longa duração foi avaliada. Ratos de meia-idade apresentaram um comprometimento total da memória de curta duração em relação aos adultos. Quando a memória de longa duração foi avaliada, os ratos de meia-idade apresentaram uma diminuição do seu desempenho em relação aos adultos, e tratamento de cafeína foi capaz de melhorar esse desempenho. Análise de Western blot do hipocampo de ratos tratados com cafeína revelou um aumento do imunoconteudo de BDNF no hipocampo em ratos de meia-idade, um efeito atenuado pelo tratamento crônico de cafeína. Além disso, o tratamento com cafeína aumentou o imunoconteúdo de pro-BDNF e CREB em ambas as idades, e, ainda, foi encontrado um aumento do imunoconteúdo de CREB em ratos de meia-idade. O imunoconteúdo de TrkB diminuiu no hipocampo de ratos de meia-idade quando comparados aos adultos, e o tratamento com cafeína foi capaz de diminuir o imunoconteúdo de TrkB em ambas as idades. Os dados encontrados indicam uma estreita associação entre a modificação do desempenho da memória e imunoconteúdo BDNF. Portanto, nossos dados sugerem que a cafeína é capaz de normalizar o desempenho da memória durante o envelhecimento e pode estar relacionada à capacidade da cafeína de normalizar os níveis de BDNF. / The beneficial effects of chronic caffeine administration in memory performance have been observed in different conditions and animal models but the underlying mechanisms remain unknown. This study was designed to investigate whether chronic caffeine administration could improve the performance in different memory tasks used in adult and middle-aged rats. In addition, the effects of caffeine on brain derived neurotrophic factor (BDNF) immunocontent was analyzed to establish a connection between the behavioral findings and BDNF, one of the neurotrophins strictly involved in memory and learning processes. Moreover, it was analyzed the immunocontent of tirosine kinase receptor (Trk B), the precursor of BDNF (proBDNF) and the transcription factor CREB. Adult (2 months old) and middle-aged (12 months old) Wistar rats received either drinking water or caffeine (1 mg/mL) during 30 days. Both groups were submitted to open field and inhibitory avoidance tasks. Middle-aged rats presented decreased locomotor activity as compared to adults and caffeine was devoid of effect at any age. In the inhibitory avoidance task, short- and long-term memory was evaluated. Middle-aged rats presented impaired performance compared to adult ones for short-term memory. When long-term memory was evaluated, middle-aged rats showed a decreased in their perfomances compared to adult rats, and caffeine treatment was able to improve it. Western blot analysis of hippocampus from caffeine-treated rats revealed that BDNF increased by aging and caffeine treatment prevented it. In addition, caffeine treatment increased the pro-BDNF and CREB immunocontent in both ages. Furthermore, CREB densities increased with aging. TrkB immunocontent was decreased in the hippocampus from middle-aged rats when compared to adult ones, and caffeine decreased the density of TrkB in both ages. The present findings indicate a close association between the modification of memory performance and BDNF immunocontent. Therefore, our data suggest caffeine normalyze memory performance upon aging and may be related to the ability of caffeine to normalyze the levels of BDNF.

Le virus T lymphotropique humain de type 1 (HTLV-1) est l’agent étiologique d’une forme rare et très agressive de leucémie de l’adulte (ATL). Le processus leucémogène a longtemps été attribué à la seule action de l’oncoprotéine Tax. Cependant, une nouvelle protéine virale, appelée HBZ (HTLV-1 bZIP factor), a été découverte en 2002. Elle est codée par le brin complémentaire du génome proviral et transcrite en antisens à partir du LTR3’. HBZ s’est avéré être un acteur clef de la prolifération et de la transformation des cellules T infectées, et donc du développement de l’ATL. La présente étude propose de nouvelles pistes quant aux mécanismes par lesquels HBZ induit la survie et la prolifération cellulaire. Nous avons montré que la protéine HBZ stimule l’expression de la neurotrophine BDNF et que les cellules de patients ATL surexpriment à la fois BDNF et son récepteur TrkB. De plus, ces patients présentent une concentration sérique anormalement élevée de la forme mature de BDNF, suggérant l’existence d’une boucle autocrine/paracrine BDNF/TrkB. L’activité de cette boucle a été confirmée in vitro et promeut la survie des cellules infectées par HTLV-1. D’autre part, nous avons découvert qu’HBZ dérégule l’expression du suppresseur de tumeur JunD dans les cellules T infectées, et induit celle de l’isoforme potentiellement oncogène ΔJunD. La production de ΔJunD résulterait d’une altération des mécanismes d’initiation de la traduction par HBZ. Nos résultats montrent aussi que ΔJunD promeut la prolifération et la transformation cellulaire en l’absence de sérum. Nous proposons donc que son expression pourrait contribuer à l’évolution des cellules T infectées en cellules leucémiques. / The human T-lymphotropic virus type 1 (HTLV-1) is associated with a rare and aggressive form of adult leukemia (ATL). For a long time, leukemogenesis was thought to mainly result from the action of the Tax oncoprotein. However, a new viral protein, called HBZ (HTLV-1 bZIP factor), was discovered in 2002. It is encoded by the minus strand of the proviral genome and transcribed in antisens from the 3’LTR. Since its discovery, HBZ came out as a key player in proliferation and transformation of infected T cells, thus contributing to ATL development. In this study we provide new leads regarding the mechanisms of HBZ-induced cell survival and proliferation. On one hand, we show that HBZ stimulates the expression of the BDNF neurotrophin and that ATL cells from patients overexpress both BDNF and its high-affinity receptor TrkB. Moreover, sera from patients exhibited abnormal levels of the mature form of BDNF, suggesting the existence of a BDNF/TrkB paracrine/autocrine loop. That loop was confirmed to be activated in vitro and to support the survival of HTLV-1-infected cells. On the other hand, we discovered that HBZ deregulates the expression of the JunD tumor suppressor in infected T cells and induces that of the ∆JunD isoform, which is potentially oncogenic. ∆JunD production would result from alteration of the translational initiation by HBZ. Our results also show that ∆JunD induces proliferation and transformation of serum starved cells. Finally, we hypothesize that HBZ-induced expression of ∆JunD may influence infected T cells to turn leukemic.

Neurotrophins BDNF and NT4 influence the development of the rodent gustatory system. Despite binding to the same receptor, TrkB, they have different roles. BDNF is chemo-attractive for gustatory neurons and regulates gustatory neuron targeting and number during development. NT4 regulates gustatory neuron number earlier in development than BDNF, but it is not chemo-attractive and does not regulate gustatory neuron targeting. To elucidate the mechanisms that regulate these processes we have examined which TrkB intracellular signalling pathways are required for specific aspects of gustatory development by studying the effect of specific point mutations in TrkB docking sites. We found that the TrkB/Shc docking site is involved in regulating the survival of geniculate ganglion neurons as a point mutation in this adaptor site (TrkbS/S) caused large losses of these neurons as early as E12.5. These losses were exacerbated throughout development until after birth. A point mutation in the TrkB/PLCγ (TrkbP/P) docking site did not cause loss of geniculate ganglion neurons at any point during development. Animals with a point mutation in both docking sites (TrkbD/D) caused a further decrease in neuron numbers compared to animals with a mutation in only one of the docking sites, similarly to what has previously been shown in Trkb null animals. We concluded that the TrkB/Shc docking site is crucial for determining the survival of geniculate ganglion neurons during mouse gustatory development, while the TrkB/PLCγ docking site does not affect the neuronal survival directly and likely plays a role in maintenance of these neurons. Examining the targeting of geniculate ganglion afferents into the tongue revealed large deficits in innervated neural bud and taste bud numbers in TrkbS/S animals both before and after birth. This was concluded to be reflecting the lack of neuronal survival in this ganglion, a result that was mirrored in TrkbD/D animals. TrkbP/P animals, on the other hand, exhibited a developmental delay in innervation. This was indicated by a low amount of innervated neural buds following the initial innervation period, which was compensated for by a large increase in the number of innervated taste buds by birth. By adulthood, the numbers of taste buds present on the tongues of TrkbP/P animals reached normal numbers compared to control animals. This suggested that the TrkB/PLCγ docking site is involved primarily in innervation. Finally, we examined the morphology of taste buds in newly born and adult animals. We found that the low amount of geniculate ganglion afferents innervating the tongue in TrkbS/S and TrkbD/D animals caused a decrease in size of taste buds. This effect was seen to be partially rescued by adulthood in TrkbS/S animals but not in TrkbD/D animals due to lack of viability. The morphology of taste buds was unaffected in TrkbP/P animals until adulthood, at which point the size of the taste buds was increased. These results are in agreement with previous findings showing dependency of taste bud morphology on the amount of innervation. Overall, our findings show a differential role of TrkB adaptor sites in gustatory development. Despite activated by the same ligands, the docking sites on this receptor are able to exert different influence on signalling pathways downstream of TrkB affecting neuronal survival, targeting and morphology of taste buds.

<p>Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.</p> / Dissertation

<p>Temporal lobe epilepsy is a common and devastating disorder that features recurrent seizures and is often associated with pathologic anxiety and hippocampal sclerosis. An episode of prolonged seizures (status epilepticus) is thought to promote development of human temporal lobe epilepsy years later. A chemical-genetic approach established proof of concept that transiently inhibiting the receptor tyrosine kinase, TrkB, following status epilepticus prevented epilepsy, anxiety-like behavior and hippocampal damage in a mouse model, providing rationale for developing a therapeutic targeting TrkB signaling. To circumvent the undesirable consequence that global inhibition of TrkB exacerbates neuronal degeneration following status epilepticus, we sought to identify both the TrkB-activated signaling pathway mediating these pathologies and a compound that uncouples TrkB from the responsible signaling effector. To accomplish these goals, we used genetically modified mice and a model of seizures and epilepsy induced by a chemoconvulsant. Genetic inhibition of TrkB-mediated phospholipase C gamma 1 (PLC gamma 1) signaling suppressed seizures induced by a chemoconvulsant, leading to design of a peptide (pY816) that inhibited the interaction of TrkB with PLC gamma 1. We demonstrate that pY816 selectively inhibits TrkB-mediated activation of PLC gamma 1 both in vitro and in vivo. Treatment with pY816 prior to administration of a chemoconvulsant suppressed seizures in a dose- and time-dependent manner. Treatment with pY816 initiated after chemoconvulsant-evoked status epilepticus and continued for just three days suppressed seizure-induction of epilepsy, anxiety-like behavior and hippocampal damage assessed months later. This study elucidates the signaling pathway by which TrkB activation produces diverse neuronal activity-driven pathologies and demonstrates therapeutic benefits of an inhibitor of this pathway in an animal model in vivo. A strategy of uncoupling a receptor tyrosine kinase from a signaling effector may prove useful in diverse diseases in which excessive receptor tyrosine kinase signaling contributes.</p> / Dissertation

<p>Understanding the mechanisms of limbic epileptogenesis in cellular and molecular terms may provide novel therapeutic targets for its prevention. The neurotrophin receptor tropomyosin-related kinase B (TrkB) is thought to be critical for limbic epileptogenesis. Enhanced activation of TrkB, revealed by immunodetection of enhanced phosphorylated TrkB (pTrkB), a surrogate measure of its activation, has been identified within the hippocampus in multiple animal models. Knowledge of the cellular locale of activated TrkB is necessary to elucidate its functional consequences. Using an antibody selective to pTrkB in conjunction with confocal microscopy and cellular markers, we determined the cellular and subcellular locale of enhanced pTrkB induced by status epilepticus (SE) evoked by infusion of kainic acid into the amygdala of adult mice. SE induced enhanced pTrkB immunoreactivity in two distinct populations of principal neurons within the hippocampus--the dentate granule cells and CA1 pyramidal cells. Enhanced immunoreactivity within granule cells was found within mossy fiber axons and giant synaptic boutons. By contrast, enhanced immunoreactivity was found within apical dendritic shafts and spines of CA1 pyramidal cells. A common feature of this enhanced pTrkB at these cellular locales is its localization to excitatory synapses between excitatory neurons, presynaptically in the granule cells and postsynaptically in CA1 pyramidal cells. Long-term potentiation (LTP) is one cellular consequence of TrkB activation at these excitatory synapses that may promote epileptogenesis.</p><p>The importance of TrkB in diverse neuronal processes, as well as its involvement in various disorders of the nervous system, underscores the importance of understanding how it is activated. The canonical neurotrophin ligand which activates TrkB is brain derived neurotrophic factor (BDNF). Zinc, however, has also been demonstrated to activate this receptor through a mechanism whereby it does not directly interact with it, known as transactivation. Presynaptic vesicles of mossy fiber boutons of stratum lucidum are particularly enriched in zinc, where it is co-released with glutamate in an activity dependent fashion, and incorporated into these vesicles by the zinc transporter, ZnT3. Given the presence of large quantities of zinc within stratum lucidum, we hypothesized that this metal may contribute to TrkB transactivation at this locale. To this end, we examined the contributions of both BDNF and synaptic vesicular zinc to TrkB activation in stratum lucidum of mouse hippocampus under physiological conditions. Utilization of mice which are genetic knockouts for BDNF and/or ZnT3 allowed us to examine TrkB activation in the absence of one or both of these ligands. This was done using an antibody for pTrkB in conjunction with confocal microscopy, assaying immunoreactivity at the cellular and synaptic locales within stratum lucidum where pTrkB was previously found to be enriched. Our results suggest that BDNF contributes to TrkB activation within stratum lucidum. Interestingly, ZnT3 mice displayed an increase in BDNF protein and TrkB activation, demonstrating that synaptic zinc regulates BDNF and TrkB signaling at this locale.</p> / Dissertation

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 146-166).

Recentemente suspeitas de que mecanismos epigenéticos poderiam estar relacionados à fisiopatologia da depressão foram levantadas. Estudos recentes indicam que as alterações na transcrição gênica, induzidas por estresse ou por drogas antidepressivas, parecem envolver mecanismos epigenéticos. Nesse sentido, resultados preliminares de nosso grupo de pesquisa indicaram pioneiramente inibição global da metilação de DNA através da administração sistêmica do agente inibidor da DNA metiltransferase (DNMTs), 5-aza-2-deoxicitidina (5-azaD), induz efeito tipo-antidepressivo, dose-dependente, no modelo animal do nado forçado em ratos(Sales et al., 2011). O córtex pré-frontal medial ventral (CPFMv) é uma estrutura límbica intimamente relacionada com a neurobiologia da depressão. Evidências recentes indicam que o efeito tipo-antidepressivo aparece associado a aumento dos níveis da neurotrofina BDNF (brain derived neurotrophic factor) e de seu receptor TrkB no CPFMv, sendo a sinalização intracelular mediada pela ativação da proteína m-TOR. Contudo, não há evidências de que esses mecanismos moleculares estariam envolvidos nos efeitos induzidos pelos inibidores da metilação do DNA. Sabe-se, no entanto, que tanto o BDNF quanto TrkB têm sua expressão regulada por metilação do DNA. Diante disso, o objetivo presente trabalho será investigar a participação da via BDNF-TrkB-mTOR do CPFMv no efeito antidepressivo induzido por inibidores da metilação de DNA. Para tanto, ratos tratados com inibidores da metilação de DNA (5-azaD ou RG-108), em dois momentos diferentes (imediatamente após o PT e 23horas após o PT) foram submetidos ao teste do nado forçado (FST). Outro grupo de animais recebeu uma injeção intra-CPMv de k252a ou Rapamicina, 40 minutos antes do teste e uma injeção de BDNF intra-CPFMv, 30 minutos antes do teste. Em outro experimento, grupos independentes de animais submetidos ao nado forçado foram tratados sistemicamente com RG-108 e receberam injeção intra-CPFMv de K252a (antagonista de Trk) ou de rapamicina (inibidor da m-Tor), a fim de investigar se o efeito dessas drogas depende da via BDNF-TrkB-mTOR no CPFMv. Um grupo independente foi tratado com RG108 e CPFM desses animais foi dissecado para posterior análise da expressão de BDNF, TrkB e m-TOR, bem como da metilação de DNA. O tratamento com RG108 e 5azaD sistêmico reduziu o tempo de imobilidade dos animais submetidos ao nado forçado nos dois tempo de administração. A administração intra-CPFMv de BDNF promoveu efeito antidepressivo no FST, e esse efeito foi bloqueado pela administração de k252a ou Rapamicina no CPFMv. No mesmo sentido, o efeito antidepressivo do RG108 sistêmico foi bloqueado pela administração intra-CPFMv de k252a ou Rapamicina. Entretanto, a medida dos níveis de metilação global no CPFMv não apresentou alteração como tratamento com RG108, e também não mostrou alteração nos níveis de BDNF presente no CPF. O tratamento com RG108 não alterou a expressão, bem como a ativação de TRkB e mTOR. Concluímos que os inibidores da metilação do DNA apresentam agudamente efeito tipo antidepressivo rápido, que necessita da funcionalidade integral da via BDNF-TRkB-mTOR. Entretanto, esse efeito parece não alterar a síntese e expressão das proteínas envolvidas nessa via no que diz respeito ao CPFmv. / Recent studies indicate that changes in gene transcription induced by stress or antidepressant drugs appear to involve epigenetic mechanisms. Accordingly, results of our research group pioneered indicated global inhibition of DNA methylation through systemic administration of an inhibitor of DNA methyltransferase (DNMTs), 5-aza-2-deoxycytidine (5-AzaD), induces antidepressant-like effect dose-dependent in the animal model of forced swimming in rats (Sales et al., 2011). The ventral medial prefrontal (vmPFC) cortex is a limbic structure closely related to the neurobiology of depression. Recent evidence indicates that the antidepressant-like effect appears associated with increased levels BDNF (Brain derived neurotrophic factor) and its receptor TrkB in vmPFC, and intracellular signaling mediated by activation of protein mTOR. However, there is no evidence that these molecular mechanisms are involved in the effects induced by inhibitors of DNA methylation. It is known, however, both as BDNF and TrkB expression is regulated by DNA methylation. Thus, the goal of this work is to investigate the role of BDNF-TRkB pathway mTOR-vmPFC in the antidepressant effect induced by inhibitors of DNA methylation. To this end, rats treated with inhibitors of DNA methylation (5-Azad or RG-108), at two different times (immediately after 23hours after the PT and PT) were subjected to the forced swim test (FST). Another group received an intra-vmPFC injection of K252a or Rapamycin 40 minutes before the test, and an injection intra-vmPFC of BDNF 30 minutes before the test. In another experiment, separate groups undergoing the forced swim were treated systemically with RG-108 and received intra-vmPFC of K252a (Trk antagonist) or injection of rapamycin (m-Tor inhibitors) in order to investigate the effect these drugs depends on BDNF-TrkB-mTOR pathway in vmPFC. A separate group was treated with RG108 and mPFC these animals were dissected for analysis of the expression of BDNF and TrkB m-TOR, as well as DNA methylation. The systemic treatment whit 5azaD and RG108 reduced the immobility time of rats subjected to FST administration in both time. The intra-vmPFC BDNF administration promoted antidepressant effect in the FST, and this effect was blocked by the administration of K252a or Rapamycin in vmPFC. Similarly, the antidepressant effect of systemic RG108 was blocked by intra-vmPFC of K252a or Rapamycin administration. However, the measurement of the levels of global methylation in CPFMv did not change as treatment with RG108, and also showed no change in the levels of BDNF present in the CPF. Treatment with RG108 did not alter the expression and activation of TrkB and mTOR. We conclude that inhibitors of DNA methylation present acutely antidepressant-like effect, it needs the full functionality of the BDNF-TrkB-mTOR pathway. However, this effect seems not to alter the synthesis and expression of proteins involved in this pathway at vmPFC.