Indiana University-Purdue University Indianapolis (IUPUI) / The serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are unequivocally useful for lowering cholesterol levels in patients with dyslipidemias. In addition to cholesterol lowering properties, statins exert a number of pleiotropic, vascular-protective effects include improvement of endothelial function, increased nitric oxide (NO) bioavailability, antioxidant properties. Since endothelial dysfunction and reactive oxygen species (ROS) are important pathophysiological determinants of essential hypertension, these actions of statins raise the possibility that statin therapy may be useful for simultaneously clinical hypertension management. However, the signaling mechanisms of statins that improve hypertension remain unclear. Our previous study showed, in the NTS, insulin may decrease blood pressure and heart rates through PI3K-Akt-eNOS pathway, and NTS integrates convergent information from peripheral baroreceptors and central cardiovascular regulatory center. Statins also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway, members of the Ras and Rac1 GTPase family are major substrates for posttranslational modification by isoprenylation and may be important targets for inhibition by statins. Statins could inhibit Rac1 isoprenylation and Rac1-mediated nicotinamide adenine dinucleotide phosphate oxidase activity attenuates reactive oxygen species production. The aim of this study was to investigate the possible signaling pathways involved in simvastatin-mediated blood pressure regulation in the nucleus tractus solitarii (NTS). Male 20-week-old spontaneously hypertensive rats (SHR) were divided into two groups: control group and intracerebroventricular injection with simvastatin group for three days. We found that systolic blood pressure measured with tail-cuff method of the simvastatin-treated rats decreased significantly, and the NO level in the NTS was significantly increased. In addition, we observed that simvastatin could lower the ROS level and increase Ras GTPase activity in the NTS. Immunoblotting and immunohistochemistry analysis further showed that simvastatin increased the phosphorylation ratio of ERK1/2, Akt, and endothelial nitric oxide synthase (eNOS) in the NTS. Taken together, these results suggest that eNOS signaling in the NTS may play an important role in simvastatin-induced blood pressure lowering effects. Keywords: statins, nucleus tractus solitarii, nitric oxide, oxidative stress, central cardiovascular regulatory, isoprenylation

A malária mata mais de um milhão de pessoas por ano, sendo uma das doenças infecciosas mais relevantes e um grande problema de saúde pública. Além disso, o surgimento de cepas resistentes aos quimioterápicos utilizados faz necessário o estudo de novos alvos para tratamentos contra esta doença. No nosso laboratório foi demonstrada a biossíntese de isoprenóides, em P. falciparum pela via MEP. Sabe-se que substâncias inibidoras da biossíntese de isoprenóides, dentre essas os terpenos, apresentam atividade antimalárica. Levando em consideração o anterior, nós avaliamos o potencial antimalárico do álcool perilico (POH) em P. falciparum e P. berghei. Nossos resultados demonstraram que o POH teve efeito inibitório contra o crescimento do P. falciparum in vitro, nas cepas 3D7 e K1 com uma IC50 de 4,8 ± 0,5 &mu;M, e 10,41±2,33 &mu;M, respectivamente. Além disso, o POH não teve efeito tóxico na linhagem celular Vero. Ainda, Comprovamos que o POH inibiu a farnesilação de proteinas entre 20 e 37 KDa de P. falciparum. Por outro lado, os experimentos in vivo não mostraram eficácia do tratamento do POH contra PbGFP em camundongos Balb/c. Em contraste, foi demostrada a eficácia do POH na de malária cerebral experimental (MCE), , indicando uma redução na taxa de incidência da MCE no grupo tratado com POH, comparado o não tratado ( P<0,05). Além disso, o POH reduziu a inflamação no cérebro dos animais tratados, uma vez que teve uma redução significativa na adesão de leucócitos aos vasos cerebrais (P<0.001), como também, o numero de hemorragias foi menor comparados com os animais não tratados. (P<0.0001). Portanto, os resultados obtidos nesta pesquisa abrem novas alternativas no estudo do mecanismo de ação do POH como um terpeno com grande potencial para tratar MC. / Malaria kills over one million people a year worldwide, and is one of the most important infectious diseases and a major public health problem. Furthermore, the emergence of resistant strains to chemotherapeutic agents used, make it necessary to study new targets for treatments against this disease. In our laboratory we have demonstrated the isoprenoids biosynthesis in P. falciparum, by the MEP pathway. It is known that the substances that inhibit isoprenoid biosynthesis, among these terpenes, have antimalarial activity in vitro and in vivo. Considering this, we evaluate the antimalarial potential of PA (POH) in P. falciparum and P. berghei. Our results showed that the POH had inhibitory effect against the growth of strains 3D7 and K1 of P. falciparum in vitro, with an IC50 of 4.8 &mu;M ± 0.5, and 10.41 ± 2.33 &mu;M, respectively. Furthermore, the POH had no toxic effect on cell line Vero. Moreover, the POH proved that inhibited proteins farnesylation from 20 to 37 kDa of P.falciparum. On the other hand, in vivo experiments did not show efficacy on treatment against POH PbGFP in BALB/c mice. In contrast, the effectiveness of POH in the experimental cerebral malaria (MCE) was demonstrated, indicating a reduction in the incidence rate of MCE in the group treated with POH, compared with of untreated animals (P <0.05). In addition, the POH reduced inflammation in the brain of treated animals, since it had a significant reduction in leukocyte adhesion to cerebral vessels (P <0.001), as also the number of bleeding was lower compared to untreated animals (P<0.0001). Therefore, the results obtained in this work provide new alternatives to study the POH\'s mechanism of action as a terpene with great potential to treat MC.

A malária mata mais de um milhão de pessoas por ano, sendo uma das doenças infecciosas mais relevantes e um grande problema de saúde pública. Além disso, o surgimento de cepas resistentes aos quimioterápicos utilizados faz necessário o estudo de novos alvos para tratamentos contra esta doença. No nosso laboratório foi demonstrada a biossíntese de isoprenóides, em P. falciparum pela via MEP. Sabe-se que substâncias inibidoras da biossíntese de isoprenóides, dentre essas os terpenos, apresentam atividade antimalárica. Levando em consideração o anterior, nós avaliamos o potencial antimalárico do álcool perilico (POH) em P. falciparum e P. berghei. Nossos resultados demonstraram que o POH teve efeito inibitório contra o crescimento do P. falciparum in vitro, nas cepas 3D7 e K1 com uma IC50 de 4,8 ± 0,5 &mu;M, e 10,41±2,33 &mu;M, respectivamente. Além disso, o POH não teve efeito tóxico na linhagem celular Vero. Ainda, Comprovamos que o POH inibiu a farnesilação de proteinas entre 20 e 37 KDa de P. falciparum. Por outro lado, os experimentos in vivo não mostraram eficácia do tratamento do POH contra PbGFP em camundongos Balb/c. Em contraste, foi demostrada a eficácia do POH na de malária cerebral experimental (MCE), , indicando uma redução na taxa de incidência da MCE no grupo tratado com POH, comparado o não tratado ( P<0,05). Além disso, o POH reduziu a inflamação no cérebro dos animais tratados, uma vez que teve uma redução significativa na adesão de leucócitos aos vasos cerebrais (P<0.001), como também, o numero de hemorragias foi menor comparados com os animais não tratados. (P<0.0001). Portanto, os resultados obtidos nesta pesquisa abrem novas alternativas no estudo do mecanismo de ação do POH como um terpeno com grande potencial para tratar MC. / Malaria kills over one million people a year worldwide, and is one of the most important infectious diseases and a major public health problem. Furthermore, the emergence of resistant strains to chemotherapeutic agents used, make it necessary to study new targets for treatments against this disease. In our laboratory we have demonstrated the isoprenoids biosynthesis in P. falciparum, by the MEP pathway. It is known that the substances that inhibit isoprenoid biosynthesis, among these terpenes, have antimalarial activity in vitro and in vivo. Considering this, we evaluate the antimalarial potential of PA (POH) in P. falciparum and P. berghei. Our results showed that the POH had inhibitory effect against the growth of strains 3D7 and K1 of P. falciparum in vitro, with an IC50 of 4.8 &mu;M ± 0.5, and 10.41 ± 2.33 &mu;M, respectively. Furthermore, the POH had no toxic effect on cell line Vero. Moreover, the POH proved that inhibited proteins farnesylation from 20 to 37 kDa of P.falciparum. On the other hand, in vivo experiments did not show efficacy on treatment against POH PbGFP in BALB/c mice. In contrast, the effectiveness of POH in the experimental cerebral malaria (MCE) was demonstrated, indicating a reduction in the incidence rate of MCE in the group treated with POH, compared with of untreated animals (P <0.05). In addition, the POH reduced inflammation in the brain of treated animals, since it had a significant reduction in leukocyte adhesion to cerebral vessels (P <0.001), as also the number of bleeding was lower compared to untreated animals (P<0.0001). Therefore, the results obtained in this work provide new alternatives to study the POH\'s mechanism of action as a terpene with great potential to treat MC.

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