BACE1 Inhibition Increases Susceptibility to Oxidative Stress by Promoting Mitochondrial Damage

BACE1 is a key enzyme facilitating the generation of neurotoxic β-amyloid (Aβ) peptide. However, given that BACE1 has multiple substrates we explored the importance of BACE1 in the maintenance of retinal pigment epithelial (RPE) cell homeostasis under oxidative stress. Inhibition of BACE1 reduced mitochondrial membrane potential, increased mitochondrial fragmentation, and increased cleaved caspase-3 expression in cells under oxidative stress. BACE1 inhibition also resulted in significantly lower levels of mitochondrial fusion proteins OPA1 and MFN1 suggesting a higher rate of mitochondrial fission while increasing the levels of mitophagic proteins Parkin and PINK1 and autophagosome numbers. In contrast, BACE2 had minimal effect on cellular response to oxidative stress. In summary, our results emphasize the importance of BACE1 in augmenting cellular defense against oxidative stress by protecting mitochondrial dynamics

Effects if Plasmodium berghei on thymus : high levels of apoptosis and premature egress of CD4+ CD8+ thymocytes in experimentally infected mice

Orientador: : Liana Maria Cardoso VerinaudDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O timo é um órgão linfóide primário, responsável pela diferenciação, maturação e desenvolvimento dos linfócitos T (ou timócitos, enquanto no timo). Este órgão apresenta complexa arquitetura tímica composta por elementos celulares e solúveis responsáveis pela migração dos precursores de linfócito T do espaço perivascular para a região cortical e subseqüente migração para a região medular. A trajetória do timócitos pelos microambientes tímicos permite que estas células recebam estímulos que os tornam linfócitos maduros, simples positivos TCD4+ ou TCD8+, prontos para migrar para os órgãos linfóides secundários. Durante a maturação, timócitos que são auto-reativos ou anérgicos são eliminados pelo processo de morte celular programada. Entretanto, a desregulação da apoptose nos linfócitos pode acarretar no desenvolvimento de doenças auto-imunes, tumores e imunodeficiências. Estudos em modelos murinos de atrofia tímica relatam que em diversas infecções parasitárias, virais, helmínticas, bacterianas e fúngicas e até mesmo em distúrbio metabólico como a diabetes experimental o timo é um órgão alvo. Nestes modelos, foi observado intenso acometimento tímico com involução do órgão acompanha por desarranjo da arquitetura tímica e depleção de todas as subpopulações de timócitos. Em estudos anteriores, observamos profundas alterações no timo durante a infecção experimental com Plasmodium berghei que incluíam severa atrofia no décimo quarto dia após inoculação do parasita, alterações histológicas, perda da delimitação cortico-medular, redução de todas sub-populações de timócitos e a presença intra-tímica do parasita. No presente trabalho avaliamos se a grande perda de timócitos observada no timo de animais infectados pelo Plasmodium berghei NK65 é devido a um aumento da ocorrência de apoptose, e/ou necrose, ou se é devida à saída prematura destas células para a periferia.A avaliação de morte celular na população de timócitos total revelou aumento no número de células Anexina-V+/PI- em animais infectados quando comparados aos controles.Ainda, a análise da apoptose em timos atróficos, no pico da parasitemia, revelou aumento de núcleos apoptóticos nas regiões cortical e medular tímica bem como aumento no número de linfócitos (CD4+, DP e DN) Anexina-V positivos. Alteração na expressão de genes pró-apoptóticos no timo de animais infectados quando comparado ao controle, sugerindo a participação de vias de ativação extrínsecas especificas da apoptose, também foram observadas. Embora 90% dos timócitos de animais infectados sejam Anexina-V positivos, as alterações na expressão dos elementos de matriz extracelular, relatadas anteriormente pelo nosso grupo, sugere também a exportação precoce destas células para a periferia do sistema imune. Nossos resultados apontam para um aumento de linfócitos imaturos (DP e DN) nos linfonodos mesentéricos de animais infectados, um indicativo de que parte dos timócitos imaturos sai do timo antes de completarem seu amadurecimento. Em conjunto, nossos dados mostram que as alterações estruturais e de componentes solúveis do timo durante a infecção com Plasmodium berghei NK65 podem favorecer tanto a deleção, por apoptose, de timócitos corticais como também a migração precoce de células imaturas para a periferia do sistema imune. O acometimento tímico na atrofia tímica induzida pelo Plasmodium berghei NK65 revela que durante uma infecção o timo sofre influências de diversos fatores que provocam profundas alterações nas subpopulações linfocitárias. Também, é possível que as alterações observadas em nosso modelo prejudique a resposta imune periférica contra o parasita contribuindo para o agravamento da doença e que a presença de timócitos prematuros nos linfonodos favoreça o desenvolvimento de doenças auto-imunesAbstract: The thymus is the primary lymphoid organ responsible for the differentiation, maturation and development of T lymphocytes (or thymocytes, as in the thymus).This organ presents a complex architecture composed by soluble and cellular elements that support the migration of lymphocyte precursors from space perivascular to the cortical region, and then their migration toward the medullar area. The trajectory of thymocytes by thymic microenvironments allows these cells to receive stimuli that make them mature into CD4+ or CD8+ T cells, which leave the thymus to populate peripheral lymphoid organs. Studies using murine models of thymic atrophy have reported that thymus is a target organ in several parasitic, viral, helminthic, bacterial and fungal infections. In these models, one can observe intense thymic involution accompanying by disruption of thymic architecture and depletion of all thymocyte subpopulations. In a previous study, our laboratory has demonstrated profound changes in the thymus during experimental infection with Plasmodium berghei, the causative agent of Malaria. Such alterations comprised severe atrophy, histopathological changes with loss of cortical-medullary delimitation, and reduction of all thymocytes sub-populations. In addition, the presence of parasites inside the thymus was detected for the first time. In another study using the same experimental model, we have demonstrated that Plasmodium-infected animals present changes in the expression of extracellular matrix elements and chemokine protein families, which are important molecules for the dynamic thymocyte migration within the thymus. In this study we evaluate whether the acute thymic atrophy observed in Plasmodium berghei-infected animals is correlated with increased apoptotic levels of thymocytes, or with their premature emigration to the periphery. Our data showed profound histological alterations, which included a very large number of cells showing nuclear condensation and karyorrhectic changes surrounded by histiocytes suggesting increased levels of apoptosis. This was confirmed by immunohistochemistry and flow cytometry techniques. Also, increased expression of proapoptotic genes was observed in the thymus of infected animals when compared with controls, suggesting the involvement of the extrinsic, or receptor-iniciated, pathway of apoptosis. Although the rate of apoptosis in the thymus from infected animals is much higher than the controls, the hypothesis that a premature emigration of thymic cells to the peripheral lymphoid organs may also account for the severe atrophy observed during Plasmodium infection could not be ruled out. In fact, we have found a consistent fraction of immature cells, mainly double-negative and double-positive thymocytes, in mesenteric lymph nodes of infected animals. Although this result serves as an evidence of premature emigration of non-mature thymocytes, the precise mechanism by which this process occurs is not clear until now. No significant differences were found in the spleen. Taken together, our data show that thymic alterations observed during Plasmodium berghei infection can favour both the deletion of thymocyte subpopulation by apoptosis and the early emigration of immature cells to the periphery of the system immune. As a consequence, an altered peripheral immune response to the parasite can be expected. Our findings provide relevant information concerning the immune involvement in malaria infection. Moreover, the murine model described here could be useful for mechanist studies of acute thymic atrophyMestradoImunologiaMestre em Genética e Biologia Molecula

Thymic Alterations Induced By Plasmodium Berghei: Expression Of Matrix Metalloproteinases And Their Tissue Inhibitors.

The thymus plays a crucial role in the generation of T-cells, and so our laboratory has been interested in the study of the intrathymic events that occur during infection diseases and may cause disruption in its functions. Previously, we showed that thymus from experimentally Plasmodium berghei-infected mice present histological alterations with high levels of apoptosis, changes in cell migration-related molecules, and premature egress of immature thymocytes to periphery. In addition, parasites were found inside the thymus. In this work we investigated alterations in the expression pattern and activity of matrix metalloproteinases MMP-2 and -9, and their tissue inhibitors, TIMP-1 and TIMP-2. Our results show enhanced expression and widespread distribution of these molecules in thymus from infected animals. Also, the presence of active MMP-2 was detected. These data are suggestive of MMPs and TIMPs importance in the earlier observed changes in the extracellular matrix during thymic alterations after plasmodium infection.27953-

Thymic alterations induced by Plasmodium berghei: Expression of matrix metalloproteinases and their tissue inhibitors

AbstractThe thymus plays a crucial role in the generation of T-cells, and so our laboratory has been interested in the study of the intrathymic events that occur during infection diseases and may cause disruption in its functions. Previously, we showed that thymus from experimentally Plasmodium berghei-infected mice present histological alterations with high levels of apoptosis, changes in cell migration-related molecules, and premature egress of immature thymocytes to periphery. In addition, parasites were found inside the thymus. In this work we investigated alterations in the expression pattern and activity of matrix metalloproteinases MMP-2 and -9, and their tissue inhibitors, TIMP-1 and TIMP-2. Our results show enhanced expression and widespread distribution of these molecules in thymus from infected animals. Also, the presence of active MMP-2 was detected. These data are suggestive of MMPs and TIMPs importance in the earlier observed changes in the extracellular matrix during thymic alterations after plasmodium infection

Thymic alterations induced by Plasmodium berghei: Expression of matrix metalloproteinases and their tissue inhibitors

The thymus plays a crucial role in the generation of T-cells, and so our laboratory has been interested in the study of the intrathymic events that occur during infection diseases and may cause disruption in its functions. Previously, we showed that thymus from experimentally Plasmodium berghei-infected mice present histological alterations with high levels of apoptosis, changes in cell migration-related molecules, and premature egress of immature thymocytes to periphery. In addition, parasites were found inside the thymus. In this work we investigated alterations in the expression pattern and activity of matrix metalloproteinases MMP-2 and -9, and their tissue inhibitors, TIMP-1 and TIMP-2. Our results show enhanced expression and widespread distribution of these molecules in thymus from infected animals. Also, the presence of active MMP-2 was detected. These data are suggestive of MMPs and TIMPs importance in the earlier observed changes in the extracellular matrix during thymic alterations after plasmodium infection27915359COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação08/58604-0

Chloroquine treatment enhances regulatory T cells and reduces the severity of experimental autoimmune encephalomyelitis.

BACKGROUND: The modulation of inflammatory processes is a necessary step, mostly orchestrated by regulatory T (Treg) cells and suppressive Dendritic Cells (DCs), to prevent the development of deleterious responses and autoimmune diseases. Therapies that focused on adoptive transfer of Treg cells or their expansion in vivo achieved great success in controlling inflammation in several experimental models. Chloroquine (CQ), an anti-malarial drug, was shown to reduce inflammation, although the mechanisms are still obscure. In this context, we aimed to access whether chloroquine treatment alters the frequency of Treg cells and DCs in normal mice. In addition, the effects of the prophylactic and therapeutic treatment with CQ on Experimental Autoimmune Encephalomyelitis (EAE), an experimental model for human Multiple Sclerosis, was investigated as well. METHODOLOGY/PRINCIPAL FINDINGS: EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) peptide. C57BL/6 mice were intraperitoneally treated with chloroquine. Results show that the CQ treatment provoked an increase in Treg cells frequency as well as a decrease in DCs. We next evaluated whether prophylactic CQ administration is capable of reducing the clinical and histopathological signs of EAE. Our results demonstrated that CQ-treated mice developed mild EAE compared to controls that was associated with lower infiltration of inflammatory cells in the central nervous system CNS) and increased frequency of Treg cells. Also, proliferation of MOG35-55-reactive T cells was significantly inhibited by chloroquine treatment. Similar results were observed when chloroquine was administrated after disease onset. CONCLUSION: We show for the first time that CQ treatment promotes the expansion of Treg cells, corroborating previous reports indicating that chloroquine has immunomodulatory properties. Our results also show that CQ treatment suppress the inflammation in the CNS of EAE-inflicted mice, both in prophylactic and therapeutic approaches. We hypothesized that the increased number of regulatory T cells induced by the CQ treatment is involved in the reduction of the clinical signs of EAE

Chloroquine administration alters the frequency of regulatory T (Treg) cells and dendritic cells (DCs), but not the proliferative capability of T cells.

<p>Briefly, mice were treated with chloroquine via i.p. for five consecutive days. Three days after the last dose mice were killed and splenic cells were analyzed by flow cytometry. Increased numbers of Treg cells (A) and reduced frequency of DCs (B) was found in mice treated with chloroquine when compared to the control group. In addition, splenic T cells proliferative response was not altered in the presence of concanavalin-A (C). Subpopulations of leukocytes showed slight changes when compared to control subjects (D). Results are representative of three independent experiments.</p

Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.</p