Introdução: Ácidos graxos de cadeia curta (AGCC) são compostos que contêm de 1 a 6 átomos de carbono. Estudos mostraram que possuem efeitos imunomoduladores, antiproliferativos e pró-apoptóticos, via ativação de receptores acoplados à proteína G ou via controle epigenético, agindo na histona acetil transferase (HAT) e na histona deacetilase (HDAC). O acetato é o AGCC encontrado em maiores concentrações nos cólons e no sangue, sendo também um intermediário em diversas reações metabólicas. Apesar disso, foi pouco estudado até o momento. As células da microglia são os macrófagos residentes no sistema nervoso central e desempenham importante papel em diversas doenças. Objetivo: estudar os efeitos do acetato na microglia esclarecendo seus efeitos na produção de mediadores inflamatórios e na viabilidade celular. Conclusão: O acetato estimula a produção de TNF-α e melhora a viabilidade de culturas da microglia ativadas por LPS. A melhora da viabilidade celular ocorre pela indução de autofagia. O mecanismo responsável pela indução da autofagia é epigenético, sendo completamente independente da ativação de receptores GPR. / Introduction: short-chain fatty acids (SCFA) are compounds containing from 1 to 6 carbon atoms. Studies have shown that these compounds have immunomodulatory, anti-proliferative and pro-apoptotic effects via activation of G-protein coupled receptors or via epigenetic control acting on histone acetyl transferase (HAT) and histone deacetylase (HDAC). Acetate is the SCFA found in highest concentrations in the colon and blood. It is also an intermediate in many metabolic reactions. Nevertheless, it was little studied so far. The microglial cells are resident macrophages of the central nervous system and play an important role in several diseases. Objective: To study the effects of acetate on microglia and clarify its effects on inflammatory mediators production and cell viability. Conclusion: Acetate stimulates TNF-α production and improves cell viability of microglial cultures activated by LPS. The improved cell viability occurs by autophagy induction. The mechanism responsible for induction of autophagy is epigenetic, being completely independent of the GRP receptor activation.

No description available.

INTRODUÇÃO: Atualmente as células da microglia têm recebido grande atenção dentro da resposta imune, isto devido ao fato de que sua ativação por citocinas inflamatórias é capaz de promover a infiltração e destruição do sistema nervoso central (SNC) durante algumas doenças, principalmente no caso da esclerose múltipla (EM). Além de seu papel pró-inflamatório, já demonstrou-se que estas também são capazes de expressar moléculas supressoras como a indoleamina-2,3-dioxigenase (IDO), capaz de suprimir a proliferação de células T. Contudo, ainda pouco se sabe sobre seu verdadeiro papel na patogenia da EM. Recentemente tem sido descrita uma população de células T chamadas Th17, capaz de secretar grandes quantidades de IL-17, IL-21 e GM-CSF possuindo uma importância fundamental na patogenia da EM e de seu modelo murino, a EAE. Nesse contexto, a relação entre as Th17 e as células da microglia pode nos fornecer dados importantes acerca dos mecanismos envolvidos nas lesões observadas no SNC. OBJETIVO: Este trabalho teve como objetivo melhor elucidar a relação existente entre a expressão das moléculas imunes por células da microglia e a ação que estas promovem sobre as células T. MÉTODOS: Utilizamos culturas de células da microglia de linhagem, chamadas C8-B4, assim como cultura primária de células da microglia obtidas a partir sistema nervoso de camundongos C57BL/6 adultos. Caracterizamos o perfil imune da microglia, avaliando a transcrição de genes para citocinas através de PCR em tempo real assim como a expressão de suas moléculas ativadoras por citometria de fluxo. A avaliação da IDO se deu através da expressão da mesma por células da microglia ativadas ou não por LPS ou IFN-?. Ja sua capacidade funcional foi medida através da atividade proliferativa de linfócitos T CD4 específicos para MOG 35-55. RESULTADOS: Nossos resultados demonstraram que as células de ambas as culturas possuem a capacidade de expressar diversas moléculas imunes, tanto pró quanto anti-inflamatórios. Dentre estas observamos TLR-4, TLR-2, IL-6, IL-10 e TGF-?. Além disso, confirmamos a expressão da enzima IDO por estas células. O bloqueio de tal enzima impede o controle que a microglia tem sobre a proliferação dos linfócitos T CD4, tanto in vitro quanto in vivo. No modelo in vivo tal efeito repercute em uma encefalomilite mais severa, onde o quadro clínico do animal não regride. CONCLUSÃO: Os resultados aqui obtidos nos dão a certeza da influência das microglias dentro do contexto inflamatório, afirmando sua capacidade de modular a resposta imune. Além disto, fica clara a importância da enzima IDO, cuja ação dentro do controle de uma autoimunidade demonstra ser altamente necessária / INTRODUCTION: Microglia cells has gained great attention recently because its activation by inflammatory cytokines can promote infiltration and destruction of Central Nervous System (CNS) during some disease, mainly in the case of Multiple Sclerosis (MS). On the other hand, these cells may also express suppressor molecules such as the indoleamine-2,3-dioxygenase (IDO), able to suppress T cell proliferation. However, still little is known about its role in MS pathogenesis. Recently it has been described a new population of T cells called Th17, able to secrete high amounts of IL-17, IL-21 and GM-CSF, with a fundamental importance on MS and its murine model, EAE. In this context, the relationship between Th17 and microglia cells can provide us important data about the mechanisms involved in the establishment of CNS lesions. OBJECTIVES: This work had the objective to better elucidate the relationship between the expression of some molecules by microglia and its role T cell activation. METHODS: Through a cellular lineage knowing as C8-B4 and primary cultures of microglia obtained from CNS of adult mice C57BL6 we investigated the transcription of several genes for cytokines and membrane expression of several pattern recognition receptors. The IDO evaluation was performed after activation with LPS or rIFN-?. Its functional capacity was measured trough its action over T cell proliferation. RESULTS: Our results demonstrated that both cells have the capacity of express several immune molecules, both pro and anti-inflammatory. Among this, we observed TLR-4, TLR-2, IL-6, IL-10 and TGF-?. We also confirmed IDO expression by these cells. The blockade of such enzyme prevents the control of microglia above T CD4 lymphocytes proliferation, both in vitro and in vivo. Using the in vivo model, IDO blocker rendered a encephalomyelitis more severe. Conclusion: The results here obtained give us the certainty of microglia influence in inflammatory context, stating its capacity of modulating the immune response

Introdução: Ácidos graxos de cadeia curta (AGCC) são compostos que contêm de 1 a 6 átomos de carbono. Estudos mostraram que possuem efeitos imunomoduladores, antiproliferativos e pró-apoptóticos, via ativação de receptores acoplados à proteína G ou via controle epigenético, agindo na histona acetil transferase (HAT) e na histona deacetilase (HDAC). O acetato é o AGCC encontrado em maiores concentrações nos cólons e no sangue, sendo também um intermediário em diversas reações metabólicas. Apesar disso, foi pouco estudado até o momento. As células da microglia são os macrófagos residentes no sistema nervoso central e desempenham importante papel em diversas doenças. Objetivo: estudar os efeitos do acetato na microglia esclarecendo seus efeitos na produção de mediadores inflamatórios e na viabilidade celular. Conclusão: O acetato estimula a produção de TNF-α e melhora a viabilidade de culturas da microglia ativadas por LPS. A melhora da viabilidade celular ocorre pela indução de autofagia. O mecanismo responsável pela indução da autofagia é epigenético, sendo completamente independente da ativação de receptores GPR. / Introduction: short-chain fatty acids (SCFA) are compounds containing from 1 to 6 carbon atoms. Studies have shown that these compounds have immunomodulatory, anti-proliferative and pro-apoptotic effects via activation of G-protein coupled receptors or via epigenetic control acting on histone acetyl transferase (HAT) and histone deacetylase (HDAC). Acetate is the SCFA found in highest concentrations in the colon and blood. It is also an intermediate in many metabolic reactions. Nevertheless, it was little studied so far. The microglial cells are resident macrophages of the central nervous system and play an important role in several diseases. Objective: To study the effects of acetate on microglia and clarify its effects on inflammatory mediators production and cell viability. Conclusion: Acetate stimulates TNF-α production and improves cell viability of microglial cultures activated by LPS. The improved cell viability occurs by autophagy induction. The mechanism responsible for induction of autophagy is epigenetic, being completely independent of the GRP receptor activation.

No description available.

Die vorliegende Arbeit beinhaltet experimentelle Untersuchungen zur neuroprotektiven Wirkung des körpereigenen Lipids L-alpha-Lysophosphatidylinositol (LPI). Die Vermittlung dieser Wirkung soll durch den zentralnervös exprimierten G-Protein-gekoppelten Rezeptor 55 (GPR55) erfolgen. Als Modelsystem diente die organotypische hippocampale Schnittkultur (OHSC) der Ratte, welche exzitotoxisch mittels N-Methyl-D-Aspartat (NMDA) geschädigt wurde, um Neurodegeneration zu initiieren. Die Inkubation mit LPI nach NMDA-Schädigung reduzierte die Anzahl toter Neurone und die der Mikroglia in der Körnerzellschicht des Gyrus dentatus. Ein Clodronat-induzierter Verlust der Mikroglia und die siRNA-vermittelte Herabregulation von Gpr55 hoben jeweils den neuroprotektiven Effekt von LPI in der OHSC auf. Diese Beobachtungen wiesen auf eine Mikroglia- und GPR55 abhängige Neuroprotektion hin. LPI wirkte zudem synergistisch und verstärkte die (bekannter Maßen) durch Cannabinoide induzierte und über den Cannabinoid Typ 1 Rezeptor vermittelte Neuroprotektion. Ferner wurde Gpr55 mittels qPCR in Mikroglia und Astrozyten nachgewiesen. LPI steuerte außerdem die Expression von Gpr55 in Mikroglia und beeinflusste deren Migrationsverhalten. Die vorliegenden Ergebnisse machen deutlich, dass LPI in einem in vitro Modellsystem zur Untersuchung des sekundären neuronalen Schadens protektiv wirkt und für die Vermittlung dieser Neuroprotektion Mikroglia und GPR55 in Frage kommen.

Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.   In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.   Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier. The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

The majority of the estimated three million traumatic brain injuries that occur each year are classified as “mild” and do not require surgical intervention. However, debilitating symptoms such as difficulties focusing on tasks, anxiety, depression, and visual deficits can persist chronically after a mild traumatic brain injury (TBI) even if an individual appears “fine”. These symptoms have been observed to worsen or be prolonged when an individual has suffered multiple mild TBIs. To test the hypothesis that increasing the amount of time between head injuries can reduce the histopathological and behavioral consequences of repeated mild TBI, a mouse model of closed head injury (CHI) was developed. A pneumatically controlled device with a silicone tip was used to deliver a diffuse, midline impact directly onto the mouse skull. A 2.0mm intended depth of injury caused a brief period of apnea and increased righting reflex response with minimal astrogliosis and axonal injury bilaterally in the entorhinal cortex, optic tract, and cerebellum. When five CHIs were repeated at 24h inter-injury intervals, astrogliosis was exacerbated acutely in the hippocampus and entorhinal cortex compared to a single mild TBI. Additionally, in the entorhinal cortex, hemorrhagic lesions developed along with increased neurodegeneration and microgliosis. Axonal injury was observed bilaterally in the white matter tracts of the cerebellum and brainstem. When the inter-injury interval was extended to 48h, the extent of inflammation and cell death was similar to that caused by a single CHI suggesting that, in our mouse model, extending the inter-injury interval from 24h to 48h reduced the acute effects of repeated head injuries. The behavioral consequences of repeated CHI at 24h or 48h inter-injury intervals were evaluated in a ten week longitudinal study followed by histological analyses. Five CHI repeated at 24h inter-injury intervals produced motor and cognitive deficits that persisted throughout the ten week study period. Based upon histological analyses, the acute inflammation, axonal injury, and cell death observed acutely in the entorhinal cortex had resolved by ten weeks after injury. However, axonal degeneration and gliosis were present in the optic tract, optic nerve, and corticospinal tract. Extending the inter-injury interval to 48h did not significantly reduce motor and cognitive deficits, nor did it protect against chronic microgliosis and neurodegeneration in the visual pathway. Together these data suggested that some white matter areas may be more susceptible to our model of repeated mild TBI causing persistent neuropathology and behavioral deficits which were not substantially reduced with a 48h inter-injury interval. In many forms of TBI, microgliosis persists chronically and is believed to contribute to the cascade of neurodegeneration. To test the hypothesis that post-traumatic microgliosis contributes to mild TBI-related neuropathology, mice deficient in the growth factor progranulin (Grn-/-) received repeated CHI and were compared to wildtype, C57BL/6 mice. Penetrating head injury was previously reported to amplify the acute microglial response in Grn-/- mice. In our studies, repeated CHI induced an increased microglial response in Grn-/- mice compared to C57BL/6 mice at 48h, 7d, and 7mo after injury. However, no differences were observed between Grn-/- and WT mice with respect to their behavioral responses or amount of axonal injury or ongoing neurodegeneration at 7 months despite the robust differences in microgliosis. Dietary administration of ibuprofen initiated after the first injury reduced microglial activation within the optic tract of WT mice 7d after repeated mild TBI. However, a two week ibuprofen treatment regimen failed to affect the extent of behavioral dysfunction over 7mo or decrease chronic neurodegeneration, axon loss, or microgliosis in brain-injured Grn-.- mice when compared to standard diet. Together these studies underscore that mild TBIs, when repeated, can result in long lasting behavioral deficits accompanied by neurodegeneration within vulnerable brain regions. Our studies on the time interval between repeated head injuries suggest that a 48h inter-injury interval is within the window of mouse brain vulnerability to chronic motor and cognitive dysfunction and white matter injury. Data from our microglia modulation studies suggest that a chronically heightened microglial response following repeated mild TBI in progranulin deficient mice does not worsen chronic behavioral dysfunction or neurodegeneration. In addition, a two week ibuprofen treatment is not effective in reducing the microglial response, chronic behavioral dysfunction, or chronic neurodegeneration in progranulin deficient mice. Our data suggests that microglia are not a favorable target for the treatment of TBI.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Microglia-mediated neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Uncontrolled microglia activation produces major proinflammatory factors including cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) that may cause dopaminergic neurodegeneration. Peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone has potent antiinflammatory property. We hypothesize pioglitazone protects dopaminergic neuron from lipopolysaccharide (LPS)-induced neurotoxicity by interacting with relevant signal pathways, inhibiting microglial activation and decreasing inflammatory mediators. First, the neuroprotection of pioglitazone was explored. Second, the signaling transductions such as jun N-terminal kinase (JNK) and the interference with these pathways by pioglitazone were investigated. Third, the effect of pioglitazone on these pathways-mediated PGE2 / nitric oxide (NO) generation was investigated. Finally, the effect of PPARγ antagonist on the inhibition of PGE2 / NO by pioglitazone was explored. The results show that LPS neurotoxicity is microglia-dependent, and pioglitazone protects neurons against LPS insult possibly by suppressing LPS-induced microglia activation and proliferation. Second, pioglitazone protects neurons from COX-2 / PGE2 mediated neuronal loss by interfering with the NF-κB and JNK, in PPARγ-independent mechanisms. Third, pioglitazone significantly inhibits LPS-induced iNOS / NO production, and inhibition of LPS-induced iNOS protects neuron. Fourth, inhibition p38 MAPK reduces LPS-induced NO generation but no effect is found upon JNK inhibition, and pioglitazone inhibits p38 MAPK phosphorylation induced by LPS. In addition, pioglitazone increases PPARγ phosphorylation, followed by the increased PI3K/Akt phosphorylation. Nevertheless, inhibition of PI3K increases LPS-induced p38 MAPK phosphorylation. Inhibition of PI3K eliminates the inhibitive effect of pioglitazone on the LPS-induced NO production, suggesting that the inhibitive effect of pioglitazone on the LPS-induced iNOS and NO might be PI3K-dependent.