Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-13T19:49:52Z No. of bitstreams: 2 DISSERTAÇÃO Giselle Machado M. Moreno .pdf: 1689109 bytes, checksum: 2d05742bbd9d111da0b3253b85d9846c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T19:49:53Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Giselle Machado M. Moreno .pdf: 1689109 bytes, checksum: 2d05742bbd9d111da0b3253b85d9846c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-03-07 / Conselho Nacional de Desenvolvimento Cientìfico e Tecnológico – CNPq / A estimulação transcraniana por corrente contínua (ETCC) consiste na aplicação de corrente direta de baixa intensidade através do crânio e tem se mostrado eficaz no tratamento de diversas desordens neurológicas e psiquiátricas. Dentre as técnicas de estimulação do sistema nervoso central a ETCC ocupa posição de destaque por ser capaz de modular a excitabilidade cortical com vantagens como: não ser invasiva, ser indolor, de baixo custo, fácil uso e fácil mascaramento na realização de estudos. No entanto, apesar de extensas pesquisas sobre os efeitos da ETCC em diversos estados patológicos, seus mecanismos básicos de ação permanecem desconhecidos. Partindo do entendimento do grande envolvimento glial e de moléculas envolvidas no crescimento axonal na dinâmica de funcionamento das sinapses e excitabilidade cortical, o objetivo deste estudo foi verificar os efeitos da ETCC anódica sobre parâmetros celulares e moleculares relacionados à plasticidade sináptica. Foram utilizados 20 ratos Wistar machos adultos, divididos aleatoriamente em dois grupos: (i) ETCC ativa anódica (E), e (ii) ETCC fictícia, sham (S). Os animais receberam ETCC anódica com intensidade de corrente igual a 400 μA, durante 10 minutos por dia, durante cinco dias consecutivos. Após o tratamento foi feita análise imunohistoquímica para reatividade microglial (Iba1) e astrocitária (GFAP), foram investigadas possíveis alterações teciduais estruturais (HE) e degeneração neuronal (FJC), bem como quantificação da expressão da proteína associada ao crescimento axonal, GAP-43. Os ratos do grupo E apresentaram aumento de ~90% na expressão da proteína GAP-43 em homogenados de todo o córtex cerebral (p = 0.032) e na reatividade microglial por uma extensa área cortical em torno da região estimulada, quando comparados ao grupo S. Não foram observadas alterações anatomopatológicas no tecido nem sinais de astrogliose ou neurodegeneração no córtex cerebral dos animais que receberam ETCC. Conclui-se que os parâmetros de estimulação utilizados no presente estudo são capazes de induzir alterações moleculares e celulares no córtex cerebral de animais saudáveis, na ausência de injúria ao tecido nervoso. É possível que tais efeitos estejam envolvidos em algumas das ações da ETCC sobre a plasticidade sináptica e excitabilidade cortical.

The last century has seen a steady increase in the extension of the average lifespan. This has concomitantly produced higher incidences of age-related chronic degenerative diseases like Alzheimer’s and Parkinson’s diseases. Age is the single greatest risk factor for the development of not just these degenerative conditions but cancer as well. The aged niche undergoes a number of maladaptive changes that allow underlying conditions to present and progress. Exactly which changes, contribute to the progression of which disease is currently an area of intense study. However, these answers often present therapeutic targets for disease prevention. Age is characterized by a progressive loss of tissue function that eventually leads to the death of the organism. At the cellular level, aged tissues are characterized by a loss of resident stem cell populations, senescence, and low-grade inflammation. While aging is heterogeneous in terms of its ultimate effect on tissue function the underlying changes have a degree of overlap. Cells often experience increased oxidative stress and a diminished activity in pathways like NRF2 whose role it is to provide resistance to such stress. Aged cells also have some change in their overall chromatin and nucleosome structure contributing to observable changes in gene expression and regulation. When these disruptions occur in tissues that can affect the larger organism such as the hypothalamus they affect the organism as a whole and contribute to syndromes seen in age such as insulin resistance. The immune system, in particular, is sensitive to both the cell-autonomous and systematic changes that occur with age. Immune and endocrine signaling pathways have a considerable amount of overlap, and mounting evidence points to the role of inflammation in the metabolic syndromes that occur with age. Immune dysfunction in the CNS has a special significance because of the dual roles of microglia. These cells exist not just to protect against foreign invasion but play vital roles in the maintenance of brain architecture and in processes central to cognition like long-term potentiation and the differentiation of stem cells in the hippocampus. The aged microglial phenotype contributes to the decline that occurs normally with age but can also be central to the progression of underlying pathologies including several degenerative diseases. Therapies targeting the maintenance of microglial function with age hold the potential to delay disease onset and possibly preserve cognitive function further into life. Top-down research approaches are well suited for the study of interactions between complex systems. The rapid improvement of mass spectrometry over the past decade has allowed researchers to examine more complex samples with fewer preparation steps and improved accuracy. This approach has thus far worked very well in the study of aging with the number of “Omics” techniques in aging models increasing rapidly. We use both label-free mass spectrometry and the more traditional real-time PCR to analyze signaling pathways and systems in both tissue homogenates and isolated cells from aged animals. By analyzing inflammatory and neurogenic pathways in animals treated with polyphenolic compounds we were able to postulate that the improved behavioral effect of these compounds is likely related to the decrease of pro-inflammatory cytokines and a restoration of WNT signaling. Proteomic analysis of aged microglia revealed widespread changes in chromatin structure and cellular machinery responsible for the regulation of transcription. In addition, we uncovered a shift in the underlying metabolic state of aged microglia and identified several pathways upstream of these changes. These upstream pathways included mTOR, a well-studied nutrient sensing pathway that plays a role in regulating microglial phenotype. Modulation of identified pathways through the use of both genetic (siRNA) and pharmacological (allosteric inhibitor) was able to recapitulate the aged phenotype in normal cells, confirming the role of these pathways in pathological changes.

No description available.

Parkinson’s disease (PD) is characterized by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Mechanisms regulating this neurodegeneration, however, are unclear. Evidence from PD pathology and models of PD, indicate mitochondrial disfunction triggers several death signalling pathways. Accordingly, in vivo and in vitro mitochondrial stress models of PD were employed to explore the role of two divergent molecular influences on dopaminergic neuronal survival. We examined neuroinflammatory and death signalling pathways arising from MPTP-induced mitochondrial stress. Interferon-gamma (IFN-ɣ) is a cytokine known to activate cellular components of inflammation, including microglia of the central nervous system (CNS). Results of a screen for cytokines in PD patient plasma revealed elevated levels of IFN-ɣ, suggesting a correlation between IFN-ɣ and PD associated DA cell death. In an MPTP mouse model of PD, germline deletion of IFN-ɣ improved survival of DA neurons and the nigrostriatal system, along with a reduction in microglia activation. Employing a survival co-culture system of neurons and microglia, it was found that neutralizing IFN-ɣ reduced DA cell loss induced by the mitochondrial complex I inhibitor, rotenone. DA cell death required localized microglia, activated through the IFN-ɣ-receptor (IFN-ɣ-R), with DA survival inversely proportional to IFN-ɣ expression, found to be up-regulated following rotenone. Investigation of the calpain-Cdk5-MEF2 signalling pathway in the MPTP model of DA cell death, motivated an examination of the nuclear orphan receptor, Nur77, following a review of potential MEF2 regulatory targets. MPTP induced a reduction in Nur77 mRNA from basal ii levels in SNc tissue, further regulated by ectopic Nur77 expression. These results strengthened our new model of MEF2 Nur77 regulation in DA neurons. In MPP+/MPTP DA survival experiments, loss in germline Nur77 expression presented an elevation in DA neuronal death both in vitro and in vivo, with a greater impairment in the nigrostriatal circuitry in comparison with normal expressing animals and cells. Dopaminergic supersensitivity related to Nur77 deficiency was attenuated with the ectopic expression of AV-Nur77 in vivo. These opposing mediators of survival yield new mechanisms by which DA neurons die, suggesting a mutitargeting approach to halt the progression of DA cell death.

Background: Gliomas are the most frequent primary brain tumours in adults with two main histological subtypes: astrocytoma and oligodendroglioma. Translocator protein (TSPO) is a pro-inflammatory molecule over-expressed predominantly in activated microglia under pathological conditions. In astrocytoma samples, TSPO has also been found to be up-regulated and correlated with the malignancy of the tumours. [11C]-(R)PK11195 is a selective radioligand for the TSPO widely applied in clinical PET studies. We used [11C]-(R)PK11195 PET to investigate in vivo cerebral TSPO expression and microglial activation in patients with gliomas of different histological subtypes and grades. Methods: 24 glioma patients and 10 healthy volunteers underwent volumetric MRI and dynamic [11C]-(R)PK11195 PET scans. Tissue time-activity curves (TACs) were extracted from tumour regions and normal grey and white matter of the brains. Parametric maps of binding potential (BPND) were generated with the simplified reference tissue model. Co-registered MRI/PET was used to guide tumour biopsies. Tumour tissue was quantitatively assessed for TSPO expression and microglial infiltration by immunohistochemistry. Results: Three types of tumour TAC were observed in gliomas (grey matter-like kinetics, white matter-like kinetics and mixed kinetics), which differed between low-grade astrocytomas and low-grade oligodendrogliomas but were independent of the tumour grade. [11C]-(R)PK11195 BPND also differed between the two subtypes of low-grade gliomas, and low-grade gliomas demonstrated lower BPND than high-grade gliomas. 4 cases of high-grade glioma with minor or no contrast enhancement on MRI showed pronounced [11C]-(R)PK11195 binding. Immunohistochemistry confirmed that expression of TSPO correlated with [11C]-(R)PK11195 BPND of the tumour. It was related mainly to expression by neoplastic cells while the contribution from tumour-infiltrating microglia was minimal. When compared with control subjects, increased [11C]-(R)PK11195 BPND was found in patients’ normal appearing cerebral structures, being more prominent in the tumour-bearing than the tumour-free hemisphere. This extra-tumoral [11C]-(R)PK11195 binding was correlated with the duration of epileptic seizures, the symptom shared by the majority of our patients. Conclusions: Gliomas show differences in [11C]-(R)PK11195 kinetics and binding that are related to histological subtype and grade. Neoplastic cells rather than activated microglia are the main cellular sources expressing TSPO and determine the [11C]-(R)PK11195 binding within the tumours. [11C]-(R)PK11195 PET has the potential to detect malignant transformation of non-enhancing gliomas and facilitate the targeting of more aggressive areas in tumour biopsy. The high extra-tumoral [11C]-(R)PK11195 binding indicates widespread microglial activation in otherwise normal appearing cerebral structures of glioma patients. It is associated with epilepsy and could open up novel therapeutic perspectives for seizure control in this patient population.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22737号 / 医博第4655号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊佐 正, 教授 林 康紀, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Les récepteurs purinergiques P2X4 (P2X4R) sont des récepteurs canaux exprimés par lesneurones et les microglies du système nerveux central et sont impliqués dans de nombreuxprocessus physiologiques et pathologiques. Des études préliminaires, menées au sein dulaboratoire, ont permis de mettre en évidence une interaction entre P2X4R etl’Apolipoprotéine E (ApoE), ainsi qu’une augmentation d’ApoE dans les macrophages et lesmicroglies provenant de souris déficientes pour P2X4R. Basée sur ces observations, lapremière partie de cette thèse a cherché à caractériser les mécanismes impliquant P2X4R danscet effet. ApoE étant un facteur de risque majeur dans la maladie d’Alzheimer, la deuxièmepartie de cette thèse a été consacrée à étudier l’implication de P2X4R dans cette pathologie.Les résultats présentés montrent que P2X4R module l’activité de la cathepsine B, enzymeresponsable de la dégradation lysosomale d’ApoE. L’utilisation de souris APP/PS1 a permisde montrer que l’absence de P2X4R conduit à une amélioration des capacités mnésiques, unediminution de la concentration de peptide Aβ soluble ainsi qu’à une augmentation d’ApoEmicrogliale.Ces résultats indiquent que P2X4R régule la dégradation d’ApoE par un mécanismedépendant de la cathepsine B, et que son invalidation permet d’améliorer les symptômescognitifs de la maladie d’Alzheimer. / P2X4 receptors (P2X4R) are purinergic ion channels expressed on neurons and microglia inthe central nervous system. They have been widely studied and have been implicated in manyphysiological and pathological processes. Previous studies conducted in the laboratoryrevealed an interaction between P2X4R and the Apolipoprotein E (ApoE), as well as anincrease in ApoE level in primary macrophages and microglia obtained from mice lackingP2X4R. Based on these results, this thesis aimed to decipher the mechanisms underlyingP2X4R regulation of ApoE levels. In addition, ApoE being a major risk factor forAlzheimer’s disease, part of this work investigated potential implications of P2X4R in thispathology.Results show that P2X4R modulates cathepsin B activity, which in turn promotes ApoElysosomal degradation. APP/PS1 mice lacking P2X4R show an increase in cognitiveperformances, a decrease in soluble Aβ peptide and an increase of microglia ApoE level.These results support that P2X4R modulates ApoE degradation in a cathepsin B-dependantmanner and that its invalidation leads to an improvement in Alzheimer’s pathology.

Thyroid hormones (THs) play a fundamental role in brain function during development and adulthood. THs are essential regulators of neurogenesis, cell maturation and migration as well myelination and synaptogenesis. Neuroglial cells, including astrocytes and microglia are targets of TH and implicated in TH regulation; however, the regulation is not properly understood at the cellular level. In this study, TH regulation was investigated in vitro using human brain cell lines of astrocytes (Svg-P12) and microglia (HMC3). The cells were exposed to TH receptor agonist (triiodothyronine; T3) and inhibitors (amiodarone/1-850), of different concentrations, followed by RNA extraction and quantitative PCR. The gene expression of known TH regulated genes was studied for a better understanding of TH signaling in astrocytes and microglia. All target genes were successfully measured in both cell types. Interestingly, the regulatory effects of TH in astrocytes and microglia exhibited differences. In astrocytes, T3 exposure resulted in an upregulation in gene expression of DDX54 (DEAD-Box Helicase 54) and KLF9 (Krüppel-like factor 9) but did not affect other genes. Also, THR inhibitor exposure resulted in n upregulation in gene expression of DDX54 (DEAD-Box Helicase 54) and KLF9 (Krüppel-like factor 9) but did not affect other genes. Also, THR inhibitor exposure resulted in downregulation in gene expression of KLF9, NES (Nestin), PTGDS (Prostaglandin D2 Synthase) and MAPT (Microtubule Associated Protein Tau). In contrast, none of the TH regulated genes demonstrated a statistical significance in T3-treated microglia compared to control cells. However, THR inhibitor exposure resulted in a downregulation in gene expression of KLF9 and DDX54 and an upregulation of NES, PTGDS and MAPT. The observed differences indicate that TH signaling and regulation is different in microglia and astrocytes. The The differential signaling suggests that T3 does not regulate all of its target genes directly; rather, the regulatory effects of T3 may be exerted through complex mechanisms with other key factors involved. It can be concluded that astrocytes and microglia play important roles as mediators of the effects of THs in CNS development and function. However, further analysis is needed to acknowledge other key factors and TH signaling mechanisms influencing the gene expression in neuroglia.