Alzheimer’s disease (AD) is a progressive neurodegenerative disease resulting in alterations in memory, language, executive function and emotional behaviour. Although it can be characterised by symptoms, by the time they arise significant pathological alterations have already emerged in the central nervous system, namely increased amyloid plaques, neurofibrillary tangles and neuronal loss. Despite known pathological hallmarks the exact aetiology of AD is poorly understood and no current treatments are available. However, there is growing interest in the role of neuroinflammation in AD, with increases observed in the early stages of disease and with disease progression. Moreover, it has been suggested that peripheral inflammation can influence neuroinflammation and worsen neurodegeneration. Using Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRS) we can non-invasively measure biomarkers of neuroinflammation and degeneration allowing multi-modal investigation of its role in normal aging and AD. Considering this the objectives of this study were to (i) Use PET and MRS to investigate neuroinflammatory and metabolite alterations in transgenic (TG) models of AD and their wildtype (WT) animals. (ii) Assess rates of cognitive decline in these models using memory based tests. (iii) Investigate relatively new TgF344AD rat as an AD model by characterising younger time-points than previously reported. (iv) Investigate the contribution of peripheral inflammation on AD progression. PET and MRS imaging was carried out longitudinally in the APPswe×PS1de9 mouse. Neuroinflammation was confirmed ex vivo and cognitive ability was assessed by behavioural tests. Results revealed significantly increase hippocampal and thalamic neuoinflammation in old TG mice as assessed by [18F]DPA-714 PET and supported by immunohistochemistry. Reduced neuronal marker N-acetlyaspartate was seen with age and was exacerbated in the TG mice. Accelerated cognitive decline was also seen in TG mice. PET and MRS imaging was carried out at 6 and 12 months in the TgF344AD model, which expresses amyloid and tau pathology as well as neuronal loss. No cognitive decline was observed in TG rats; however increased anxiety behaviour was seen. Increased [18F]DPA-714 PET was observed as an effect of gene in the thalamus at 6 months and the hypothalamus at 12 months. Increases in glutamate were seen with age in the TG rats but not the WTs. Increased inflammation and metabolite alterations were seen with aging. The effect of peripheral urinary tract infection (UTI) on cognition and imaging out was assessed. Imaging was carried out prior to and after re-current UTI. Infection induced cognitive decline in infected TG but not WT rats. Infection had an increasing effect on hypothalamic neuroinflammation in WT rats but a decreasing effect on TG rats, which masked the original gene differences. This thesis is set out in the alternative format with each experimental study represented as a chapter. Results in this thesis implicate neuroinflammation in AD development and progression. In addition, we report systemic infection-CNS interactions accelerating cognitive decline in AD and highlight the importance of understanding the effects of comorbidities in disease.

Submitted by Caio Sergio Galina Spilla (caiospilla@hotmail.com) on 2018-06-13T12:51:18Z No. of bitstreams: 1 Dissertação - Mestrado, SPILLA, C. S. G..pdf: 2498178 bytes, checksum: f7d9be339f1c3e5b2aacdf4025b377c3 (MD5) / Approved for entry into archive by Satie Tagara (satie@marilia.unesp.br) on 2018-06-13T15:34:17Z (GMT) No. of bitstreams: 1 spilla_csg_me_mar.pdf: 2498178 bytes, checksum: f7d9be339f1c3e5b2aacdf4025b377c3 (MD5) / Made available in DSpace on 2018-06-13T15:34:17Z (GMT). No. of bitstreams: 1 spilla_csg_me_mar.pdf: 2498178 bytes, checksum: f7d9be339f1c3e5b2aacdf4025b377c3 (MD5) Previous issue date: 2018-05-25 / Não recebi financiamento / Reações imunes geradas no organismo materno durante o período gestacional podem provocar alterações no desenvolvimento do feto. O LPS, endotoxina lipopolissacarídea presente na parede das bactérias gram-negativas, é capaz de gerar a produção de citocinas, mimetizando dessa forma um quadro de inflamação pré-natal quando administrado na fêmea prenhe durante o período gestacional. A prole de ratas prenhes expostas a esta endotoxina pode apresentar assim diversos problemas comportamentais e/ou cognitivos que refletem alterações ocorridas no sistema nervoso central (SNC) durante o seu desenvolvimento. Entre as diversas patologias que podem resultar de um transtorno no neurodesenvolvimento está o transtorno do espectro autista (TEA). Ainda sem uma causa definida em humanos, estima-se que cerca de uma a cada 150 crianças nascidas atualmente são acometidas por essa patologia. Distúrbios de comportamento e de comunicação/interação social constituem a díade que caracteriza o TEA e dependendo do grau de acometimento é comum encontrar também nestes e de forma geral em indivíduos com transtornos de neurodesenvolvimento problemas atencionais, de aprendizado e memória. O quadro inflamatório no período de gestação causa uma série de alterações no organismo materno, sendo que algumas destas alterações alcançam o feto em desenvolvimento. Uma destas alterações pode ser a redução no conteúdo de melatonina na circulação materna e fetal. A melatonina, hormônio produzido pela glândula pineal, apresenta funções sincronizadora, antioxidante e neuroprotetora que podem ser importantes durante o neurodesenvolvimento e a alteração na síntese e liberação desse hormônio no organismo materno ao longo da gestação vem sendo correlacionada a casos de TEA. Além disso, a exposição ao LPS durante o desenvolvimento do SNC em um modelo animal resulta em uma prole que apresenta comportamentos autísticos. Apesar destes dados, os efeitos desse quadro pré-natal no comportamento, na morfologia e na neuroquímica de áreas encefálicas da prole ainda não foram totalmente esclarecidos e sua elucidação pode contribuir como base de conhecimento para estudos que investiguem prevenção e terapias farmacológicas ou comportamentais nessas patologias. O presente trabalho teve como objetivos verificar se o quadro inflamatório pré-natal induzido por LPS: 1) Altera a concentração plasmática de melatonina materna, 2) altera a expressão de comportamentos que dependem da função de memória espacial e 3) induz mudanças morfométricas e neuroquímicas no hipocampo. Para atingir o primeiro objetivo foi coletado o sangue de ratas prenhe 3 h (fase de claro) e 16 h (fase de escuro) após à exposição ao LPS ou a solução salina e a dosagem de melatonina foi realizada por meio do método ELISA. Para alcançarmos o segundo e terceiro objetivos as proles de ratas expostas ao LPS e de ratas controles expostas a solução salina no dia gestacional 9,5 foram avaliadas por meio de teste comportamental de alternância espontânea no labirinto em T. Em seguida, os animais foram perfundidos e os encéfalos processados para o estudo do volume hipocampal por estereologia, para análise de expressão neuronal das proteínas ligantes de cálcio calretinina e parvalbumina e para análise de expressão glial da proteína glial fibrilar ácida (GFAP) e da proteína de associação de ligação ao cálcio ionizada – 1 (IBA-1) por meio da técnica de imunohistoquímica. Os resultados mostraram que houve queda no conteúdo plasmático de melatonina noturno e aumento no conteúdo diurno após injeção com LPS nas fêmeas prenhes. A prole do grupo controle apresentou melhor desempenho no teste comportamental quando comparado a prole do grupo LPS. O hipocampo da prole do grupo LPS não apresentou diferença de volume total do desta estrutura em comparação ao grupo controle. Também não houve alteração na expressão de GFAP em astrócitos hipocampais no grupo LPS em relação ao controle. Por outro lado, ocorreu aumento na expressão de IBA-1, marcador de microglia, nas regiões do CA1, CA2 e CA3 no hipocampo da prole do grupo LPS em relação a prole do grupo controle. A prole do grupo LPS, considerada um modelo animal de autismo também apresentou variações na expressão das proteínas ligantes de Cálcio no hipocampo com menor expressão da proteína parvalbumina e maior expressão da proteína calretinina que o grupo controle. Os resultados mostraram que a exposição materna ao LPS foi capaz de alterar a concentração de melatonina plasmática circulante e que a prole exposta a esse ambiente inflamatório pré-natal apresentou alterações de comportamento dependente da memória espacial. Além disso, os resultados nos permitem concluir que tais alterações na memória espacial não são coincidentes com alterações no volume do hipocampo ou na reatividade astrocitária, mas sim com a ativação microglial e com alterações na neuroquímica dos neurônios gabaérgicos hipocampais que expressam as proteínas ligantes de cálcio e que conhecidamente controlam as conexões excitatórias e inibitórias envolvidas nos fenômenos de memória. / Immune reactions generated in the maternal organism during the gestational period may cause changes in the fetal development. The endotoxin lipopolysaccharide (LPS) present on the wall of Gram-negative bacteria is able to induce the production of cytokines, mimicking a state of prenatal inflammation when administered in the pregnant female during the gestational period. The offspring of pregnant rats exposed to this endotoxin may present several behavioral and/or cognitive problems that reflect changes in the central nervous system (CNS). The autism spectrum disorder (ASD) is one of this neurodevelopmental conditions, and although without a definite cause in humans, is related to be present in one to every 150 children born. Behavior and communication/social interaction disorders constitute the dyad that characterizes the ASD. Besides these, depending on the degree of involvement it is common to find in these individuals‟ attention, learning and memory problems. Melatonin, a hormone produced by the pineal gland, presents several functions during neurodevelopment and the alteration in the synthesis and release of this hormone in the maternal organism throughout pregnancy has been correlated to cases of ASD. Investigations about the effects of a prenatal inflammatory condition in behavior, in the morphology and neurochemistry of brain areas can contribute for future pharmacological or behavioral studies in these patients. Considering that exposure to LPS during the development of CNS in an animal model results in offspring presenting autistic behaviors, the present work aimed to check whether the prenatal inflammatory condition: 1) Changes the maternal melatonin plasma concentration, 2) alters behaviors that depend on the spatial memory function and 3) induces morphometric and neurochemical changes in the hippocampus. For this, blood from pregnant rats 03h and 16h after LPS exposure was collected and the melatonin dosage was performed using the ELISA method. Offsprings of rats exposed to the LPS (autism model) or saline (control) on the 9.5 gestational day were evaluated in the T-maze spontaneous alternation test. After, the animals were perfused with 4% paraformaldehyde and the brains were processed for the hippocampal volume quantification and for the analysis of the calretinin, parvalbumin, glial fibrillary acidprotein (GFAP) and ionized calcium binding association protein – 1 (IBA-1) expression by immunohistochemistry. There was difference in the plasma melatonin dosage in the day and night periods of the pregnant females of the control group and the LPS group. The results showed that the control group presented better performance in behavioral testing when compared to the autism model. The hippocampal volume showed no difference between the groups, as well as the expression of the GFAP in astrocytes. There was an increase in IBA-1 expression in all regions of the hippocampus except in the dentate gyrus in the autism model group in relation to the control group.The autism model presented lower expression of the parvalbumin and greater expression of de calretinin than the control group. The results showed that maternal exposure to LPS was able to alter maternal plasma melatonin concentration and that the offspring exposed to prenatal inflammatory environment showed deficits in behaviors dependent of spatial memory and indicate that these behaviors deficits are not coincident with changes in the hippocampal volume or in astrocytic reactivity, but but with microglial activation and with changes in the calcium binding proteins expression in gabaergic neurons of the hippocampus which are known to control excitatory and inhibitory sinapses involved in the memory phenomena.

O Transtorno Bipolar (TB) é uma doença psiquiátrica grave, altamente incapacitante que está associada com diversas comorbidades médicas e altas taxas de suicídio. Embora sua fisiopatologia não esteja completamente elucidada, inúmeros estudos têm mostrado alterações no sistema imune de indivíduos com TB. A resposta crônica destes indivíduos ao estresse parece gerar um aumento da inflamação sistêmica bem como da neuroinflamação. A micróglia ativada devido aos estímulos inflamatórios contínuos deve ocasionar diferentes prejuízos tanto bioquímicos quanto funcionas. Os macrófagos, primeira linha de defesa, são células de característica plástica de extrema importância do sistema imune e podem ser estimulados a polarizar para diferentes formas com liberação de fatores pró e antiinflamatórios, estimulando ou mantendo a homeostase no ambiente agredido de alguma forma. Desta forma, nosso trabalho buscou investigar a resposta fenotípica dos macrófagos contra o meio ambiente pró-inflamatório sistêmico observado no plasma de pacientes bipolares eutímicos, maníacos e depressivos em comparação aos controles. A amostra incluiu 5 controles saudáveis, 8 pacientes bipolares remetidos, 5 pacientes maníacos e 5 pacientes depressivos. As citocinas e quimiocinas de RNAm em células U937 tratadas com plasma mostraram um padrão de expressão diferente relativo entre controles saudáveis e pacientes com TB. As citoquinas inflamatórias tais como IL-1β e TNF-α, em pacientes bipolares maníacos e depressivos demonstram maiores quantidades de IL-1β mRNA do que os pacientes eutímicos e pacientes depressivos induziram maiores quantidades de RNAm de TNF-α do que os pacientes eutímicos em células U937. Já a expressão das quimiocinas CXCL9 e CXCL10 no plasma de pacientes com TB depressivos, demostraram ser de menor expressão significativa no grupo de pacientes maníacos quando comparados a controles e pacientes bipolares eutímicos. Nossos resultados sugerem que as citocinas periféticas devem modular a polarização M1 ou M2 de macrófagos no TB. / Bipolar Disorder (BD) is a severe and highly incapacitating psychiatric disorder which is associated with the presence of medical comorbidities. The progression of BD is related to an important cognitive deficit and also to biological and clinical manifestations that lead to treatment resistance and worse prognosis. Immune disturbances have been widely observed and investigated in BD patients. Chronic inflammatory responses induce neuroinflammation, mainly by pro-inflammatory microglial activation, and result in biochemical and functional impairment. Macrophages are the first line of defense of the immune system and exhibit cell plasticity. As well, microglia represents the resident macrophage of the central nervous system been responsible for its protection. Both cells can be stimulated to polarize into two different phenotypes, mainly pro- and anti-inflammatory, maintaining the homeostasis under physiologic and pathologic conditions. Therefore, we aimed to investigate macrophages phenotypical response when submitted to BD patients plasma in different episodes, which is considered a pro-inflammatory environment, and healthy controls plasma. Subjects included healthy controls (n=5), remitted BD patients (n=8), manic patients (n=5) and depressive patients (n=5). The mRNA expression of chemokynes and cytokines from U937 cells treated with BD patients plasma were different from those submitted to healthy controls plasma. Higher mRNA expression of IL-1β was observed in those cells submitted to manic and depressive BD patients plasma when compared to euthymic patients. Also, depressive BD patients plasma induced higher expression of TNF-α compared to euthymic patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. Inflammatory cytokines such as IL-1β and TNF-α in bipolar manic and depressive patients demonstrate higher amounts of IL-1β mRNA that euthymic patients and depressive patients induced higher amounts of TNF-α mRNA levels than the patients in euthymic U937. Since the expression of CXCL9 and CXCL10 chemokines in plasma from patients with depressive TB, proved less significant expression in the group of manic patients when compared to controls and euthymic bipolar patients.

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.

L’accident vasculaire cérébral (AVC) ischémique est une principale cause de décès et d’handicap en Europe. L’éthanol est une drogue largement consommée et la consommation chronique d’éthanol est un facteur participant à la survenue d’AVC ischémique (Reynolds et al., 2003). La consommation chronique et excessive d’éthanol est associée à un risque accru de mortalité et de morbidité suite à un AVC ischémique (Zhang et al., 2014). Ce facteur aggraverait les lésions ischémiques cérébrales dans les modèles animaux (Zhao et la 2010 ; Lemarchand et al., 2015). Le stress oxydatif et l’excitotoxicité glutamatergique peuvent jouer un rôle important dans l’exacerbation de l’infarctus cérébral après une consommation chronique et excessive d’éthanol (Zhao et la 2010 ; Zhao et la 2011). Le premier objectif de la thèse est de vérifier que la consommation chronique et excessive d’éthanol aggrave les dommages cérébraux ischémiques chez les patients et dans un modèle animal d’ischémie cérébrale. Le deuxième objectif est d'étudier l'inflammation post-ischémique dans le cerveau et dans le foie à court et moyen termes. Des rats mâles Wistar ont été soumis à l'administration chronique d'éthanol (10% ou 35% v/v, 5 ml / kg, deux fois par jour, quatre semaines avant l'opération) ou d’eau (vehicule), suivie d’une occlusion de l'artère cérébrale moyenne (OACM). Nous avons déterminé les effets de l'ingestion d’éthanol sur le volume de l'infarctus, des déficits neurologiques et moteurs, à 24 heures (J1) et à 7 jours (J7) de reperfusion sanguine. Nous avons quantifié le nombre de microglies activées dans l'hémisphère ipsilatéral. Nous avons mesuré le nombre de neutrophiles et les taux d’ARNm d’ICAM-1 et de VCAM-1 dans l'hémisphère ipsilatéral et dans le foie. Nous avons examiné la stéatose et l'état inflammatoire dans le foie des rats non ischémiés, pour évaluer l'état physiologique hépatique dans les 3 groupes à J1 et J7. Nous avons recruté 435 patients ayant eu une ischémie cérébrale supratentorielle dans les 48 heures après l'apparition des symptômes. La consommation excessive et chronique d’éthanol est définie par une prise hebdomadaire ≥300 g d'éthanol et les AVC ischémiques sévères par un score National Institutes of Health Stroke Scale (NIHSS) ≥6. Le score NIHSS a été évalué dans les 48 heures. Nous avons effectué des mesures des taux de transferrine déficiente en carbohydrates (CDT, biomarqueur de la consommation chronique excessive d'éthanol) et de marqueurs inflammatoires. Être un consommateur excessif et avoir un taux plasmatique élevé de neutrophiles étaient indépendamment associés à des déficits neurologiques plus sévères chez les patients ayant subi un AVC ischémique ou accident ischémique transitoire, dans les 48 heures. La consommation excessive et chronique d'éthanol chez le rat non ischémié a induit une stéatose hépatique et une augmentation d'un état inflammatoire dans le cortex, le striatum et le foie par l'intermédiaire d'augmentation de l'expression de protéines d'adhésion. Toutefois, aucune infiltration des neutrophiles n’a été notée dans le foie ou dans le cerveau. Dans le modèle OACM, la consommation chronique d’éthanol 35 ° aggravait le volume des lésions de l'AVC ischémique et les déficits moteurs, comparativement aux rats non exposés à l’éthanol. L'aggravation des déficits neurologiques et fonctionnels a été expliquée par une augmentation de l'inflammation post-ischémique dans le foie et le cerveau, via l'activation de la microglie, l’infiltration des neutrophiles, et l’expression des protéines d’adhésion leucocytaire à court et moyen termes. / Ischaemic stroke is a major cause of disability and death in Europe. Ethanol is a widely consumed drug and chronic ethanol consumption is a participating factor in ischaemic stroke (Reynolds et al., 2003). Chronic and excessive ethanol consumption is associated with an increased risk of mortality and morbidity from ischaemic stroke (Zhang et al., 2014). It may increase consequences of ischaemic brain injury in animals (Zhao et al., 2010, Lemarchand et al., 2015). Oxidative stress and glutamatergic excitotoxicity may play an important role in exacerbating ischaemic damage following chronic consumption of ethanol (Zhao et al., 2010; Zhao et al., 2011). The primary aim of my thesis was to assess whether chronic excessive ethanol consumption has a deleterious effect on ischaemic brain damage both in human and in a rat model. The secondary aim was to study the post-ischaemic inflammation in the brain and in the liver at short and intermediate terms. Wistar male rats were subjected to chronic administration of ethanol (10% or 35% v/v, 5ml/kg, twice per day, 4 weeks prior operation) or water (vehicle), followed by middle cerebral artery occlusion (OACM). The effects of ethanol ingestion on infarct volume, neurologic and motor deficits were determined at 24 hours (J1) and at 7 days (J7) of reperfusion. We quantified the number of activated microglia in the ipsilateral hemisphere and additionally measured the number of neutrophils and levels of ICAM-1 and VCAM-1 mRNA in the ipsilateral hemisphere and liver. Further, we examined the steatosis by comparing oil-red coloration of J1, J7 and non-ischemic rats to assess the physiologic liver status in the 3 groups. Patients with supratentorial cerebral ischaemia were recruited within 48 hours of symptom onset. Heavy drinkers were defined by a weekly consumption of ≥300 g ethanol and severe ischaemic strokes (score≥6 according to the National Institutes of Health Stroke Scale, NIHSS). The NIHSS score was evaluated within 48 hours. We performed measurements of carbohydrate-deficient transferrin (CDT, biomarker of chronic excessive ethanol consumption) and inflammatory markers plasmatic levels. Being a heavy drinker and having a higher plasma level of neutrophils were independently associated with a higher baseline severity of the neurological deficit in patients with supratentorial ischemic stroke or transient ischemic attack within 48 hours. Excessive and chronic ethanol consumption in non-ischaemic rats conferred an increased hepatic steatosis and an inflammatory condition in the cortex, the striatum and the liver, observed as increased expression of adhesion proteins. However, neutrophil infiltration was not observed in the liver or in the brain. In the OACM model, chronic consumption of 35% ethanol worsened ischemic stroke lesions and motor deficits, compared to non-ethanol-exposed rats. Neutrophil infiltration and the mRNA levels of VCAM-1 and ICAM-1 are increased in the brain and in the liver of ischaemic rats exposed to 35% ethanol, compared to control ischaemic rats, at J1 and J7. The aggravation of neurologic and functional deficits was associated with increased post-ischaemic inflammation in both the liver and brain, as observed by microglial activation, neutrophil infiltration and leukocyte adhesion at short and intermediate terms.

Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ) and tau pathologies, insulin resistance, neuro-inflammation and dysregulation of cholesterol homeostasis, all of which play a role in neuro-degeneration. The main aim of this study was to determine possible relationships between insulin signaling, cholesterol biosynthesis and their effects on Aβ, and inflammatory response in vitro. Insulin treatment increased cholesterol synthesis in human Neuroblastoma SH-SY5Y (SHY) and mouse neuroblastoma 2a (N2a) and N2a transfected with human APP (N2a-APP) by up-regulating biosynthesis enzymes including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3methyl-glutaryl-CoA reductase (HMGCR) through sterol regulatory element binding protein-2 (SREBP2) up-regulation. Aβ caused insulin resistance in N2a-APP cells by phosphorylating IRS-1 at Ser612, inhibiting signaling to downstream targets. Aβ1-42-treated SHY exhibited similar IRS-1 phosphorylation at Ser612 and inflammatory response of JNK activation. Aβ1-42 caused down-regulation of neuro-protective/anti-inflammatory DHCR24, and an increase in HMGCR levels indicating dysregulation of cholesterol homeostasis in SHY cells. Insulin resistance, Aβ toxicity, neuro-inflammation and dysregulation of cholesterol homeostasis appear to be intertwined processes in AD that should be studied simultaneously.

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.

Schizophrenia is primarily treated with the use of antipsychotic medications. However, antipsychotics used have severe, dose-dependent side effects in schizophrenia patients. Therefore, there is a need for new adjunctive drugs that lower the effective dose of first line schizophrenia drugs and improve patient symptoms. Neuroinflammation is associated with microglial activation in schizophrenia, and increased tumor necrosis factor-alpha (TNF) has shown to be associated with Metabolic Syndrome in schizophrenia patients. A newly developed anti-neuroinflammatory, PD2024, reduces TNF-alpha action in vitro and in vivo, and has been shown to be well-tolerated in rat and dog studies with no adverse effects. The purpose of this research is to evaluate the effect of PD2024 in two well-defined schizophrenia models in rats. The neonatal quinpirole model has been established through administration of the dopamine D2-like agonist quinpirole (NQ) or saline control (NS) postnatally from days 1-21. NQ treatment results in increases of dopamine D2 receptor sensitivity throughout the animal’s lifetime without changing receptor number, mimicking a hallmark of schizophrenia. The polyinosinic:polycytidylic acid (Poly I:C) model is based on mimicking an increase immune response during early brain development, which has been shown to increase the prevalence of schizophrenia. Poly I:C (2 mg/kg) was administered during the neonatal period at postnatal days (P)5-7 to produce this effect. Both models were given PD2024 at 10mg/kg orally through the diet from P30-67. Prepulse inhibition (PPI) was used to test sensorimotor gating deficits in the rats. PPI has past research showing its use as a quantitative phenotype for evaluating schizophrenia-associated behavioral and neurobiological deficits. In our PPI test, rats are exposed to three different, randomly ordered noise trials. The trials included a pulse trial with a 120-decibel startle pulse, a prepulse trial with an auditory click at 73, 76, or 82-decibels, and a no stimulus trial without any additional noise. The rats were given 25 randomized trials, comprised of 5 pulse, 15 prepulse (5 each of 73, 76, and 82dB) and 5 no stimulus trials. Background noise was 70dB, and the rats were tested during adolescence (days 45-46) and adulthood (60-65). In NQ adolescent rats, PPI was significantly improved in the PD2024-treated compared to NQ controls. NQ-PD2024 and NS rats were statistically equivalent throughout the trials. These results were reflected in the NQ adult model as well. The Poly I:C adolescents treated with PD2024 also demonstrated improved PPI performance compared to Poly I:C controls. This improvement was also shown in the adult Poly I:C rats. Overall, the PPI deficits in both models improved between 15 to 30% in adolescence and adulthood. These results indicate that PD2024 is effective in treating schizophrenia-associated behaviors.

Alcohol use can negatively impact financial, cognitive, and psychiatric aspects of human life. In the brain, alcohol can have many devastating effects. Alcohol is a well-known cytotoxic agent that can cause specific brain pathology in humans; however, the exact biological mechanisms are not well-elucidated. Animal models are invaluable tools to investigate potential novel treatments in a substance abuse model. Mice studies can be used to screen for negative outcomes prior to human trials. We hypothesize that the C1q tumor necrosis factor-related protein, CTRP3, overexpression in mice reduces neuroinflammation from ethanol consumption that has been coupled with a high fat diet when compared to control mice. The CTRP family of proteins are adipokines and CTRP3 specifically influences cell viability, metabolism, and peripheral inflammation levels. Antibody specific immunoblotting is used to probe protein expression changes in neuroinflammatory markers in mouse cerebellum brain tissue in an overexpression mouse model of CTRP3 when compared to high-fat ethanol exposed mice and baseline control mice. The two proteins examined are MAG and GFAP. Myelin associated glycoprotein, or MAG, is a protein expressed by oligodendrocytes that mediate axonal growth and myelin interactions with neurons in the brain. Oligodendrocytes are extremely sensitive to oxidative stress to which cognitive deficits in ethanol exposure is thought to be attributed. Glial fibrillary acidic protein, or GFAP, is a marker of astrocyte reactivity. Astrocytes are cells in the brain that are responsible for environmental stabilization and actively participate in neurotransmission. Currently, GFAP alterations in ethanol-exposed animals are dose and age dependent. We chose to use young adult mice where GFAP reactiveness is increased during chronic ethanol exposure. The proposed studies are essential in determining CTRP3’s relationship to detrimental neuroinflammatory effects of alcohol and high fat diet in mice. The data obtained from these studies will provide compelling evidence for future clinical trials to investigate CTRP3 as a therapeutic agent in people with a high fat diet that use alcohol chronically.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22310号 / 医博第4551号 / 新制||医||1040(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 渡邉 大, 教授 万代 昌紀, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM