Introdução: A Doença de Alzheimer (DA) é uma desordem neurodegenerativa e a forma mais comum de demência. Processos inflamatórios parecem desempenhar importante papel na fisiopatologia da DA. A neuroinflamação é caracterizada pela ativação da microglia e a liberação de citocinas inflamatórias, tais como IL-1β, IL-6 e TNF-α. Porém, não se sabe qual é a real contribuição destes marcadores inflamatórios no desenvolvimento da DA. Objetivos: A proposta deste estudo é avaliar a possível relação entre marcadores inflamatórios no liquido cefalorraquidiano de indivíduos com comprometimento cognitivo leve amnéstico (CCL-a), com 60 anos ou mais, e comparar com controles saudáveis da mesma faixa etária. Métodos: Foram examinadas as concentrações de IL-1β, IL-6 e TNF-α no líquido cefalorraquidiano de sujeitos com CCL-a e em controles pelo método ELISA. Diagnósticos de CCL-a foram baseados na anamnese e nos critérios de Petersen, corroborados pela escala CDR. Para avaliar a função cognitiva o teste de memória e reconhecimento de palavras do CERAD e o Teste do Relógio foram aplicados aos participantes. Para a avaliação de sintomas depressivos usou-se o GDS. Resultados: Este estudo demonstrou diminuição significativa nos níveis de IL-1β (13.735 vs 22.932 pg/mL; p <0.001) e TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), mas não nos níveis da IL-6 (4.178 vs 5.689 pg/mL; p: 0.106), entre casos e controles. Indivíduos com IL-1β < 17 pg/mL possuem 7.2 (CI: 1.5-36; p: 0.016) mais chances de evoluírem à CCL-a. Além disso, houve correlação positiva entre IL-1β e a pontuação da lista de palavras do CERAD (rs: 0.299; p: 0.046). A análise de regressão linear mostrou que os níveis de IL-1β podem explicar 13.7% (β: 24.545; p: 0.012) da variância da pontuação do CERAD, o que sugere uma dependência linear direta. Conclusões: A neuroinflamação, mediada pela IL-1β e pelo TNF-α, provavelmente possui importante papel na prevenção de CCL-a. / Introduction: Alzheimer Disease (AD) is a neurodegenerative disorder and the most common form of dementia. Inflammatory processes may play a significant role at the pathophysiology of AD. Neuroinflammation is characterized by activation of microglia and the release of inflammatory cytokines, such as IL-1β, IL-6 and TNF-α. Although, it is unknown what is the real contribution of these inflammatory markers in the development of AD. Aims: The purpose of this study is to assess the possibly relationship between inflammatory markers in CSF of amnestic MCI subjects, with sixty years or older, and compare to aged healthy controls. Methods: We examined concentrations of IL-1β, IL-6 and TNF-α at CSF of amnestic MCI subjects and controls by ELISA. MCI diagnoses were based on anamnesis and Petersen criteria, corroborated by CDR. To assess the cognitive function the word list memory test and word recognition of CERAD and Clock Drawing Test were applied to subjects, and to evaluated depression symptoms the GDS was used. Results: This study demonstrated significant diminish in the levels of IL-1β (13.735 vs 22.932 pg/mL; p <0.001) and TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), but not IL-6 (1.913 vs 2.627 pg/mL; p: 0.002), between cases and controls. Individuals with IL-1β < 17 pg/mL were at a 7.2 (CI: 1.5-36; p: 0.016) increased odds of aMCI. Furthermore, there was a positive correlation between IL-1β and the CERAD word list score (rs: 0.299; p: 0.046). The linear regression analysis showed that IL-1β levels can explain 13.7% (β: 24.545; p: 0.012) of the variance on this CERAD subscore, suggesting a direct linear dependence. Conclusion: Neuroinflamation mediated by IL-1β and TNF-α may play an important role in preventing aMCI.

Introdução: A Doença de Alzheimer (DA) é uma desordem neurodegenerativa e a forma mais comum de demência. Processos inflamatórios parecem desempenhar importante papel na fisiopatologia da DA. A neuroinflamação é caracterizada pela ativação da microglia e a liberação de citocinas inflamatórias, tais como IL-1β, IL-6 e TNF-α. Porém, não se sabe qual é a real contribuição destes marcadores inflamatórios no desenvolvimento da DA. Objetivos: A proposta deste estudo é avaliar a possível relação entre marcadores inflamatórios no liquido cefalorraquidiano de indivíduos com comprometimento cognitivo leve amnéstico (CCL-a), com 60 anos ou mais, e comparar com controles saudáveis da mesma faixa etária. Métodos: Foram examinadas as concentrações de IL-1β, IL-6 e TNF-α no líquido cefalorraquidiano de sujeitos com CCL-a e em controles pelo método ELISA. Diagnósticos de CCL-a foram baseados na anamnese e nos critérios de Petersen, corroborados pela escala CDR. Para avaliar a função cognitiva o teste de memória e reconhecimento de palavras do CERAD e o Teste do Relógio foram aplicados aos participantes. Para a avaliação de sintomas depressivos usou-se o GDS. Resultados: Este estudo demonstrou diminuição significativa nos níveis de IL-1β (13.735 vs 22.932 pg/mL; p <0.001) e TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), mas não nos níveis da IL-6 (4.178 vs 5.689 pg/mL; p: 0.106), entre casos e controles. Indivíduos com IL-1β < 17 pg/mL possuem 7.2 (CI: 1.5-36; p: 0.016) mais chances de evoluírem à CCL-a. Além disso, houve correlação positiva entre IL-1β e a pontuação da lista de palavras do CERAD (rs: 0.299; p: 0.046). A análise de regressão linear mostrou que os níveis de IL-1β podem explicar 13.7% (β: 24.545; p: 0.012) da variância da pontuação do CERAD, o que sugere uma dependência linear direta. Conclusões: A neuroinflamação, mediada pela IL-1β e pelo TNF-α, provavelmente possui importante papel na prevenção de CCL-a. / Introduction: Alzheimer Disease (AD) is a neurodegenerative disorder and the most common form of dementia. Inflammatory processes may play a significant role at the pathophysiology of AD. Neuroinflammation is characterized by activation of microglia and the release of inflammatory cytokines, such as IL-1β, IL-6 and TNF-α. Although, it is unknown what is the real contribution of these inflammatory markers in the development of AD. Aims: The purpose of this study is to assess the possibly relationship between inflammatory markers in CSF of amnestic MCI subjects, with sixty years or older, and compare to aged healthy controls. Methods: We examined concentrations of IL-1β, IL-6 and TNF-α at CSF of amnestic MCI subjects and controls by ELISA. MCI diagnoses were based on anamnesis and Petersen criteria, corroborated by CDR. To assess the cognitive function the word list memory test and word recognition of CERAD and Clock Drawing Test were applied to subjects, and to evaluated depression symptoms the GDS was used. Results: This study demonstrated significant diminish in the levels of IL-1β (13.735 vs 22.932 pg/mL; p <0.001) and TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), but not IL-6 (1.913 vs 2.627 pg/mL; p: 0.002), between cases and controls. Individuals with IL-1β < 17 pg/mL were at a 7.2 (CI: 1.5-36; p: 0.016) increased odds of aMCI. Furthermore, there was a positive correlation between IL-1β and the CERAD word list score (rs: 0.299; p: 0.046). The linear regression analysis showed that IL-1β levels can explain 13.7% (β: 24.545; p: 0.012) of the variance on this CERAD subscore, suggesting a direct linear dependence. Conclusion: Neuroinflamation mediated by IL-1β and TNF-α may play an important role in preventing aMCI.

Introdução: A Doença de Alzheimer (DA) é uma desordem neurodegenerativa e a forma mais comum de demência. Processos inflamatórios parecem desempenhar importante papel na fisiopatologia da DA. A neuroinflamação é caracterizada pela ativação da microglia e a liberação de citocinas inflamatórias, tais como IL-1β, IL-6 e TNF-α. Porém, não se sabe qual é a real contribuição destes marcadores inflamatórios no desenvolvimento da DA. Objetivos: A proposta deste estudo é avaliar a possível relação entre marcadores inflamatórios no liquido cefalorraquidiano de indivíduos com comprometimento cognitivo leve amnéstico (CCL-a), com 60 anos ou mais, e comparar com controles saudáveis da mesma faixa etária. Métodos: Foram examinadas as concentrações de IL-1β, IL-6 e TNF-α no líquido cefalorraquidiano de sujeitos com CCL-a e em controles pelo método ELISA. Diagnósticos de CCL-a foram baseados na anamnese e nos critérios de Petersen, corroborados pela escala CDR. Para avaliar a função cognitiva o teste de memória e reconhecimento de palavras do CERAD e o Teste do Relógio foram aplicados aos participantes. Para a avaliação de sintomas depressivos usou-se o GDS. Resultados: Este estudo demonstrou diminuição significativa nos níveis de IL-1β (13.735 vs 22.932 pg/mL; p <0.001) e TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), mas não nos níveis da IL-6 (4.178 vs 5.689 pg/mL; p: 0.106), entre casos e controles. Indivíduos com IL-1β < 17 pg/mL possuem 7.2 (CI: 1.5-36; p: 0.016) mais chances de evoluírem à CCL-a. Além disso, houve correlação positiva entre IL-1β e a pontuação da lista de palavras do CERAD (rs: 0.299; p: 0.046). A análise de regressão linear mostrou que os níveis de IL-1β podem explicar 13.7% (β: 24.545; p: 0.012) da variância da pontuação do CERAD, o que sugere uma dependência linear direta. Conclusões: A neuroinflamação, mediada pela IL-1β e pelo TNF-α, provavelmente possui importante papel na prevenção de CCL-a. / Introduction: Alzheimer Disease (AD) is a neurodegenerative disorder and the most common form of dementia. Inflammatory processes may play a significant role at the pathophysiology of AD. Neuroinflammation is characterized by activation of microglia and the release of inflammatory cytokines, such as IL-1β, IL-6 and TNF-α. Although, it is unknown what is the real contribution of these inflammatory markers in the development of AD. Aims: The purpose of this study is to assess the possibly relationship between inflammatory markers in CSF of amnestic MCI subjects, with sixty years or older, and compare to aged healthy controls. Methods: We examined concentrations of IL-1β, IL-6 and TNF-α at CSF of amnestic MCI subjects and controls by ELISA. MCI diagnoses were based on anamnesis and Petersen criteria, corroborated by CDR. To assess the cognitive function the word list memory test and word recognition of CERAD and Clock Drawing Test were applied to subjects, and to evaluated depression symptoms the GDS was used. Results: This study demonstrated significant diminish in the levels of IL-1β (13.735 vs 22.932 pg/mL; p <0.001) and TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), but not IL-6 (1.913 vs 2.627 pg/mL; p: 0.002), between cases and controls. Individuals with IL-1β < 17 pg/mL were at a 7.2 (CI: 1.5-36; p: 0.016) increased odds of aMCI. Furthermore, there was a positive correlation between IL-1β and the CERAD word list score (rs: 0.299; p: 0.046). The linear regression analysis showed that IL-1β levels can explain 13.7% (β: 24.545; p: 0.012) of the variance on this CERAD subscore, suggesting a direct linear dependence. Conclusion: Neuroinflamation mediated by IL-1β and TNF-α may play an important role in preventing aMCI.

Studies presented in this dissertation were conducted to gain more insight into the role of phospholipid asymmetry and amyloid-β (Aβ)-induced oxidative stress in brain of subjects with amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD). AD is a largely sporadic, age-associated neurodegenerative disorder clinically characterized by the vast, progressive loss of memory and cognition commonly in populations over the age of ~65 years, with the exception of those with familial AD, which develop AD symptoms as early as ~30 years-old. Neuropathologically, both AD and FAD can be characterized by synapse and neuronal cell loss in conjunction with accumulation of neurofibrillary tangles and senile plaques. Elevated levels of oxidative stress and damage to brain proteins, lipids, and nucleic acids are observed, as well. Likewise, aMCI, arguably the earliest form of AD, displays many of these same clinical and pathological characteristics, with a few exceptions (e.g., no dementia) and to a lesser extent. Studies in this dissertation focused on the contributions of oxidative stress to the exposure of phosphatidylserine (PtdSer) to the outer-leaflet of the lipid membrane, how and when PtdSer asymmetric collapse contributes to the progression of aMCI, AD, and FAD, and the role played by methionine-35 (Met-35) of Aβ in oxidative stress and damage, as measured in a transgenic mouse model of Aβ pathology. Normally, the PtdSer is sequestered to the cytosolic, inner-leaflet of the bilayer by the adenosine triphosphate (ATP)-dependent, membrane-bound translocase, flippase, which unidirectionally transports PtdSer inward against its concentration gradient. Oxidative stress-induced modification of flippase and/or PtdSer, however, leads to prolonged extracellular exposure of PtdSer on the outer membrane leaflet, a known signal for both early apoptosis and selective recognition and mononuclear phagocytosis of dying cells. Within the inferior parietal lobule (IPL) of subjects with aMCI and AD, a significant collapse in PtdSer asymmetry was found in association with increased levels of both pro- and anti-apoptotic proteins, Bax, caspase-3, and Bcl-2. Moreover, a significant collapse in PtdSer asymmetry was also found in whole brain of human double-mutant knock-in mouse models of Aβ pathology, together with significantly reduced Mg2+ATPase activity, representing flippase activity, and increased levels of pro-apoptotic caspase-3. Significant PtdSer externalization corresponded to the age at which significant soluble Aβ(1-42) deposition occurs in this particular mouse model (9 months), and not of plaque deposition (12 months), suggesting that elevated levels of Aβ(1-42), together with increasing oxidative stress and apoptosis, may contribute to altered PtdSer membrane localization. Also in this dissertation, transgenic mice carrying Swedish and Indiana mutations on the human amyloid precursor protein (APPSw,In) and APPSw,In mice carrying a Met35Leu mutation on Aβ were derived to investigate the role of Met-35 in Aβ(1-42)-induced oxidative stress in vivo. Oxidative stress analyses revealed that Aβ-induced oxidative stress requires the presence of Met-35, as all indices of oxidative damage (i.e., protein carbonylation, nitration, and protein-bound 4-hydroxy-2-trans-nonenal [HNE]) in brain of Met35Leu mice were completely prevented. Moreover, immunohistochemical analyses indicated that the Met35Leu mutation influences plaque formation, as a clear reduction in Aβ-immunoreactive plaques in Met35Leu mice was found in conjunction with a significant increase in microglial activation. In contrast, behavioral analyses suggested that spatial learning and memory was independent of Met-35 of Aβ, as Met35Leu mice demonstrated inferior water-maze performance compared to non-transgenic mice. Differential expression and redox proteomic analyses to pinpoint proteins significantly altered by the APPSw,In and Met35Leu mutations was performed, as well. Expression proteomics showed significant increases and decreases in APPSw,In and Met35Leu mouse brain, respectively, in proteins involved in cell signaling, detoxification, structure, metabolism, molecular chaperoning, protein degradation, mitochondrial function, etc. Redox proteomics found many of these same proteins to be oxidatively modified (i.e., protein carbonylation and nitration) in both APPSw,In and Met35Leu mouse brain, providing additional insights into the critical nature of Met-35 of Aβ for in vivo oxidative stress in a mammalian species brain, and strongly suggesting similar importance of Met-35 of Aβ(1-42) in brain of subjects with aMCI and AD. Taken together, studies presented in this dissertation demonstrate the role of oxidative stress-induced alteration of PtdSer asymmetry and Met-35 in Aβ-induced oxidative stress in aMCI, AD, and FAD brain.