The processing of amyloid precursor protein (APP) by - and -secretases produces amyloid (A) peptide, the principal component of the neuritic plaques found in Alzheimers disease (AD) pathology. The enzyme -secretase is a multimeric protein consisting of presenilins-1/2 (PS1/PS2), nicastrin, anterior pharynx defective 1 (APH-1) and presenilin enhancer-2 (PEN-2). Recently it was discovered that p23, a transmembrane protein involved in intracellular protein trafficking, negatively regulates -secretase activity. In the present study, I evaluated the levels/expression of the nicastrin and p23 and their possible colocalization with PS1 in normal adult and developing brains. Additionally, I have studied the alterations of p23 levels in both animal model of neurodegeneration and in postmortem AD brains. Nicastrin and p23 were widely distributed throughout the brain and colocalized in all brain regions with PS1. The levels of nicastrin and p23 were relatively high at the early stages of postnatal development and then declined gradually as age increased. Interestingly, p23 level/expression was found to be altered following kainic acid-induced neurodegeneration in the adult rat brain. Additionally, p23 levels were reduced in the brains of individuals with AD. These results, taken together, suggest that both nicastrin and p23 are expressed in neurons throughout the brain and their levels decline gradually during development to reach an adult profile. Additionally, my results indicate that a decreased level of p23 may contribute to AD pathogenesis by increasing the production of A-related peptides.

No description available.

Submitted by Isac Macêdo (isac@ioc.fiocruz.br) on 2013-10-29T13:11:14Z No. of bitstreams: 1 Dissertação de Mestrado - Carolina C B Gruszkowski.pdf: 2705318 bytes, checksum: f07dc28b794bcd1bd613cf1fad937d4a (MD5) / Made available in DSpace on 2013-10-29T13:11:14Z (GMT). No. of bitstreams: 1 Dissertação de Mestrado - Carolina C B Gruszkowski.pdf: 2705318 bytes, checksum: f07dc28b794bcd1bd613cf1fad937d4a (MD5) / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Os métodos de diagnóstico usados hoje para a Doença de Chagas, embora simples e de baixo custo, podem apresentar baixas sensibilidade e especificidade ou reações cruzadas com outros patógenos. Da mesma forma, os medicamentos usados no tratamento apresentam severos efeitos colaterais. Dentre os vários alvos metabólicos sugeridos, as proteases têm sido apontadas como moléculas candidatas devido às suas particularidades.Recentemente o nosso grupo identificou duas aspartil proteases em T. cruzi: uma solúvel (Cruzipsina-Il) e outra membranar (Cruzipsina-I); entretanto, suas funções biológicas permaneceram desconhecidas. Os nossos resultados demonstram, pela 1 a vez, a existência de um complexo proteolítico membranar em T. cruzi com propriedades similares ao complexo y-secretase de outras células eucarióticas. Usando ferramentas de bioinformática localizamos em banco de dados as seqüências primárias de 3 das 4 proteínas do complexo (presenilina, nicastrina e Aph-l). Esta informação foi importante para caracterizarmos através de síntese paralela de peptídeos os epitopos B lineares das 3 proteínas usando soros de pacientes. A presenilina apresentou 10 epitopos majoritários, a nicastrina, 5 e a Aph-l, 10. Alguns epitopos foram selecionados por vários critérios e anticorpos anti-peptídeos obtidos em coelhos. O soro anti-peptídeo PRE-2 de presenelina identificou por immunoblotting a banda de 48 kDa na fração CZP-I como sendo presenelina, enquanto o soro anti-peptideo NIC-l de nicastrina apresentou também uma marcação na mesma posição. A análise por microscopia confocal em epimastigotas localizou as proteínas predominantemente nas regiões anterior e mediana das células. Além disso, as marcações mostraram-se mais fortes em células permeabilizadas, sugerindo a co-localização de ambas as proteínas nas membranas internas da célula. Um teste de ELISA empregando 4 peptídeos sintéticos da presenilina e nicastrina de T. cruzi apresentou sensibilidade e especificidade de 71,59% e 70,53%, respectivamente. Utilizando a extração diferencial com detergente e análise por EGPA-SDS após cromatografia de afinidade, confirmamos na cepa CL Brener de T. cruzi o perfil de bandas presentes na cepa Y, de 240, 56, 48 e 34 kDa na fração CZP-I (detergente) e 56, 52, 37 e 34 kDa na fração CZP-I1 (solúvel). Ensaios enzimáticos usando o inibidor L-685,458 e moduladores alostéricos DAPT e Composto XXIIE da presenilina humana sugeriram que aparentemente o sítio catalítico da presenilina de T. cruti possui uma constituição semelhante. ao da presenilina humana, enquanto diferenças significativas puderam ser observadas no sitio alostérico. O inibidor de sítio ativo L-685,458 foi capaz de inibir 100% da atividade enzimática em 5!-lM, enquanto os moduladores DAPT e Composto XXIIE não apresentaram uma inibição significativa. Portanto, os nossos estudos sugerem que devido as suas funções enzimáticas e localização celular a presenilina do T. cruzi é um bom alvo quirnioterapêutico e imunológico. / The diagnostic methods used today for Chagas disease, although simple and unexpensive, may present low sensitivity and specificity or cross-reactions with other pathogens. Likewise, drugs used nowadays to treat Chagas disease show severe side effects. Among the various metabolic targets suggested, proteases have been identified as candidate molecules due to its particularities. Recently our group identified two aspartyl proteases in T. cruzi: a soluble (Cruzipsin-I1) and a membrane-bound (Cruzipsin-l); however, their biological functions remained unknown. Our results demonstrate for the 1 st time, the existence of a membrane proteolytic complex in T. cruzi with similar properties to the y-secretase complex of other eukaryotic cells. Using bioinformatics tools, the primary sequences of three of four complex proteins (presenilin, nicastrin and Aph-l) where localized at a protein database. This information was important for parallel synthesis of peptides and the identification of linear epitopes of proteins using sera from eight patients. Presenilin presented 10 major epitopes, nicastrina,5 and Aph-l, 10. Some epitopes were selected by different criteria and anti-peptide sera were obtained in rabbits. Serum anti-peptide PRE-2 of presenelin .identified by immunoblotting a 48 kDa band in fraction CZP-I as presenelin, while the anti-peptide NIC-I of nicastrin also presented a marking in the same position. Analysis by confocal microscopy of epimastigotes localized the proteins mainly at anterior and middle portions of the cells. ln addition, the markings were found to be stronger in permeabilized cells, suggesting co- localization of both proteins in the inner membranes of the cell. An ELISA employing four synthetic peptides for T. cruzi presenilin and nicastrin presented sensitivity of 71.59% and specificity and 70.53%. USlng differential detergent extraction and analysis by SDS-PAGE after affinity chromatography confirmed that the profile bands present in the strain CL Brener were like those of strain Y: 240, 56, 48 and 34 kDa in fraction CZP-I (detergent), and 56, 52, 37 and 34 kDa in CZP-I1 fraction (solub1e). Enzyme assays using the inhibitor L-685,458 and allosteric modulators DAPT and Compound XXI/E of human presenilin apparently suggested that the catalytic site of T. cruri presenilin has a constitution similar to the human presenilin, while significant differences were observed in the allosteric site. The active site inhibitor L-685,458 was able to inhibit 100% of enzyme activity at 5 !J.M, while the modulators DAPT and Compound XXI/E showed no significant inhibition. Therefore, our studies suggest that due to their enzymatic functions and cellular localization the presenilin of T. cruzi is a good target for chemotherapeutic and for immune system.

O Mal de Alzheimer é o maior causador de demência em idosos: acomete 10% da população mundial com idade em torno dos 65 anos e atinge cerca de 50% dos indivíduos com mais de 85 anos. A progressão dos sintomas da doença está associada a modificações estruturais nas sinapses colinérgicas em determinadas regiões cerebrais. A maior característica fisiopatológica do AD é a deposição de placas neuríticas extracelulares em áreas cerebrais relacionadas à memória, placas constituídas pelo peptídeo ?-amiloide 40/42, que é formado pela clivagem da Proteína Precursora Amiloide, durante seu metabolismo pela via amiloidogênica, que começa com a enzima ?-secretase. O objetivo do trabalho foi o planejamento e avaliação de novos inibidores de ?-secretase. Para isso, foram utilizadas diferentes técnicas de modelagem molecular e planejamento de moléculas, tendo como base os inibidores da ?-secretase descritos na literatura cujas estruturas estão depositadas no PDB. Posteriormente, foi realizada a avaliação in vitro da atividade inibitória de algumas destas moléculas, onde observou-se que três são capazes de satisfatoriamente inibir a atividade da ?-secretase na concentração de 1 µM. / The Alzheimer\'s disease is the major cause of elderly dementia: it affects 10% of global population with 65 years old and about 50% of individuals with 85 years old or more. The evolution of the disease symptoms is associated with structural changes in cholinergic synapses at certain brain regions. The major pathophysiological feature of AD is the deposition of extracellular neuritic plaques in areas of the brain related to memory. The ?-amyloid peptide 40/42 constitutes the plaques. It\'s formed by cleavage of the amyloid precursor protein during its metabolism by the amyloidogenic pathway, which starts with the ?-secretase enzyme. The goal of this project was the planning and evaluation of new ?-secretase inhibitors activity. For this, we used different molecular modeling and drug design techniques, based on the ?-secretase inhibitors described in the literature, whose structures are deposited in the PDB, with subsequent in vitro evaluation of this molecules activity. The in vitro assays showed three molecules able to inhibit ?-secretase at 1 µM.

In certain neurodegenerative diseases such Dementia with Lewy Bodies (DLB), it is hypothesised that misfolded α-synuclein (α-syn) and β-amyloid both contribute to pathology. α-Syn and β-amyloid have been suggested to synergistically promote one another’s accumulation and aggregation, but the mechanisms are unknown. β-Amyloid is generated from β-/γ-secretase-mediated processing of the amyloid precursor protein (APP). This study investigated how α-syn overexpression in cells affects β-amyloid production from APP, using multiplex assays, luciferase reporter assays, and western blotting. Wildtype α-syn expression induces β-amyloid generation from APP in SH-SY5Y human neuroblastoma cells, and similar changes to APP processing occur in another neuronal cell model. Dominant-negative overexpression of α-syn mutants revealed that disrupting the N-terminal domain can increase APP amyloidogenic processing. Secretase enzymes that perform APP processing were next investigated. γ-Secretase activity, measured by a luciferase reporter, was not increased by α-syn overexpression. A higher ratio of β- to α-secretase processing was hypothesised, which led to expression and activity studies of the major β- and α-secretases, BACE1 and ADAM10 respectively. It was shown that the BACE1 protein expression is post-transcriptionally upregulated in α-syn cells, with increased APP cleavage in cells. ADAM10 protein expression is transcriptionally suppressed in wild-type α-syn cells, reducing total levels of catalytically active enzyme. However the change in ADAM10-mediated APP processing may be negligible since, critically, plasma membrane expression of ADAM10 appears to be maintained. To aid understanding of the mechanism that connects α-syn to APP processing, BACE1 expression was used in pharmacological studies of cell stress signalling. This approach revealed that in α-syn cells BACE1 lysosomal and/or proteasomal degradation may be disturbed. Additionally, BACE1 expression is induced by translational de-repression mediated by eIF2α ser-51 phosphorylation, which was increased in α-syn cells. Although preliminary, the data suggests a role for oxidative stress mediating the increased BACE1 expression in wild-type α-syn cells.

O Mal de Alzheimer é o maior causador de demência em idosos: acomete 10% da população mundial com idade em torno dos 65 anos e atinge cerca de 50% dos indivíduos com mais de 85 anos. A progressão dos sintomas da doença está associada a modificações estruturais nas sinapses colinérgicas em determinadas regiões cerebrais. A maior característica fisiopatológica do AD é a deposição de placas neuríticas extracelulares em áreas cerebrais relacionadas à memória, placas constituídas pelo peptídeo ?-amiloide 40/42, que é formado pela clivagem da Proteína Precursora Amiloide, durante seu metabolismo pela via amiloidogênica, que começa com a enzima ?-secretase. O objetivo do trabalho foi o planejamento e avaliação de novos inibidores de ?-secretase. Para isso, foram utilizadas diferentes técnicas de modelagem molecular e planejamento de moléculas, tendo como base os inibidores da ?-secretase descritos na literatura cujas estruturas estão depositadas no PDB. Posteriormente, foi realizada a avaliação in vitro da atividade inibitória de algumas destas moléculas, onde observou-se que três são capazes de satisfatoriamente inibir a atividade da ?-secretase na concentração de 1 µM. / The Alzheimer\'s disease is the major cause of elderly dementia: it affects 10% of global population with 65 years old and about 50% of individuals with 85 years old or more. The evolution of the disease symptoms is associated with structural changes in cholinergic synapses at certain brain regions. The major pathophysiological feature of AD is the deposition of extracellular neuritic plaques in areas of the brain related to memory. The ?-amyloid peptide 40/42 constitutes the plaques. It\'s formed by cleavage of the amyloid precursor protein during its metabolism by the amyloidogenic pathway, which starts with the ?-secretase enzyme. The goal of this project was the planning and evaluation of new ?-secretase inhibitors activity. For this, we used different molecular modeling and drug design techniques, based on the ?-secretase inhibitors described in the literature, whose structures are deposited in the PDB, with subsequent in vitro evaluation of this molecules activity. The in vitro assays showed three molecules able to inhibit ?-secretase at 1 µM.

Un tiers de la population européenne est touché par au moins un trouble du cerveau. En effet, la maladie d’Alzheimer, et les gliomes, représentent respectivement le syndrome de démence et les tumeurs cérébrales les plus fréquentes chez l’homme. Plusieurs études épidémiologiques ont montré l’existence d’une corrélation inverse entre le risque de développer une maladie neurodégénérative et un cancer cérébral. Ceci suggère la présence de dénominateurs moléculaires communs entre ces pathologies. Dans les deux cas, un dysfonctionnement mitochondrial est rapporté, représentant une caractéristique partagée par ces deux troubles neurologiques. La protéine kinase mitochondriale PINK1 responsable, lorsqu’elle est mutée, d’une forme précoce et familiale de Parkinson, est particulièrement impliquée dans les processus de maintien de l’homéostasie mitochondriale. Par conséquent, les mécanismes moléculaires régulant PINK1 ainsi que leurs impacts au cours des désordres mitochondriaux répertoriés dans la maladie d’Alzheimer et les tumeurs cérébrales, ont suscité un intérêt central, lors de ma thèse. Au cours de ce travail, nous avons examiné certaines des fonctions mitochondriales de PinK1, associées au maintien de l’homéostasie mitochondriale dans un contexte « Alzheimerisé ». Nous mettons en évidence le rôle de la γ-secretase dans la physiologie mitochondriale en contrôlant la régulation transcriptionnelle de PINK1 par l’AICD, le fragment généré conjointement avec le peptide amyloïde toxique Aβ, à partir du précurseur βAPP. Nous montrons de surcroît, l’initiation de cette régulation par la parkine. / One third of the European populations is affected by a brain disorder. Thus, Alzheimer’s disease and gliomas represent the most frequent human brain dementia syndrome and tumor type, respectively. Several epidemiological studies have shown an inverse relationship between the risk of developing a neurodegenerative disease and a brain tumor, suggesting the existence of common molecular denominators between these pathologies. Interestingly, both pathologies are characterized by a mitochondrial dysfunction. The mitochondrial kinase associated to autosomal recessive Parkinson’s disease, PINK1, is particularly implicated in the control of mitochondrial homeostasis. The main objective of my thesis was to study the molecular mechanisms underlying PINK1 gene regulation and their link with the mitochondrial dysfunction observed in either Alzheimer’s disease or gliomas. Thus, during my thesis we have examined the ability of PINK1 to control mitochondria homeostasis in an Alzheimer’s pathological context. We demonstrate that AICD, a cleavage product of the trans-membrane protein βAPP by γ-secretase, impacts mitochondrial physiology via its ability of positively controlling PINK1 transcription. In addition, we show that the signaling cascade linking γ-secretase and PINK1 is initiated by parkin transcriptional regulation of presenilins, the main component of γ-secretase catalytic complex. Finally, we also establish that the tumor suppressor p53 can negatively regulate PINK1 transcription in vitro and in vivo suggesting that the misregulated autophagic response associated to brain tumors development may be caused by defective p53-PINK1 interplay.

Alzheimer disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of ß-amyloid (Aß) peptides as plaques in the brain. Central to this AD pathology is mismetabolism of the amyloid precursor protein (APP). Recent studies suggest that flavonoids, a class of secondary plant metabolites, may be useful for the prevention and treatment of a variety of neurodegenerative diseases. The studies detailed herein, investigate the ability of two such classes of flavonoids, green tea derived catechins and 5,7-dihydroxyflavones, to modulate APP metabolism in "Swedish" mutant APP (APPsw) models of AD. Studies showed that green tea derived (-)-epigallocatechin-3-gallate (EGCG) effectively reduced Aß generation and resultant amyloidosis both in vitro and in vivo. In concert with these findings, EGCG markedly promoted non-amyloidogenic APP proteolysis via activation of the putative a-secretase, a-disintegrin-and-metalloprotease-10 (ADAM10). Furthermore, luteolin and various related 5,7-dihydroxyflavones, effectively reduced Aß generation and resultant amyloidosis both in vitro and in vivo, as well. Data revealed that luteolin decreased amyloidogenic γ-secretase APP proteolysis via presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Elucidation of these flavonoids' cellular/molecular mechanisms also revealed their potential for opposing neurofibrillary tangle (NFT) pathology, another hallmark of AD. These data raise the possibility that flavonoid administration to AD patients may prove to be viable and effective prophylactic strategy.

<p>Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects over 5 million people in the United States alone. This number is predicted to triple to by the year 2050 due to both increasing life expectancies and the absence of disease-attenuating drugs. The etiology of AD remains unclear, and although there are multiple theories implicating everything from oxidative stress to protein misfolding, misregulated metal ions appear as a common thread in disease pathology. </p><p>Chelation therapy has shown some effectiveness in clinical trials, but to date, there are no FDA-approved metal chelators for the treatment of AD. One of the biggest problems with general chelators is their inability to differentiate between the metal ions involved in disease progression verses those involved in normal metabolic function. To address this problem, we have developed a prochelator approach whereby the prochelator (SWH) does not bind metals with significant biological affinity. However, once activated to the chelator (CP) via enzymatic hydrolysis, the molecule is able to bind copper and reduce its toxicity both in vitro and in a cellular model of Alzheimer's Disease. </p><p>Central to this strategy is the site-specificity provided by enzymatic activation of the prochelator. In our system, SWH to CP conversion is mediated by beta-secretase, an enzyme involved in A-beta generation. However, in order to render SWH capable of hydrolysis in cells, we modified the prochelator to contain a dihydrocholesterol membrane anchor attached via a polyethylene glycol linker. From this construct, we created beta-MAP, which is an SWH-based FRET probe to demonstrate beta-secretase-mediated conversion of SWH to CP. beta-MAP was also used to confirm the efficacy of a known beta-secretase inhibitor without the need to for mutated cells lines or expensive antibodies. beta;-MAP and the associated microscopy method represent a significant advancement to the currently available ELISA assays for beta-secretase activity.</p><p>While activation of the prochelator by an enzyme in cells is encouraging, non-specific hydrolysis of the peptide prevents significant accumulation of the chelator on the cell membrane. Furthermore, attachment of the polyethylene glycol and sterol units induce cell toxicity not seen with the native CP peptide. These drawbacks prevent the current prochelator from effectively protecting cells from AD conditions. Structural modifications to overcome these problems, including implementation of a new peptide sequence are planned for future experiments.</p> / Dissertation

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.