Alzheimer’s disease (AD) is a multifactorial disease that results in progressive neurodegeneration. Brain regions are differentially affected in AD; some are more vulnerable to degeneration than others. There is an age-dependent effect on beta-amyloid (Aβ) accumulation and neuroinflammation as disease progresses. In the TgCRND8 APP transgenic mouse model, levels of various Aβ species and cytokines were determined as a function of brain region and age. Aβ was found to accumulate in the brain prior to the sequential elevation of IL-1β and CXCL1. Levels of Aβ, IL-1β and CXCL1 were elevated in regions that are severely affected in AD patients. It has been shown for the first time in an APP transgenic model that CXCL1 elevation occurs following IL-1β elevation.

Die Alzheimersche Erkrankung (AD) ist die häufigste Demenzerkrankung bei älteren Menschen in den westlichen Industriestaaten mit ständig wachsender Zahl der Erkrankten. Trotz angestrengter wissenschaftlicher und medizinischer Forschung ist u. a. noch keine Möglichkeit der klinischen Frühdiagnose dieser Erkrankung etabliert. In der vorliegenden Arbeit wurden am transgenen Mausmodell Tg2576 mit beta-Amyloidplaque-Pathologie cholinerge und adrenerge Parameter sowie Einfluss-größen des Energiestoffwechsels untersucht, um transgen-induzierte neuro-chemische bzw. neuromorphologische Veränderungen im Hirngewebe zu erkennen. Außerdem sollte die Möglichkeit einer ex vivo Markierung solcher Moleküle getestet werden, von denen eine besondere Bindungsaffinität an beta-Amyloidablagerungen bereits bekannt ist. Das Ziel der vorliegenden Arbeit war, diese Mauslinie weiterhin hinsichtlich ihres Modellcharakters für die Alzheimersche Erkrankung zu beschreiben und potenzielle Plaque-assoziierte Markermoleküle aufzufinden, die einen in vivo Nachweis der b-Amyloidablagerungen erlauben. Die so gewonnenen Erkenntnisse könnten zur Entwicklung neuer Strategien zur Frühdiagnostik der Alzheimerschen Erkrankung beitragen. Genutzt wurden dazu v. a. die biochemischen Methoden der Rezeptorautoradiografie und der Immunhistochemie sowie der in situ Hybridisierung als molekularbiologische Methode. Weiterhin wurde eine radiochemische Methode zur ex vivo Darstellung der Azetylcholinesterase getestet. Bei der Untersuchung des cholinergen Systems konnte eine signifikante Verringerung in der [3H]Hemicholinium-3-Bindung (als Marker des hoch affinen Cholintransporters) bei den fünf Monate alten transgenen Mäusen im Vergleich zu deren nicht transgenen Wurfgeschwistern festgestellt werden. Es wird hier von einem modulatorischen Effekt des löslichen beta-Amyloids ausgegangen, da die jüngeren Tiere noch keine Plaqueablagerungen aufweisen, die Ursache solcher Veränderungen sein könnten. Beim vesikulären Azetylcholintransposter (VAChT) konnte eine signifikante Erhöhung in der [3H]Vesamicol-Bindung bei 17 Monate alten transgenen Mäusen im Vergleich zu nicht transgenen Geschwistertieren beobachtet werden, obwohl der gegenteilige Fall erwartet wurde. Das Ergebnis wird als Ausdruck einer erhöhten Vesikeldichte interpretiert. Die immunhistochemische Untersuchung der a4- und a7-Untereinheiten der nikoti-nischen Azetylcholinrezeptoren ergab, dass die beta-Amyloidplaqueablagerung keinen Einfluss auf morphologische Veränderungen der Neuronen hatte, die diese Rezep-toren tragen. Es gab keine Hinweise, dass solche Neuronen degenerieren. Hinsichtlich der untersuchten Parameter des Glukosestoffwechsels konnten keine Veränderungen zwischen transgenen Tieren und nicht transgenen Wurfgeschwistern festgestellt werden. Bei der Verteilung der Hirnkapillaren konnte eine verringerte Dichte in unmittelbarer Umgebung der b-Amyloidplaques, verglichen mit weiter entfernt liegenden Gebieten, ermittelt werden. Dieser Befund bedarf weiterer Unter-suchungen, da er Relevanz für die in vivo Diagnostik und Therapie besitzen könnte. Im Vergleich mit den zwar oft nicht einheitlichen Befunden bei Alzheimerpatienten wird deutlich, dass das Mausmodell Tg2576 als Modell der Alzheimerschen Erkrankung nicht alle Aspekte der Pathogenese simuliert. Übereinstimmungen ergeben sich bei dieser transgenen Maus hinsichtlich der beta-Amyloidproduktion und -ablagerung im Hirn und den auch beim Menschen vorkommenden entzündlichen Reaktion um die Plaques. Damit kann sie als geeignetes Modell zum Studium der Amyloidogenese und damit verbundener inflammatorischer Prozesse angesehen werden. / Alzheimer´s Disease (AD) is the most common form of dementia among the elderly in the Western world, with growing prevalence. In spite of intensive scientific and medical research, no possibility of early clinical diagnosis for this disease has yet been established. In this thesis cholinergic and adrenergic parameters, as well as energy metabolism, were studied in the transgenic mouse model Tg2576, to reveal transgene-induced neurochemical and neuromorphological changes in the brain tissue of these animals. Also, the ex vivo labelling of marker molecules with a known high affinity for beta-amyloid was to be tested. The objective was to further characterize the Tg2576 mouse strain as a model of AD, and to find plaque-associated marker molecules which could be used for the in vivo detection of b-amyloid plaques. Such findings could contribute to the development of new stategies for the early diagnosis of AD. Quantitative receptor autoradiography, immunohistochemistry and in situ hybridization were the main biochemical and molecular biological methods employed. Furthermore, a radiochemical method was used for ex vivo labelling of acetylcholinesterase. The 5 month-old transgenic mice, with no significant plaque load, demonstrated reduced [3H]hemicholinium-3 binding to choline uptake sites in anterior brain regions, as compared to non-transgenic littermates. This provides evidence of the modulatory effect of soluble beta-amyloid on high affinity choline uptake sites. However, a significant increase of [3H]vesamicol receptor binding to the vesicular acetylcholine transporter was detected in 17 month-old transgenic animals, as compared to non-transgenic mice. Even though the opposite was expected, the result could be interpreted as an elevated vesicle density. The immunhistochemical studies of a4 and a7 subunits of the nicotinic acetylcholine receptor revealed that neurons expressing these receptors do not undergo morphological changes in close proximity to beta-amyloid plaques. There was no sign of degeneration in these neurons. Concerning the examined parameters of glucose metabolism, no changes between transgenic animals and non-transgenic littermates were detected. This observation is in accordance with the data available in the literature, but in contrast to findings in AD patients. The density of brain capillaries in close vicinity of beta-amyloid plaques, compared to a more distant surrounding, is reduced. This finding needs further examination because it could be relevant for in vivo diagnosis and therapy. Comparing the transgenic Tg2576 mouse model to AD patients, it becomes apparent that the mouse model does not simulate all aspects of the pathogenesis of this disease. Consistent with the human disease this model is characterized by b-amyloid plaque production and deposition in the brain, as well as inflammatory processes around the plaques, which are also known in humans. Therefore, it represents a suitable model to study amyloidogenesis and inflammation.

Alzheimer’s disease (AD) is characterized by cognitive decline and hallmark neuropathology, including β-amyloid (Aβ). Therapeutic strategies for AD are focusing on reducing Aβ. Canines develop Aβ neuropathology and cognitive decline with age similar to AD patients. In previous studies, immunization with Aβ1-42 (VAC) in aged canines decreased brain Aβ but did not improve cognition. Behavioral enrichment (ENR) improved cognition without reducing brain Aβ. We hypothesized that VAC combined with ENR would provide cognitive benefits and reduce Aβ neuropathology, as compared individual VAC and ENR treatments. Aged beagles were placed into groups: control, VAC with fibrillar Aβ1-42, ENR, and combination treatment (VAC+ENR) for 18 months. Learning and memory was evaluated throughout the study. Serum IgG antibody titers, cerebral spinal fluid (CSF) and brain Aβ were measured. Serum anti-Aβ1-42 IgG increased significantly in VAC animals. ENR but not VAC significantly increased CSF Aβ1-40. No cognitive improvements were observed in any group. VAC significantly reduced brain Aβ1-40 and 1-42, as well as reduced plaque load. An overall slowing of plaque accumulation was seen in the ENR group. VAC and ENR were able to modify pathology when used as separate treatments; however, the combination treatment did not succeed in further reducing Aβ or improving cognition. Previous AD clinical trials using immunotherapy yielded similar outcomes to our study showing reduced Aβ pathology but little to no cognitive improvements. In combination these results suggest that future studies should focus on prevention approaches both in the canine model and in human clinical trials.

abstract: Alzheimer's disease (AD) is the most common type of dementia, affecting one in nine people age 65 and older. One of the most important neuropathological characteristics of Alzheimer's disease is the aggregation and deposition of the protein beta-amyloid. Beta-amyloid is produced by proteolytic processing of the Amyloid Precursor Protein (APP). Production of beta-amyloid from APP is increased when cells are subject to stress since both APP and beta-secretase are upregulated by stress. An increased beta-amyloid level promotes aggregation of beta-amyloid into toxic species which cause an increase in reactive oxygen species (ROS) and a decrease in cell viability. Therefore reducing beta-amyloid generation is a promising method to control cell damage following stress. The goal of this thesis was to test the effect of inhibiting beta-amyloid production inside stressed AD cell model. Hydrogen peroxide was used as stressing agent. Two treatments were used to inhibit beta-amyloid production, including iBSec1, an scFv designed to block beta-secretase site of APP, and DIA10D, a bispecific tandem scFv engineered to cleave alpha-secretase site of APP and block beta-secretase site of APP. iBSec1 treatment was added extracellularly while DIA10D was stably expressed inside cell using PSECTAG vector. Increase in reactive oxygen species and decrease in cell viability were observed after addition of hydrogen peroxide to AD cell model. The increase in stress induced toxicity caused by addition of hydrogen peroxide was dramatically decreased by simultaneously treating the cells with iBSec1 or DIA10D to block the increase in beta-amyloid levels resulting from the upregulation of APP and beta-secretase. / Dissertation/Thesis / M.S. Chemical Engineering 2014

Metal ions are ubiquitously found in all living systems and play vital roles in supporting life forms by performing an array of biological activities. Such biological activities include binding and transforming organic molecules, and also acting as active centers and cofactors for catalysis of various acid-base and redox reactions in biological system. The main focus in bioinorganic chemistry is to elucidate the structural and functional roles of metals in biological systems. Among all transition metal ions, Cu2+ and Fe3+ are especially versatile and important due to their abilities to go through redox efficiently. This dissertation can be divided into four main chapters. The bioinorganic chemistry of Cu- and Fe-containing proteins were briefly discussed in Chapter one. The next chapter focuses on bacitracin, a cyclic peptide-based antibiotic produced by soil bacteria Bacillus subtilis. Bacitracin is a metalloantibiotics that can coordinate with many transition metal ions and exhibit different biological activities. In the first part of Chapter two, the aim is to explore the chemicals interactions in soil micro-ecology by investigating the interactions of different flavonoids and Cu(II)-bacitracin complex. The second part of chapter two demonstrated the binding and oxidation activity of iron(III)-bacitracin. Metal-mediated oxidative stress plays a crucial role in the development of different neurodegenerative diseases. In chapter 3, various synthetic and natural compounds were used to inhibit the oxidation chemistry mediated by Cu(II)-beta-amyloid complex associated with Alzheimer’s disease. Many proteins incorporate copper ions at their active sites for different functions, and among all of the chemistry copper-containing-proteins can perform, one of the most interesting aspect is the ability to bind and activate O2. Therefore, the biomimetic of two different Cu(II) complexes were investigated. In all studies, a combination of kinetic and different spectroscopic methods (UV-vis, NMR and resonance Raman spectroscopy) were used to study their metal binding and activity.

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No description available.

The classification of neurodegenerative disorders is based on the major component of the protein aggregates in the brain. The most common altered proteins associated with neurodegeneration are Hyperphosphorylated tau (HPt), beta amyloid (Aβ), alpha-synclein (αS) and transactive response DNA binding protein 43 (TDP43). In this study we assessed the incidence and the neuroanatomical distribution of proteins associated with neurodegeneration in the brain tissue of cognitively unimpaired subjects. We demonstrated the early involvement of the Locus Coeruleus (LC) with HPt pathology in cognitively unimpaired mid aged subjects, a finding which supports the notion that LC is an initiation site of HPt pathology. This may suggest that development of clinical assessment techniques and radiological investigations reflecting early LC alterations may help in identifying subjects with early stages of neurodegeneration. Furthermore, we studied a large cohort of cognitively unimpaired subjects with age at death ≥50 years and we applied the National Institute on Aging –Alzheimer’s disease (AD) Association (NIA-AA) guidelines for the assessment of AD related neuropathological changes. Interestingly, a considerable percentage of the subjects were classified as having an intermediate level of AD pathology. We also showed that the altered proteins;  HPt , Aβ, αS, and TDP43 are frequently seen in the brain of cognitively unimpaired subjects with age at death ≥50 years, the incidence of these proteins increased significantly with age. This finding suggests that neurodegeneration has to be extensive to cause functional disturbance and clinical symptoms. Moreover, we investigated the correlation between AD related pathology in cortical biopsies, the AD / cerebrospinal fluid (CSF) biomarkers and the Mini Mental State examination (MMSE) scores in a cohort of idiopathic Normal Pressure Hydrocephalus (iNPH) patients. We demonstrated that AD/ CSF biomarkers and MMSE scores reflect AD pathology in the cortical biopsies obtained from iNPH patients.  In conclusion, this study shows that the altered proteins associated with neurodegeneration are frequently seen in the brain tissue of cognitively unimpaired aged subjects. This fact should be considered while developing diagnostic biomarkers for identification of subjects at early stages of the disease, in order to introduce therapeutic intervention prior to the occurrence of significant cognitive impairment.

Durante a sepse ocorre liberação de mediadores inflamatórios, que contribui para o comprometimento do sistema nervoso central e alterações na secreção dos hormônios hipofisários. Contudo, não está elucidado se essas alterações persistem em animais sobreviventes à sepse. Nesse contexto, o objetivo deste trabalho foi estudar as alterações neuroimunoendócrinas em animais sobreviventes à sepse polimicrobiana experimental. A sepse foi induzida pelo método de ligadura e perfuração cecal (CLP) (uma perfuração com agulha 14G) e os animais foram observados durante cinco ou dez dias. Os animais naive ou sobreviventes foram submetidos ao desafio osmótico intraperitoneal com salina hipertônica (2M), ou desafio imune intravenoso com LPS (1.5 mg/kg), ou ao estresse por imobiliza- ção. Após 30 minutos do desafio osmótico ou 60 minutos para os demais estímulos, os animais foram decapitados para coleta de sangue e do hipotálamo. O sangue foi coletado para dosagem de nitrato, citocinas e hormônios (vasopressina, ocitocina, ACTH e corticosterona); e o hipotálamo para a quantificação dos níveis de citocinas e dos conteúdos de sinaptofisina e beta-amilóide. No presente estudo, nós observamos um aumento periférico dos mediadores inflamatórios: nitrato e IL-1?, e centralmente da citocina IL-6 no hipotálamo de animais sobreviventes à sepse. Além disso, dependendo do estímulo, observou-se uma atenuada (desafio osmótico) ou exacerbada (desafio imune) secreção da vasopressina e/ou ocitocina em animais sobreviventes à sepse. Adicionalmente, também encontramos uma diminuição no conteúdo hipotalâmico de sinaptofisina, e nenhum sinal de acúmulo de beta-amilóide. Concluímos que as alterações na secreção de vasopressina e ocitocina observadas podem estar associadas à neuroinflamação sustentada e disfunção sináptica com evidências de processo neurodegenerativo. Este modelo de sepse pode fornecer informações importantes para a compreensão das alterações fisiopatológicas à longo prazo. / Inflammatory mediators released during sepsis contribute to central nervous system impairment and alterations in the secretion of pituitary hormones. However, it is unclear whether these changes persist in sepsis survivor animals. In this context, the objective of this work was to study the neuroimunoendocrine alterations in sepsis survivor animals. Sepsis was induced by the cecal ligation and perforation (CLP) method and the animals (perforated once with a 14G needle) were observed for five or ten days. Naive or sepsis survivors were submitted to intraperitoneal osmotic challenge with hypertonic saline (2M), or intravenous immune challenge with LPS (1.5 mg/kg), or immobilization stress. After 30 minutes of the osmotic challenge or 60 minutes for the other stimuli, the animals were decapitated for collection of blood and the hypothalamus. Blood was collected for the determination of nitrate, cytokines and hormones (vasopressin, oxytocin, ACTH and corticosterone); and the hypothalamus for quantification of cytokine levels, and synaptophysin and beta-amyloid contents. In the present study, we observed a peripheral increase of inflammatory mediators: nitrate and IL-1?, and centrally of the IL-6 cytokine in the hypothalamus of sepsis survivor animals. In addition, depending on the stimulus, an attenuated (osmotic challenge) or exacerbated (immune challenge) secretion of vasopressin and/or oxytocin was observed in sepsis survivor animals. In addition, we also found a decrease in the hypothalamic content of synaptophysin, and no signal of beta amyloide accumulation. We conclude that alterations in vasopressin and oxytocin secretion may be associated with sustained neuroinflammation and synaptic dysfunction with evidence of neurodegenerative process. This model of sepsis may provide important informations for understanding long-term pathophysiological alterations.

A acetilcolinesterase (AChE) é uma enzima essencial que encerra a transmissão colinérgica através de uma rápida hidrólise do neurotransmissor, acetilcolina (ACh). Uma ampla série de evidências mostraram que os inibidores da AChE podem interferir com a progressão da doença de Alzheimer (DA). O desenvolvimento bem sucedido de compostos inibidores da AChE foi baseado na teoria de que o declínio nas funções cognitivas e mentais associadas a DA está relacionado com a perda da neurotransmissão cortical colinérgica, sendo assim, esses compostos podem ser usados para tratar as deficiências colinérgicas. Uma coleção de moléculas orgânicas foi escaneada para ser avaliada a capacidade dessas moléculas em inibir a atividade enzimática da AChE com o objetivo de se encontrar compostos líderes para posteriores otimizações, conduzindo a fármacos com aumento da eficácia e/ou menores efeitos adversos. As estratégias aplicadas incluem o screening ou triagem virtual baseado na estrutura e também no ligante, modelagem farmacofórica, docking molecular e buscas por similaridade (forma e eletrostática). Os estudos foram também concentrados na descoberta de novas classes de inibidores da AChE, tendo como molécula de referência o fármaco donepezil, o qual inaugurou uma nova classe de inibidores da AChE com a ação mais longa e mais seletiva com efeitos adversos manejáveis. Do total de compostos triados, 50 foram selecionados com adequadas propriedades físico-químicas e ADME/Tox. Em geral, esses compostos possuem substancial interação com o sítio periférico aniônico (PAS) da AChE e a maioria deles faz interações adicionais com o sítio catalítico (CAS) e com outros resíduos de aminoácidos importantes ao longo da enzima. Destes 50 compostos, oito foram comercialmente adquiridos e os ensaios enzimáticos revelaram que estes compostos exibem uma alta afinidade pela AChE. Os resultados apontam, ainda, que o composto entitulado ZINC30019441 exibiu a mais potente atividade inibitória para a AChE, com 1,8 micromolar de concentração, e os demais ficaram ainda situados em baixo micromolar, de 2 a 3 micromolar de concentração. Estes resultados, além das modificações químicas ora propostas in silico para estes inibidores protótipos, apontam para o desenvolvimento de uma nova e promissora série de potentes anticolinesterásicos, contendo propriedades de fármacos, as quais são ainda apropriadas para atuarem no Sistema Nervoso Central e interagirem com o peptídeo beta-amiloide, com vistas ao tratamento quimioterápico da doença de Alzheimer. A perspectiva imediata inclui os ensaios de anti-agregação do peptídeo com os oito inibidores já testados com a Acetilcolinesterase. / Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). A wide range of evidence shows that AChE inhibitors may interfere with the progression of Alzheimer\'s disease (AD). The successful development of AChE inhibitor compounds was based on the theory that the decline in cognitive and mental functions associated with AD is related to the loss of cortical cholinergic neurotransmission, thus, these compounds can be used for treating cholinergic deficiencies. A collection of organic molecules has been scanned to evaluate the ability of these molecules to inhibit the enzymatic activity of AChE in order to find lead compounds for further optimization, leading to drugs with increased efficacy and/or fewer adverse effects. The strategies applied include structure-based virtual screening and also in ligand-based virtual screening, pharmacophoric modeling, molecular docking and similarity searches (shape and electrostatic). Studies have also focused on the discovery of new classes of AChE inhibitors, having as a reference molecule the drug donepezil, which has opened a new class of AChE inhibitors with longer and more selective action with manageable side effects. 50 of the compounds screened, were selected with appropriate physical and chemical properties and ADME/Tox. In general, these compounds possess substantial interaction with the peripheral anionic site (PAS) of AChE and most of them make further interact with the catalytic site (CAS) and other key amino acid residues throughout the enzyme. Out of these 50 compounds, eight were commercially purchased and the enzyme assays have shown that these compounds exhibit a high affinity for AChE. The results show also that the compound titled ZINC30019441 exhibited the most potent inhibitory activity for AChE, with 1.8 micromolar concentration, and the rest were still located in low micromolar, 2-3 micromolar concentration. These results as well as chemical modifications herein proposed for these prototypes, indicate the development of a new and promising series of potent anticholinesterase containing drug properties, which are still suitable to act on the central nervous system and interact with the ?-amyloid peptide, for chemotherapy treatment of Alzheimer\'s disease. The immediate prospect includes the peptide anti-aggregation assays with the eight inhibitors already tested against acetylcholinesterase