Thèses sur le sujet « Amyloid-beta peptide (A-beta) »

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with a strong focus on developing imaging modalities. AD is accompanied by two pathological hallmarks in the brain: extracellular neuritic plaques composed of the beta-amyloid peptide (Aβ) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. Evidence of Aβ as the driving force behind the progression of AD (the amyloid cascade hypothesis) was first published by Hardy & Higgins in 1992, and this peptide has been the focus of therapeutic and diagnostic testing for decades. Significant technological advances in recent years now allow imaging of amyloid pathology in vivo. These methods evaluate Aβ burden in a living person, and could potentially serve as both a biomarker, and as a diagnostic tool to detect disease. Pittsburgh Compound B (PiB) is currently the best studied of these imaging agents, however, our current knowledge of the quantitative relationship between PiB binding and amyloid pathology in the brain is limited. A better understanding of how these variables relate to one another is essential for the continued development of reliable diagnostic biomarkers for AD. We analyzed increasingly insoluble pools of Aβ to quantify their relative contributions to the overall Aβ burden, and to determine if any of these measures could be used to predict disease status. We found that the amount of PiB binding in a cortical region of the brain could distinguish cases of mild cognitive impairment (MCI) when corrected to the amount of PiB binding in the cerebellum. As the Aβ peptide ages, the amino acid aspartate may spontaneously convert to an isoaspartate residue through a succinimide intermediary. The presence of iso-Asp Aβ has been used to indicate the presence of aged plaques in AD and Down syndrome cases. We sought to investigate the potential relationship between levels of ‘aged’ Aβ in the plasma as indicated by iso-Asp Aβ and disease state, as a potential biomarker for the presence of AD pathology. We found that AD cases had lower levels of all forms of Aβ in plasma when standardized to the group average, and that plasma levels of Aβ and iso-Asp Aβ were reversed between disease groups. A follow up study is required, however, these initial data are a promising step towards utilizing aged iso-Asp Aβ plasma levels as a potential biomarker to indicate disease state.

The aim of this thesis was to study the pathogenetic mechanisms underlying Alzheimer s disease (AD), a neurodegenerative disorder affecting the elderly and characterized by memory loss, personality changes and cognitive dysfunction leading to dementia. I will discuss the main projects in which I participated aimed at understanding the role of the main molecular interactors involved in AD pathogenesis, i.e. Amyloid-beta peptide and tau protein, on hippocampal synaptic plasticity and memory in animal models. After reviewing the pathophysiological models that have been developed so far, our general purpose was to study novel aspects of Amyloid-beta peptide and tau involvement in physiological and pathological conditions to give a different interpretation of the disease.

Amyloid-β (Aβ) peptide is generated after sequential cleavage of the constitutively expressed amyloid precursor protein (APP) by γ and β secretases, and is recognized as the primary causative agent underlying the neuropathogenesis of Alzheimer’sDisease (AD). Once generated, monomeric Aβ demonstrates a high propensity to aggregate into toxic Aβ oligomers (AβO) of various sizes, which eventually accumulate in the brain in the form of amyloid plaques. Mutations in either the gene for APP or one or both of its processing genes, presenilin-1 (PS1) and presenilin-2 (PS2) of the secretases complex leading to accumulation of Aβ and early-onset familial AD. Late onset AD is modulated by mutations in the gene for apolipoprotein E (apo-E), with the isoform apo-E4 leading to an approximate eight-fold increase in risk for AD, and by environmental and life style factors. The Alzheimer’s disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. A major roadblock to the management of AD is the inability to definitively diagnose AD until post-mortem examination. It is therefore imperative to develop methods that permit safe, early detection and monitoring of disease progression. Magnetic resonance imaging (MRI) is a non-invasive way to detect and monitor AD progression and therapy, but so far MRI contrast has been obtained only using Gd(III) based contrast agents. Fluorene compounds have garnered attention as amyloid imaging agents. Our lab has developed a spin labeled fluorene (SLF) compound that contains a fluorene moiety with known affinity for Aβ and a pyrroline nitroxyl spin-label moiety. We hypothesized that the SLF compound will specifically coat assemblies of amyloid beta in the brain and, by establishing a boundary of magnetic field inhomogeneity, produce MRI contrast in tissues with elevated levels of the Aβ peptide. I found that labeling of brain specimens with the SLF compound produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of SLF into live mice resulted in good brain penetration, with the compound able to generate contrast 24-hr post injection. (Abstract shortened by ProQuest.)

Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia associated with amyloid-beta peptide deposition and loss of mitochondrial function and regulation. Currently, there is no cure for AD, thus, there is a need to continuously develop therapeutic strategies that could address the complex multifactorial causes of AD development. Due to this necessity, this study has investigated the role of vitamin B2 as a disease modifying drug for AD by employingamyloid-beta and mitochondrial based AD therapeutic strategies. Using a transgenic C. elegans AD worm model expressing amyloid-beta (Aβ1-42) in muscle cells at temperature upshift to 25°C, we screened for protective effect of dose-dependent concentrations of active forms of vitamin B2, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), against amyloid-beta mediated paralysis. Protective concentrations were then assayed for improvement of mitochondrial metabolic functions by performing ATP, oxygen consumption and reactive oxygen species (ROS) production assays. Consequently, we investigated for drug protective mechanisms of FMN and FAD using RNAi genetic screening technique. FMN and FAD significantly delayed amyloid-beta mediated paralysis and improved mitochondrial metabolic functions at final concentrations of 0.74mM and 0.74µM respectively. More so, both compounds induced activation of stress response FOXO transcription factor, daf-16. Specifically, FMN treatment induced mitochondrial unfolded protein response (UPRmt) pathway through ubiquitin-like protein (ubl-5) activation as well as other stress response pathway signature such as Activating Transcription Factor Associated with Stress (atfs-1). This study will be useful in understanding the importance of micronutrients such as vitamin B2 in normal cellular function as related to neurodegenerativediseases and aging. Therefore, vitamin B2 supplementation could be an important source of Alzheimer’s disease therapeutic strategy.

Alzheimer’s disease (AD) is associated with amyloid-beta peptide deposition and loss of mitochondrial function. Using a transgenic C. elegans AD worm model expressing amyloid-beta in body wall muscle, we determined that supplementation with either of the forms of vitamin B2, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) protected against amyloid-beta mediated paralysis. FMN and FAD were then assayed to determine effects on ATP, oxygen consumption, and reactive oxygen species (ROS) with these compounds not significantly improving any of these mitochondrial bioenergetic functions. Knockdown of the daf-16/FOXO transcriptional regulator or the FAD synthase enzyme completely abrogated the protective effects of FMN and FAD, while knockdown of the mitochondrial unfolded protein response factors ubl-5 or atfs-1 also blocked the protective effects. Therefore, vitamin B2 supplementation could lead to the activation of conserved signaling pathways in humans to delay the onset and progression of neurodegenerative diseases such as AD.

High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.

The Amyloid Cascade Hypothesis states that fibrillation of the amyloid beta (Aβ) peptide is the primary cause of Alzheimer’s pathology. The trigger for the fibrillation is a subject of much debate, although it is clear, oxidative stress is a key feature of Alzheimer’s aetiology. This thesis explores a possible role of oxidation of Aβ, in particular the effect of histidine and methionine side-chain oxidation, on Aβ fibril growth rates. Within chapters 2 and 3 of this thesis is a discussion of various approaches to chemical synthesis of 2-oxo-histidine with a view to the incorporation of the oxidised amino acids into Aβ peptide using Fmoc approaches. Chapter 2 describes attempted chemical transformation of (protected) L-histidine into L-oxohistidine. Dimethyldioxirane oxidised Boc-His-OMe yielded products containing isopropylidene groups, while oxidation using a Cu(II)/ascorbate generated 2-oxo-histidine but gave very low yields. Within chapter 3, a successful synthesis of protected 2-oxo-histidine is described, via the known imidazolin-2-one-4-carboxylic. Chapter 4 analyses Aβ(1-40) fibrillation kinetics by treating the intact peptide with various oxidants. Contrary to previous reports, hydrogen peroxide alone did not slow fibrillation rates. Cu(II)/Cu(I)- catalysed oxidation increased the likelihood of amorphous aggregation over fibrillation. This thesis shows oxidation of Aβ has a profound influence on fibril growth and that incorporation of a stable oxidised histidine into Aβ is a realisable goal.

Various types of nanoparticles (NPs), such as Liposomes, Solid Lipid NPs and Polymeric NPs, are being extensively explored for their potentialities in the medical field. NPs are attractive tools in biomedical applications thanks to their biocompatibility, non-immunogenicity, non-toxicity, biodegradability, high physical stability, possibility of drug loading and releasing, and higher probability for surface functionalization. The project is devoted to the synthesis of NPs functionalized with amyloid-beta ligands (Aβ-ligands), imaging tools and/or blood brain barrier-transporters (BBB-transporters) for the cure and the diagnosis of Alzheimer’s disease (AD). Amyloid β (Aβ) aggregates are considered as possible targets for therapy and/or diagnosis of Alzheimer disease (AD). It has been previously shown that some small molecules targets Aβ plaques and, among them, curcumin interacts with their precursors, suggesting a potential role for the prevention or the treatment of AD. Herein, a chemoselective ligation procedure was used to generate NPs decorated with a curcumin derivative, designed to maintain all the features required for interaction with Aβ. In summary, this thesis describes the preparation and characterization of new curcumin derivatives and NPs, with affinity for Aβ peptide. They could be exploited as ligands and/or vectors for the targeted delivery of new diagnostic and therapeutic molecules for AD. The NPs preparations and the biological results were obtained in collaboration with scientists involved in a joint European project: NAD - Nanoparticles for therapy and diagnosis of Alzheimer’s Disease - 2008-2012, FP7-NMP-2007-LARGE-1-Large-scale integrating project NMP-2007-4.0-4 Substantial innovation in the European medical industry: development of nanotechnology-based systems for in-vivo diagnosis and therapy.

Les mécanismes de type prion sont désormais reconnus comme sous-tendant la plupart des maladies neurodégénératives humaines, avec en premier lieu la maladie d’Alzheimer (MA) au niveau de ses 2 marqueurs spécifiques, l’amyloïde β (Aβ à l’origine de l’hypothèse étiopathogénique de la cascade amyloïde) et la protéine Tau phosphorylée. Par ailleurs la protéine du prion (PrPC) est décrite comme interagissant à de multiples niveaux avec le métabolisme de l’Aβ sans que les mécanismes physiopathologiques sous-jacents n’aient pu être expliqués. Pour sortir de l’impasse actuelle concernant le développement d’approches thérapeutiques efficaces pour la MA, l’industrie pharmaceutique a besoin de modèles expérimentaux innovants. En effet, à ce jour aucun modèle in vivo, en dépit des progrès réalisés avec les souris transgéniques, n’arrive à refléter la complexité cérébrale humaine ni à mimer une MA clinique. Les cultures in vitro en 2D sont quant à elles très éloignées des situations conduisant à l’accumulation d’agrégats protéiques pathologiques. Le but de notre thèse a été d’utiliser dans le domaine des neurosciences les nouvelles perspectives de recherche ouvertes par les technologies des cellules souches pluripotentes induites (cellules iPS) en développant un modèle de différentiation en 3D pour obtenir des organoïdes cérébraux humains (OC) (mini cerveaux). Leur capacité d’auto-organisation en 3D de tissu neuroectodermique nous a permis de recréer un système complexe mimant différentes structures cérébrales humaines dans lesquelles nous avons pu caractériser les marqueurs attendus. L’étude de l’expression des protéines d’intérêt APP et PrPC pendant la différentiation neurale a permis de caractériser la modulation des niveaux des deux protéines en fonction du temps de culture. Afin d’orienter le modèle vers des mécanismes d’accumulation protéique de type MA, nous avons testé différents inducteurs chimiques dont l’Aftin-5 qui est capable de moduler les voies post-traductionnelles de l’APP. Plusieurs stratégies de traitement ont été adoptées pour induire le clivage de l’APP et la génération d’Aβ. La production des fragments solubles Aβ38, Aβ40, Aβ42 a été mise en évidence par ELISA. Les niveaux générés sont reproductibles et l’augmentation du ratio Aβ42/Aβ40 est cohérente avec les données extrapolées des modèles murins et humains, ce qui a permis de valider notre modèle. Les niveaux d’expression génique et protéique de PrPC et de APP suite au traitement ont été analysés afin de mieux déterminer le rôle de l’interaction entre ces deux facteurs. L’objectif à long terme consiste à améliorer ce modèle, dont les limites actuelles sont notamment l’absence de vascularisation et le niveau de maturation du tissu neural. Le défi majeur dans le cadre de la culture des OC consiste donc à favoriser l’intégration du système vasculaire, et par ailleurs à accélérer le vieillissement in vitro pour l’étude de maladies neurodégénératives. La perspective de pouvoir automatiser le système de culture des OC permet d’envisager l’utilisation de ce modèle à plus grande échelle dans le cadre de test de cytotoxicité et/ou de criblage pharmacologique à haut débit pour identifier de nouvelles molécules thérapeutiques pour la MA
Prion-like mechanisms are known to underlie most of human neurodegenerative diseases including Alzheimer’s disease (AD), which is characterized by two important pathological markers, β amyloid (or Aβ at the origin of the etiopathogenic amyloid cascade hypothesis) and phosphorylated tau protein. Furthermore, the prion protein (PrPC) interacts at multiple levels with the metabolism of Aβ, by mechanisms which are not well understood. To overcome the current limits in the development of efficient strategies to treat AD, the pharmaceutical industry requires innovative experimental models. However, even if a lot of progress has been achieved by using transgenic mouse models, to date no in vivo model can reflect the complexity of human brain or reproduce a clinical context. 2D in vitro cell culture models are unable to allow the aggregation and accumulation of pathological proteins as observed in vivo. The aim of this study consists in taking advantage of the research prospects offered by induced pluripotent stem cell (iPSCs) in the field of neurosciences. iPSCs can be used to generate 3D models of differentiation also called human cerebral organoids or mini-brains (MBs). Their ability to self-organise in 3D neuroectodermic tissue leds to a complex system that mimics different human cerebral structures in which we were able to characterize the expected markers. The study of the two proteins of interest (APP and PrPC) during neural differentiation has allowed us to follow the modulation of protein expression level occurring during the in vitro development of the human MBs. In order to use this model to reproduce the protein accumulation mechanisms seen in AD, we have tested chemical inductors such as Aftin-5 in order to modulate the APP post-transcriptional pathway towards a pathological outcome. Many strategies of treatment are adopted to lead APP cleavage and Aβ generation. The production of soluble fragments Aβ38, Aβ40, Aβ42 in the supernatant of organoids has been showed using ELISA technique. The levels generated are reproducible and the increase of Aβ42/Aβ40 ratio is consistent with extrapolated data from mouse and human models thus validating our model. Analysis at the gene and protein level has been assessed in order to understand the interaction between PrPC and APP after treatment. The long-term goal consists in improving this model which is notably hampered by the absence of vascularization and the low level of maturation of the neural tissue. The main challenge in MB culture thus consists in the integration of the vascular system, and also in increasing the speed of ageing process in vitro for the study of neurodegenerative diseases. In the long term, the prospect of automating the culture of MBs would allow the use of the system for cytotoxicity testing and/or high throughput screening for the discovery of new drugs for AD

One of the main difficulties for the success of tissue and organ transplantation is the deficiency of techniques that ensure regular and stable vascularisation. The knowledge of mechanisms involved in the formation of new vessels could allow application of clinic strategies to facilitate tissue and organ transplantation on the one hand and the reduction of excessive vascular growth in many pathologies on the other. Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new vessels (Bouïs et al, 2006). It occurs in both physiological (embryo development, menstrual cycle, pregnancy, wound healing) and pathological conditions (inflammation, psoriasis, metastasis growth). In the last years an increasing amount of evidence suggests that Alzheimer's disease (AD) is not only a neurodegenerative disease characterized by neuronal degeneration and loss of synaptic connections but it is also a vascular pathology. In fact cerebral microvessel vasoconstriction, degeneration of vessel smooth muscle cells and alteration of the basal membrane have been observed in cerebral capillary AD brains. In particular the possible connection between angiogenesis and AD starts from the observations of increased vascular density close to senile plaques in AD patients in comparison to older non-demented people (Vagnucci and Li, 2003). Senile plaques (extracellular and vascular deposits of proteinaceous material where the major component is β-amyloid peptide, Aβ) together with neuro fibrillary tangles (NFT) (composed of iperphophorylated form of τ protein) represent the main neuropathological alterations founded in AD brains (Robertson et al., 2003). A large amount of in vivo, ex-vivo and in vitro experiments show contradictory data on Aβ peptide angiogenic capability (Paris et al., 2004; Cantara et al., 2004). The aim of the present research is the attempt to understand some of the mechanisms that favour angiogenesis. The research deonstrates that Aβ peptides are pro-angiogenic in vivo, ex-vivo and in vitro on human cerebral endothelial cells while they are anti-angiogenic on rat cerebral endothelial cells, probably as consequence of their cytotoxic effect on these cells. Moreover Aβ1-42 peptide does not affect the release of VEGF-A and the expression of mRNA of VEGF-A on rat cerebral endothelial cells and rat hippocampal astrocytes cultures. Astrocytes release factors that induce angiogenesis and release of IL-6 on rat cerebral endothelial cells independently of the presence of Aβ1-42 peptide. Finally the presence of hVEGF165 inhibits toxic and anti-angiogenic effects of the peptide probably taking away Aβ1-42 through the binding A?-hVEGF165. Other experiments are necessary to understand mechanisms connected to AD although it seems that the result could depend on numerous different cell types in response of Aβ effects. Nevertheless capillary alteration and the hypoxia seem to contribute to the process.
Uno dei principali impedimenti alla ricostruzione di tessuti ed organi nell'ingegneria tessutale è la mancanza di strategie che assicurino costante e stabile vascolarizzazione. L'acquisizione di nuove conoscenze sui meccanismi che intervengono nel processo angiogenico potrebbe permettere una efficace applicazione clinica nella ricostruzione di tessuti e organi da una parte e nella riduzione della eccessiva crescita vascolare in patologie angiogenesi-dipendente dall'altra. L'angiogenesi è un processo complesso in cui numerosi cellule e mediatori cellulari interagiscono per stabilire un microambiente specifico per la crescita di nuovi vasi sanguigni (Bouïs et al, 2006). Questo processo si verifica sia in condizioni fisiologiche (sviluppo embrionale, ciclo mestruale, gravidanza, guarigione di ferite) che patologiche (processi infiammatori, psoriasi, crescita di metastasi). Negli ultimi tempi si sta affermando l'ipotesi che la demenza di Alzheimer (AD) non sia solamente una malattia neurodegenerativa caratterizzata da degenerazione neuronale e perdita delle connessioni sinaptiche ma anche una patologia vascolare. Numerose infatti sono le alterazioni riscontrabili a livello dei capillari cerebrali nei soggetti AD: vasocostrizione dei microvasi cerebrali, degenerazione delle cellule muscolari lisce vasali e alterazione della membrana basale. In particolare desta vasto interesse il possibile legame tra angiogenesi e AD derivante dall'osservazione di una aumentata densità  vascolare in prossimità delle placche senili nei pazienti AD rispetto a soggetti anziani non dementi (Vagnucci and Li, 2003). Le placche senili, depositi extracellulari e vascolari di materiale proteico costituite in prevalenza dal peptide β-amiloide (Aβ), assieme ai grovigli neurofibrillari (NFT), costituiti dalla forma iperfosforilata della proteina τ, rappresentano le principali alterazioni neuro-patologiche riscontrabili nel cervello di soggetti AD (Robertson et al., 2003). Numerosi studi in vivo, ex-vivo e in vitro riportano risultati contraddittori sulla capacità  del peptide Aβ di stimolare il processo angiogenico (Paris et al., 2004; Cantara et al., 2004). Scopo di questo lavoro di ricerca è il tentativo di comprendere alcuni meccanismi che favoriscono questo processo. Da questo lavoro di ricerca è emerso che il peptide Aβ è pro-angiogenico in vivo, ex-vivo e in vitro su cellule endoteliali cerebrali umane e anti-angiogenico su cellule endoteliali cerebrali di ratto, probabilmente come conseguenza dell'effetto citotossico effettuato dal peptide su queste ultime. Inoltre il Aβ1-42 non interferisce sulla via di espressione e rilascio di VEGF da parte di cellule endoteliali cerebrali e di astrociti di ippocampo di ratto in coltura. Gli astrociti secernono sostanze che inducono angiogenesi e rilascio di IL-6 da parte delle cellule endoteliali cerebrali di ratto in coltura indipendentemente dal trattamento con Aβ1-42. Infine la presenza di hVEGF165 inibisce l'effetto tossico e anti-angiogenico del peptide probabilmente in seguito al sequestramento del Aβ1-42 ad opera del legame Aβ1-42-hVEGF165. Altri studi sono necessari al fine di comprendere i meccanismi di angiogenesi correlata con la AD sebbene sembri evidente che essa possa derivare dall'interazione di diversi tipi cellulari in risposta all'effetto del peptide Aβ su di esse. Sembra tuttavia che l'alterazione dell'endotelio capillare e la conseguente ipossia contribuiscano all'attivazione di tale processo.

Alzheimer’s disease (AD) is characterized by the aggregation of amyloid beta peptide (Aβ). Aβ derives from the amyloid precursor protein (APP), which can undergo two mutually exclusive pathways. The amyloidogenic pathway involves BACE and γ-secretase activities and leads to Aβ formation. While, the non-amyloidogenic pathway involves ADAM10, a disintegrin and metalloproteinase 10, which cleaves APP within the domain corresponding to Aβ, thus precluding Aβ production. Recently, we identified a new ADAM10 binding partner, named AP2, which is responsible for ADAM10 internalization, therefore affecting its activity. Interestingly, ADAM10/AP2 interaction is significantly increased in AD patients' brain compared to healthy control subjects, suggesting a role of ADAM10/AP2 in AD pathogenesis. In this framework, we have recently developed a cell permeable peptide (named PEP3) capable of interfering with ADAM10/AP2 association. The intraperitoneal administration of this CPP to a mouse model of AD for two weeks is safe and effective in impairing ADAM10 endocytosis and, thereby, in increasing ADAM10 synaptic localization. At late stages of disease, the PEP3 administration is able to change biochemical parameters, as Aβ levels and the molecular composition of the synapses without ameliorating the cognitive deficits of these mice. On the other hand, at early stage of the pathology the 14-days administration of the PEP3 rescues the cognitive impairment of the AD mice. Further investigations revealed that the synaptic levels of the NMDA receptor subunit GluN2A are increased upon the treatment and that previously observed shrinkage and dendritic spine loss in AD mice were improved after CPP treatment. These results are mediated by an increase in endogenous sAPPα. These positive results point to ADAM10 internalization as a potential target mechanism for the development of an effective AD therapy.

A 20 weeks project at Karolinska Institutet (KI), Huddinge, Sweden is in this master thesis summarized. Alzheimer’s disease is the most common form of dementia in the world. One of the pathological hallmarks seen in AD patients consists of amyloid plaques assembled of beta amyloid (Aβ) peptide aggregates. A lot of research has been done on Aβ40 and Aβ42 but not on the longer variant with 43 residues. An earlier study by Welander et al, quantified the Aβ43 peptide from amyloid plaque cores with high-performance liquid chromatography coupled to mass-spectrometry (HPLC-MS/MS)1. Here, I present the initial development of an enzyme-linked immunosorbent assay (ELISA) with the goal to quantify Aβ43 peptides in soluble fractions of human brain tissue. An ELISA method with the possibility to quantify Aβ43 peptides from cerebral spinal fluid might have the prospect to serve as a diagnostic tool for AD in the future. Commercial ELISA kits coated with antibodies against all Aβ species was not suitable for detecting Aβ43 in soluble brain tissue from human AD patients. This is due to the high amount of Aβ40 (and in some extent Aβ42) in the samples, which will bind to the same epitope as Aβ43 on the capturing antibody. These shorter Aβ species will be in excess and bind to the capturing antibody thereby ousting Aβ43 from binding in. A better way for quantifying Aβ43 with ELISA might instead be to coat a polystyrene plate with α-Aβ43 antibodies, which are c-terminal specific to Aβ43. This will abolish the competition between the different Aβ species and function as an immunoprecipitation of unwanted species. This yielded adequate quantification of Aβ43 (2.64 pM) from tris-buffer saline (TBS) fractions from a human brain sample from AD.

Amyloid-beta peptide (Aβ) is strongly linked to the aetiology of Alzheimer’s disease (AD). Aβ is the main component of the amyloid plaques found in the brain of AD patients, however, Aβ is also present in the brain of healthy humans. It has been described that for this peptide to be neurotoxic, aggregation is needed. The accumulation of Aβ causes aggregation from low order oligomers through different intermediate species up to the formation of amyloid fibrils. However, is not the presence of the fibrils what correlates the harmfulness of the disease, but the concentration of soluble oligomeric intermediate species. Nowadays, is accepted that the neurotoxicity of these oligomers is produced on the membrane. From this point, the laboratory of Dr. Carulla developed the beta-barrel Pore Forming Oligomer (βPFO). βPFO, was produced with Aβ42, the most neurotoxic version of Aβ. It was the first described example of a stable, well-defined and homogeneous membrane oligomer with the ability to form pores on lipid membranes. In this thesis, we aim to advance in the characterization of this βPFO and the validation of βPFO as a druggable target for AD. First, as βPFO was described using detergents as a biomimetic membrane environment, we aimed to move towards a more native environment using natural lipids. By using lipid-detergent micelles we studied βPFO. In this work we demonstrated that βPFO is not able to reconstitute into the common 1,2-dihexanoyl-sn-glycero-3-phosphocholine - 1,2-dimyristoyl-sn-glycero-phosphatidylcholine (DHPC-DMPC) bicelles. Therefore, we described a new type of bicelles using dodecylphosphocholine (DPC) and DMPC, not described in the literature in these conditions until then. We showed that βPFO was able to reconstitute into DPC-DMPC bicelles preserving its overall structure and pore-forming function. Then, to advance towards βPFO validation, we immunized an alpaca with βPFO in order to generate Nanobodies. A Nanobody is a fragment of the single-chain antibodies produced by camelids, with many different properties from conventional antibodies, their reduced size, their cavity specificity, their ease of modification and production, etc. Upon the Nanobodies generation, we selected the ones specific against βPFO obtaining 11 different Nanobodies. Using enzyme-linked immunosorbent assay (ELISA) we showed that they had both, a high specificity for βPFO compared to monomeric and fibrillar Aβ42, and a high affinity for them. Moreover, we showed that these generated Nanobodies, were binding to βPFO in different manners affecting differently the protection they could cause to proteolysis. Finally, we demonstrated that upon Nanobody binding on membrane-inserted βPFO, some of the Nanobodies did not affect the current pass across the bilayer while others reduced the current pass and two of them completely blocked the pore formed. In the future, these Nanobodies could serve, not only as a tool to validate as a player βPFO in the context of AD, but also as possible therapeutics.

Peptidomimetics are synthetic oligomers that resemble the activities of peptides. Their advantages over peptides include high stability towards proteolysis and enormous chemical diversity. Over the past two decades, there have been extensive efforts to develop peptide mimics, such as beta-peptides, peptoids, D-peptides, etc. The research on peptidomimetics have led to many important applications in both medicinal and material science. In order to explore new functions, the discovery of peptidomimetics with novel frameworks is essential. We reported the synthesis and evaluation of a new class of peptidomimetics, termed as gamma-AApeptides. Previous studies of gamma-AApeptides have revealed that gamma-AApeptides are highly resistant to proteolysis, and are highly amendable to chemical diversification. However, new biological activities and folding properties of gamma-AApeptides still need to be explored. In order to expand the potential of gamma-AApeptides in chemical biology and medicinal chemistry, I have been focusing on the development of new methods to synthesize linear and cyclic gamma-AApeptides, development of one-bead-one-compound (OBOC) gamma-AApeptide libraries for the discovery of inhibitors against beta-amyloid aggregation, exploring new helical foldamers for the rational design of protein-protein interaction (PPI) inhibitors, and studying cyclic gamma-AApeptides for antimicrobial development.

La maladie d'Alzheimer représente un défi mondial pour la société. Il n'y a pas à ce jour de traitement efficace pour traiter ou ralentir les symptômes de la maladie d'Alzheimer. Cette maladie est caractérisée par une perte de synapses, une augmentation du nombre de plaques extracellulaires d'Abêta et une augmentation de Tau hyperphosphorylée agrégée intracellulaire (neurodégénérescence fibrillaire). Il est communément admis que la maladie d'Alzheimer est principalement liée à l’oligomérisation et à la fibrillation de peptides amyloïdes bêta, et que les oligomères Abêta solubles et les fibres sont des espèces neurotoxiques. Une stratégie pour réduire la présence de ces espèces toxiques de Abêta est l'utilisation de peptides ou de peptidomimétiques pour inhiber l'agrégation de Abêta, soit par interaction avec les oligomères d'Abêta pour empêcher davantage son agrégation en fibres ou en stabilisant les conformations hélicoïdales transitoires de Abêta pour empêcher sa transition vers des structures en feuillets bêta. Dans un premier temps, sur la base des résultats encourageants de glycopeptides contenant un élément polaire casseur de feuillets bêta pour moduler l'agrégation du peptide Abêta, et des propriétés uniques du groupe trifluoromethylhydroxyle, nous avons conçu et synthétisé des mimes de pentapeptides contenant la séquence (Boc) Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF3 Thr et (2S, 3S)-CF3 Thr), pour interagir avec le site de nucléation du peptide Abêta dans sa forme monomérique ou oligomérique de manière à perturber l'auto-assemblage en oligomères toxiques. Des études conformationnelles par RMN 2D et par modélisation moléculaire ont indiqué que ces peptides adoptent des conformations étendues dans des solvants polaires (eau et méthanol). Nous avons constaté expérimentalement et théoriquement que les pentapeptides contenant l’acide aminé non naturel (2S, 3S)-CF3-thréonine sont plus étendus que les pentapeptides contenant les acides aminés naturels L-sérine et L-thréonine. Nous avons également observé que les pentapeptides (2S, 3S)-CF3-Thr ont une propension à s’auto-associer en formant des brins bêta intermoléculaires. La capacité de ces pentapeptides à inhiber la formation de fibres amyloïdes a été évaluée sur les peptides Abêta 1-42 et IAPP (impliqué dans le diabète de type II) par des essais de fluorescence à la thioflavine T (ThT). Il a été constaté qu'aucun de ces pentapeptides ont un effet d'inhibition de l'agrégation des peptides Abêta 1-42 et IAPP. Au contraire, certains composés ont montré un effet d'accélération de l'agrégation de IAPP. Accélérer l'agrégation est moins intuitif mais cette stratégie a plus récemment suscité un intérêt. Il pourrait être intéressant d'étudier plus en détail l’intérêt de cette accélération de l'agrégation de IAPP par des techniques complémentaires.Dans une deuxième partie de cette thèse, nous avons assemblé une unité amino acide basée sur un motif CF3-1,4 disubstituted-1,2,3-triazole en homo-oligomères (trimères et tétramères) et en peptidomimétiques. Des études conformationnelles de ces oligomères fluorés ont été menées par RMN 2D et par des simulations de modélisation moléculaire. Nos études préliminaires de modélisation moléculaire prévoient des structures hélicoïdales avec des trimères et des tétramères d'acides aminés à base de ces CF3-triazoles. L'analyse par RMN 2D du tétramère affiche des corrélations NOE très intéressantes, ce qui indique une structure repliée. La capacité de ces oligomères fluorés à moduler la formation de fibres amyloïdes des peptides Abêta 1-42 et IAPP a été évaluée par test de fluorescence à la ThT. Nous avons constaté que le trimère est un faible inhibiteur de l'agrégation de Abêta 1-42, mais aussi un promoteur d'agrégation de IAPP. Le tétramère a été trouvé capable de moduler l'agrégation de Abêta 1-42 et de IAPP, mais non d'une manière classique d'inhibition ou de promotion
It has been widely recognized that Alzheimer’s disease (AD) represents an unsettling, worldwide challenge for society. By far, there has been no effective cure for AD. Pathologically, AD is characterized by a loss of synapses, an increase in the number of extracellular Abeta plaques and an increase in intracellular aggregated hyperphosphorylated Tau (neurofibrillary tangles). It is commonly believed that AD is primarily linked to oligomerization and fibrillization of amyloid beta peptides, and the soluble Abeta oligomers and fibrils are neurotoxic species.One strategy to reduce the Abeta fibrils is the use of peptides or peptidomimetics to inhibit the Abeta aggregation either by interaction with the Abeta oligomers to prevent its further aggregation into fibrils, or by stabilizing the transient Abeta α-helical conformations to prevent its transition to beta-sheet structures.In the first direction, based on the encouraging results of the glycopeptides containing the polar sugar beta-sheet breaker element to modulate Abeta peptide aggregation and the unique properties of trifluoromethylhydroxyl group, we designed and synthesized pentapeptide mimics with the sequence (Boc) Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF3-Thr and (2S, 3S)-CF3-Thr) to interact with the nucleation site of Abeta peptide in its monomeric or oligomeric form so as to disrupt the self-assembly into toxic oligomeric form. Both 2D NMR and molecular modelling studies indicated that these peptides in polar solvent (water and methanol) adopt mainly extended backbone conformations. It is found both experimentally and theoretically that, the (2S, 3S)-CF3-threonine-containing pentapeptides are more extended than the L-serine- and L- threonine-containing pentapeptides. It is also observed that the (2S, 3S)-CF3-Thr pentapeptides have a propensity to self-associate of by forming intermolecular beta-strand contacts. The ability of these pentapeptides to inhibit amyloid fibril formation was evaluated on Abeta1-42 and IAPP peptides by ThT fluorescence assay. It was found that none of these pentapeptides have any inhibition effect in Abeta1-42 peptide and IAPP aggregation. On the contrary, some compounds showed an acceleration effect in IAPP aggregation. Accelerating the aggregation pathways is less intuitive but this strategy has more recently aroused interest. It could be interesting to further study the effect of accelerating IAPP aggregation by complementary techniques.In the other direction, we have assembled novel a CF3-1,4-triazole-based amino acid mimic into homo-oligomers (trimer and tetramer) and peptidomimetics. The fluorinated oligomers were investigated both by NMR conformational studies and molecular modelling simulations. Our preliminary modelling studies predict helical structures with trimer and tetramers of CF3-1,4-disubstituted- 1,2,3- triazole-based amino acid. NMR analysis of tetramer displayed very interesting NOE correlations, indicating a folded structure. The ability of these fluorinated oligomers to modulate the amyloid fibril formation of Abeta1-42 and IAPP peptides were evaluated by ThT fluorescence assay. It was found that trimer was a weak inhibitor of Abeta1-42 aggregation but also a promoter of IAPP aggregation. The tetramer was found able to modulate the aggregation of Abeta1-42 and IAPP but not in a classical inhibition or promotion manner

Interactions between peptides and biological lipid membranes play a crucial role in many cellular processes such as in the mechanism behind Alzheimer’s disease where amyloid-beta peptide (Abeta)is thought to be a key component. The initial step of binding between a surface active peptide and its target membrane or membrane receptor can involve a non specific electrostatic association where positively charged amino acid residues and a negatively charged membrane surface interact. Here, the use of high resolution MAS NMR provides a highly sensitive and non perturbing way of studying the electrostatic potential present at lipid membrane surfaces and the changes resulting from the association of peptides. The interaction between pharmacologically relevant peptides and lipid membranes can also involve incorporation of the peptide into the membrane core and by complementing the NMR approach with differential scanning calorimetry (DSC) the hydrophobic incorporation can be studied in a non invasive way. By using 14N MAS NMR on biological lipid systems for the first time, in addition to 31P, 2H NMR and differential scanning calorimetry (DSC), gives a full picture of the changes all along the phospholipid following interactions at the membrane interface region. Being able to monitor the full length of the phospholipid enables us to differentiate between interactions related to either membrane surface association or hydrophobic core incorporation. This approach was used to establish that the interaction between nociceptin and negatively charged lipid membranes is electrostatic and hence that nociceptin can initially associate with a membrane surface before binding to its receptor. Also, it was found that Abeta can interact with phospholipid membranes via two types of interactions with fundamentally adverse effects. The results reveal that Abeta can associate with the surface of a neuronal membrane promoting accelerated aggregation of the peptide leading to neuronal apoptotic cell death. Furthermore it is also shown that Abeta can anchor itself into the membrane and suppress the neurotoxic aggregation of Abeta.

La maladie d'Alzheimer (MA) est l'un des principaux défis de santé publique du 21ème siècle et son développement repose sur l'hypothèse amyloïde qui stipule que la formation extracellulaire de plaques amyloïdes et l'accumulation intracellulaire d'enchevêtrements neurofibrillaires Tau (NFTs) sont causées par l'agrégation de peptides β-amyloïdes (Aβ). Plusieurs techniques biophysiques ont été employées pour étudier le processus d'agrégation des peptides Aβ, comme le dosage de la thioflavine T (ThT), la diffusion dynamique de la lumière (DLS), l'électrophorèse capillaire (CE), la microscopie électronique (EM) et la microscopie à force atomique (AFM), mais malgré les informations utiles qu'elles fournissent, toutes ne sont pas adaptées au suivi des premières étapes du processus agrégatif. L'objectif principal de cette thèse et d’évaluer l'analyse de dispersion de Taylor (TDA) pour le suivi des mécanismes d'agrégation des peptides Aβ. Le TDA est une technique moderne qui permet de déterminer le rayon hydrodynamique et de quantifier des espèces en solution pour des objets moléculaires dont la tailles est comprise entre 0,1 nm et centaines de nm. Jusqu'à présent, la TDA n'a pas encore été employée pour un suivi en temps réel de l'agrégation des peptides Aβ. La TDA a révélé que le processus d'agrégation des isoformes Aβ(1-40) et Aβ(1-42) se produit selon des mécanismes distincts. Ces résultats ont été corrélés avec le test ThT et la DLS. La co-agrégation des mélanges Aβ(1-40):Aβ(1-42) a aussi été explorée conjointement par TDA et AFM, mettant en évidence l'influence de la composition du mélange sur la cinétique et la formation d'espèces oligomériques potentiellement toxiques. Enfin, le processus d'agrégation des peptides Aβ par TDA a été réalisé à l'aide d'une détection simultanée UV-LIF utilisant des peptides fluorescents marqués FITC. Cette étude a démontré que les voies d'agrégation des peptides Aβ natifs sont modifiées par la présence du fluorophore. En conclusion, la TDA permet une spéciation des espèces solubles (monomères, oligomères, protofibriles) lors de l'agrégation des peptides Aβ, ce qui apporte des informations très précises sur le mécanisme d’agrégation.Mots-clés: Maladie d'Alzheimer ; peptides β-amyloïdes ; analyse de dispersion de Taylor ; études d'agrégation ; microscopie à force atomique ; ThT assay; diffusion dynamique de la lumière
Alzheimer Disease (AD) is one of the major public health challenges of the 21st century and its development is centered around the amyloid hypothesis which states that extracellular formation of amyloid plaques and the intracellular accumulation of neurofibrillary Tau tangles (NFTs) are caused by the aggregation of β-amyloid (Aβ) peptides. Several biophysical techniques have been employed for studying the aggregation process of Aβ peptides such as thioflavin T (ThT) assay, dynamic light scattering (DLS), capillary electrophoresis (CE), electron microscopy (EM) and atomic force microscopy (AFM). Despite the useful information these methods provide, not all of them are suitable for monitoring the early stages of the process. The main objective of this thesis is to apply Taylor dispersion analysis (TDA) for the monitoring of the Aβ peptide aggregation mechanism. TDA is a modern technique that can size and quantify soluble species ranging from 0.1 nm to a few hundred nm. TDA has yet been employed for a real-time monitoring of the Aβ peptide aggregation. TDA revealed that the aggregation process of Aβ(1-40) and Aβ(1-42) isoforms occurs through distinct pathways. These results have been correlated with ThT assay and DLS. The co-aggregation of Aβ(1-40):Aβ(1-42) mixtures was further explored by TDA and AFM, highlighting the influence of the peptide ratios on the kinetics and the formation of potentially toxic oligomeric species. Finally, the aggregation process of Aβ peptides by TDA was conducted using a simultaneous UV-LIF detection in the presence of FITC-tagged Aβ peptides. This study demonstrated that the aggregation pathways of the native Aβ peptides are altered by the presence of the fluorophore. In conclusion, TDA provided a complete speciation of the different soluble species (monomer, oligomers, protofibrils) during Aβ aggregation, which brings valuable information on the mechanism of aggregation.Keywords: Alzheimer disease; β-amyloid peptides; Taylor dispersion analysis; aggregation studies; atomic force microscopy; ThT assay; dynamic light scattering

Orientadora: Profa. Dra. Giselle Cerchiaro
Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, Santo André, 2018.
Em doencas neurodegenerativas amiloidais o estresse oxidativo tem um papel importante juntamente com a proteina ¿À-amiloide (A¿À), associada a formacao de placas senis na Doenca de Alzheimer. Tais condicoes demonstraram desbalanco de metais, como cobre e zinco, tanto na concentracao celular e quanto nos processos antioxidantes. A Cu,Zn-Superoxido Dismutase (SOD1), em condicoes neurodegenerativas, pode demonstrar alteracoes estruturais e funcionais, tendo menor afinidade pelo cobre e pelo zinco. Diante destas condicoes, o objetivo principal desta tese foi em condicoes oxidativa (H2O2) e neurodegenerativa (A¿À1-42) avaliar os danos a biomoleculas, concentracao metais e a influencia da enzima SOD1 em linhagens de celulas neuronais (NSC-34 e mHippoE2). Diferentes respostas quanto a sensibilidade das linhagens neuronais foi observada durante as condicoes oxidativas e neurodegenerativa. Quanto os danos ao DNA a linhagem NSC-34 demonstrou maior sensibilidade a condicao oxidativa, com aumento de danos ao DNA, lesoes oxidativas em bases nitrogenadas que indicaram a presenca de lesoes tipo 8-oxo-G, corroborando com anormalidades nucleares e inibicao do processo de divisao celular. Nesta mesma linhagem quantidades aumentadas de Cu foram observadas, juntamente com a presenca da enzima SOD1 a nivel citoplasmatico e nuclear na condicao oxidativa (H2O2), alem de resultados significantes para danos permanentes ao DNA (anormalidades nucleares e quebras cromossomicas). A linhagem mHippoE2 apresentou aumentos significativos mediante a condicao oxidativa e neurodegenerativa, como oxidacao de proteinas e lipidios, demonstrando tambem alteracoes morfologicas citoplasmaticas. O tratamento com A¿À1-42 demonstrou aumento de danos ao DNA, lesoes oxidativas 8-oxo-G e tambem em bases purinicas. Podemos observar nesta mesma linhagem a forte influencia do Zn na condicao neurodegenerativa, atividade da SOD1 em ambas condicoes e tambem danos permanentes ao DNA mediante condicao neurodegenerativa. Dentre os resultados obtidos salientamos a relevancia dos achados na condicao neurodegenerativa ocasionada pelo peptideo A¿À1-42 nos ensaios para avaliacao genotoxica e mutagenica. Tal condicao demonstrou a presenca de danos importantes a bases nitrogenadas, tanto purinicas quando pirimidinicas, apontando tambem para possiveis efeitos mutagenicos detectados pelos eventos de quebras cromossomicas associados as anormalidades nucleares, bem como a presenca da enzima SOD1 no nucleo das celulas.
In neurodegenerative diseases, oxidative stress plays an important role associated with â-amyloid protein (Aâ), associated with the formation of amyloid plaques in Alzheimer's Disease (AD). In AD condition it has been demonstrated an imbalance of essential metals, such as copper and zinc, their cellular concentration and antioxidant processes alterations. The antioxidant enzyme Cu, Zn-Superoxide Dismutase (SOD1), under neurodegenerative conditions has structural and functional changes, such as lower affinity for copper and zinc. According to these conditions, the main objective of this thesis was to investigate how the oxidative (H2O2) and neurodegenerative (Aâ1-42) conditions cause biomolecules damage, metal alteration and SOD1 location in neuronal cell lines (NSC-34 and mHippoE2). Different responses in neuronal cell lines were observed during the conditions evaluated. For DNA damage, the NSC-34 cells demonstrated greater sensitivity to the oxidative condition, with increased DNA damage, oxidative lesions on nitrogenized bases indicating the presence of 8-oxo-G type lesions. In this same cell line we observed an increase of Cu amount, together with the presence of the SOD1 enzyme at the cytoplasmic and nuclear level in the oxidative condition (H2O2). The mHippoE2 cell line presented increased protein oxidation through the oxidative and neurodegenerative condition. Treatment with Aâ1-42 demonstrated increased DNA damage in this cell, 8-oxo-G oxidative lesions and also purine bases. We observed, in this same cell line, the strong influence of Zn on the neurodegenerative condition, SOD1 activity in both conditions and it was observed permanent damages to DNA in the neurodegenerative condition. Among the results, we highlight the relevance of the findings in the neurodegenerative condition caused by the Aâ1-42 peptide in the genotoxic and mutagenic evaluation trials. This condition demonstrated the presence of important damages to nitrogenated bases, both purine and pyrimidine, also pointing to possible mutagenic effects detected by the events of chromosomal breaks associated with nuclear abnormalities, as well as the translocation of the SOD1 enzyme to nuclei.

碩士
國立陽明大學
生命科學暨基因體科學研究所
97
Alzheimer’s disease (AD) is a neurodegenerative disease which leads to progressive dementia and neuronal death. The main histopathological hallmarks of AD are the senile plaques within the cerebral cortex and the neurofibrillary tangles within the nerve cells. The primary component of senile plaques is beta-amyloid peptide (Ab).�nRecent studies have suggested that Ab has neurotoxic properties in an aggregated state. Thus, knowing the molecular mechanism of the aggregation process of Ab may facilitate the design and development of fibrillogenesis inhibitors. Previously, we have characterized structural and aggregation properties of a rationally mutated Ab40, Ab40(F17A/L19A), by using NMR spectroscopy in our lab. In SDS environment, the aggregation rate of Ab40(F17A/L19A) is much slower than that of wild-type Ab40. The alpha-helical propensity of Ab40(F17A/L19A) is higher than that of wild-type Ab40 in the region from Q15 to D23. In aqueous solution, the cell toxicity and aggregation rate of Ab40(F17A/L19A) are also lower than those of wild-type Ab40. These characteristics suggest that Ab40(L17A/F19A) may aggregate at a lower rate in a lipid environment. In this study, we will take advantage of these characteristics and apply NMR spectroscopy to investigate the aggregation mechanism of Ab40(L17A/F19A) in lipid environment from the structural point of view. The results may help us gain more insight to the aggregation mechanism of wild-type Ab40.

Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.

博士
國立中央大學
化學學系
103
Extracellular deposits of amyloidbeta (Abeta) aggregates in brain is the hallmark of Alzheimer’s disease (AD). Abeta peptide is produced from the amyloid precursor protein (APP) within membrane by the proteolytic action of thebeta- and γ-secretases. Therefore, it is crucial to determine the configuration of Abeta peptide within a membrane to provide insights at atomic levels for the aggregation mechanism and toxicity. In this thesis, we employed replica-exchange molecular dynamics in conjunction with an implicit membrane model to investigate the mutation and sequence effects of short Abeta(25-35) fragment and familial mutations (E22Q (Dutch), D23N (Iowa) and E22Q/D23N (Dutch/Iowa) mutants) of full length Abeta(1–40) peptide on their configurations within a membrane. Our work on Abeta(25-35) and its mutants correlated the configurations of peptides within membrane with their toxicities complementing the hydrophobicity-toxicity relationship. In the work of familial mutations of Abeta, we find that the central hydrophobic core (CHC) (residues 17-21) of familial mutants is less protected and thus more exposed to the solvent than that of Abeta(1–40) peptide due to their weal long-range contacts with the N-terminal (residues 1-16). Moreover, for the familial mutants, their energy barriers for the inter-conversion of the CHC configurations between membrane and water phases is lower than that of Abeta(1–40). Therefore, it is faster for the familial mutants to convert their CHCs from membrane (less surface-exposed) to water phase (highly surface-exposed), when their CHC populations in the water phase are decreased (e.g. aggregated). As the amyloid fibril is deposited on membrane, these findings are helpful to understand the experimental observations that the familial Abeta peptides aggregate much faster than that of Abeta(1–40). This study provides the insight for the rational drug design to prevent amyloid associated diseases.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
Alzheimer’s Disease (AD) is the most prevalent neurodegenerative disease and also represents the major common type of dementia, affecting mainly individuals over 65 years. It is an extremely disabling disease that is characterized, fundamentally, by a progressive decline in cognitive capabilities, which ultimately leads to dependence on others to perform the tasks of daily routine.The neurodegeneration that occurs mainly at the hippocampus and cerebral cortex, the presence of senile plaques, made up of aggregates of peptides β-amyloid, and neurofibrillary tangles, made up of hyperphosphorylated tau protein are the most characteristic neuropathological marks of AD.Several studies have shown that abnormal aggregates of peptides Aβ and hyperphosphorylated tau protein, mitochondrial dysfunction, oxidative stress, excitotoxicity, neuroinflammation and cerebrovascular dysfunction play a key role in the degeneration and neuronal death that occurs in AD. However, currently, the etiology of AD is still enigmatic, remaining largely unclear what triggers such mechanisms of neurodegeneration. Moreover, the pharmacological therapy is clearly ineffective, because it has no curative effect, only relieving the symptoms of the disease and improving the quality of life of patients and their caregivers. Therefore, it is urgent the development of new therapeutic strategies able to effectively to prevent and/or limit the neurodegeneration. In this context, it is important to note that dietary polyphenols, due to their ability to modulate cell signaling pathways and gene transcription, have been the target of numerous studies and, they are considered promising molecules for the development of new therapies for the treatment of AD.The main goal of this monography is the elucidation of several molecular mechanisms involved in the neurodegeneration that occurs in AD and its modulation by some dietary polyphenols.
A Doença de Alzheimer (DA) é a doença neurodegenerativa mais prevalente e representa, também, o tipo mais comum de demência, afetando principalmente indivíduos acima dos 65 anos. Trata-se de uma doença extremamente incapacitante que se caracteriza, fundamentalmente, por um declínio progressivo das capacidades cognitivas, o que acaba por levar à dependência de terceiros para a realização das tarefas da rotina diária. A neurodegeneração que ocorre, principalmente, ao nível do hipocampo e córtex cerebral, a presença de placas senis, constituídas por agregados de peptídeos β-amilóide, e as tranças neurofibrilares, compostas pela proteína tau hiperfosforilada são as marcas neuropatológicas mais características da DA. Vários estudos têm evidenciado que aglomerados anormais de peptídeos Aβ e de proteína tau hiperfosforilada, a disfunção mitocondrial, o stresse oxidativo, a excitotoxicidade, a neuroinflamação e a disfunção cerebrovascular desempenham um papel chave na degeneração e morte neuronal que ocorre na DA. No entanto, atualmente, a etiologia da DA é ainda enigmática, permanecendo largamente por esclarecer o que despoleta tais mecanismos de neurodegeneração. Além do mais, a terapêutica farmacológica existente é ineficaz, pois não tem efeito curativo, atuando apenas no alívio da sintomatologia da doença e na melhoria da qualidade de vida dos doentes e seus cuidadores. Tal facto, torna urgente o desenvolvimento de estratégias terapêuticas capazes de, eficazmente, prevenirem e/ou travarem a neurodegenerescência. Neste contexto, importa referir que os polifenóis da dieta, devido, essencialmente, à sua capacidade de modular vias de sinalização celular e a transcrição de genes, têm sido alvo de inúmeros estudos e são, atualmente, considerados moléculas promissoras para o desenvolvimento de novas terapêuticas para o tratamento da DA. A presente monografia tem como principal objetivo a elucidação de vários mecanismos moleculares envolvidos na neurodegeneração que ocorre na DA e a sua modulação por alguns polifenóis da dieta.

Alzheimer’s Disease (AD) is a chronic progressive neurological condition, clinically characterized by memory deficits, cognitive and physical impairment, and personality changes. Traditionally, AD was considered a type of protein folding disorder. Here, the concept of AD as an autoimmune disease of the innate immune system was developed. After exploring evolutionary connections between the AD peptide β-amyloid (Aβ) and known antimicrobial peptides (AMPs), and elucidating the structural similarities between Aβ and AMPs, a mechanism of action for Aβ’s antimicrobial activity is proposed that is based on the compromise of bacterial membranes. Following these theoretical considerations, experimental evidence is presented for the production of Aβ by cells in response to infection, and for Aβ’s antibacterial and antiviral activity. Rooted in similarities of the cell membranes of neuronal and bacterial cells in terms of lipid composition and transmembrane potential, it is hypothesised that Aβ’s neurotoxicity is caused by its misguided attack on neurons as an AMP. In reversing the concept of Aβ as an AMP, the similarity of AMPs to Aβ is demonstrated in experiments revealing the neurotoxicity of two AMPs, LL 37, and cecropin A. To determine a mechanism for the progressive nature of AD, it was shown that, although apoptosis may be involved in AD, it is actually necrosis that is responsible for the propagation of neuronal cell death so characteristic of AD. With the Vicious Cycle of AD, a scheme was devised, integrating the results obtained here with data and research from other groups, which explains the chronic and progressive nature of AD as a result of Aβ’s physiological role as an AMP and innate immune system effector. Borne from Aβ’s activity as an AMP and its central role in the Vicious Cycle of AD, a question was investigated: do antibiotics, such as penicillin, that cause release of bacterial endotoxins due to their mechanism of action, trigger the Vicious Cycle of AD and thus lead to the development of AD? Preliminary evidence supporting this notion was presented.