Thèses sur le sujet « AMYLOID, APP, ABETA, ALZHEIMER »

Amyloid-β precursor protein (APP) mutations cause familial Alzheimer’s disease with virtually complete penetrance. We found an APP mutation (A673V) that causes disease only in the homozygous state, while heterozygous carriers were unaffected, consistent with a recessive Mendelian trait of inheritance. The A673V mutation affected APP processing, resulting in enhanced amyloid β (Aβ) production and formation of amyloid fibrils in vitro. Co-incubation of mutated and wild-type peptides conferred instability on Aβ aggregates and inhibited amyloidogenesis and neurotoxicity. The highly amyloidogenic effect of the A673V mutation in the homozygous state and its anti-amyloidogenic effect in the heterozygous state account for the autosomal recessive pattern of inheritance, and have implications for genetic screening and the potential treatment of Alzheimer’s disease.

Une des caractéristiques histopathologiques de la maladie d'Alzheimer (AD) est la présence de plaques amyloïdes formées par les peptides amyloïdes β (Aβ) de 40 et 42 résidus, qui sont les produits de clivage par des protéases de l'APP. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, les peptides APP_TM4K ont été étudiés dans la bicouche lipidique en utilisant l’ATR-FTIR et ssNMR. Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, hétérogénéité de conformation et d'orientation a été observée pour le site de clivage γ et , que peuvent avoir des implications dans le mécanisme de clivage et donc dans la production d’Aβ. Les peptides Aβ s'agrègent pour produire des fibrilles et aussi de manière transitoire d'oligomères neurotoxiques. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. Ces résultats nous ont amené à conclure que les Ca2+ stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans l’AD. Les positions et une orientation précise de deux nouveaux médicaments puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen  dans la bicouche lipidique, ont été obtenus à partir des expériences des ssNMR. Ces résultats indiquent que le mécanisme probable de modulation du clivage par la y-sécrétase est une interaction directe avec le domaine TM de l’APP
A histopathological characteristic of Alzheimer’s disease (AD) is the presence of amyloid plaques formed by amyloid β(A) peptides of 40 and 42 residues-long, which are the cleavage products of APP by proteases. To understand the role of structural changes in the TM domain of APP, APP_TM4K peptides were studied in the lipid bilayer using ATR-FTIR and ssNMR. While the overall secondary structure of the APP_TM4K peptide is helical, conformational and orientational heterogeneity was observed for the y- and for the -cleavage sites, which may have implications for the cleavage mechanism and therefore the production of Aβ. Starting from its monomeric form, Aβ peptides aggregate into fibrils and / or oligomers, the latter being the most neurotoxic. We found that in the presence of Ca2 +, Aβ (1-40) preferably forms oligomers, whereas in the absence of a2 + Aβ (1-40) aggregates into fibrils. In samples without Ca2 +, ATR-FTIR shows conversion from antiparallel β sheet conformation of oligomers into parallel β sheets, characteristic of fibrils. These results led us to conclude that Ca2 +stimulates the formation of oligomers of Aβ (1-40), that have been implicated in the pathogenesis of AD. Position and precise orientation of two new drugs  powerful modulators of γ-secretase  benzyl-carprofen and carprofen sulfonyl  in the lipid bilayer were obtained from neutron scattering and ssNMR experiments. These results indicate that carprofen-derivatives can directly interact with APP. Such interaction would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by β -secretase, diminishing or greatly reducing Aβ42 production

Un ensemble des données cohérentes de la littérature plaide en faveur d'une relation entre une baisse d'activité de la voie de signalisation de la vitamine A, des altérations de la plasticité synaptique et des déficits mnésiques spécifiques associés au vieillissement. Une diminution de l'activité de cette voie de signalisation est également évoquée dans les processus neurodégénératifs caractéristiques de la maladie d'Alzheimer. Dans ce contexte, les objectifs de ce travail étaient de mieux comprendre les conséquences neuro-anatomiques et fonctionnelles d'une baisse d'activité de la voie de signalisation de la vitamine A. Notre approche expérimentale a mis en œuvre 2 modèles animaux, un modèle de carence vitaminique A qui induit spécifiquement une baisse d'activité de sa voie de signalisation et un modèle d'hypothyroïdie dont il a été montré qu'il induit aussi une hypoactivité de la voie de signalisation de la vitamine A. La démarche expérimentale conduite chez les rats carencés en vitamine A comporte deux volets : (i) un volet mettant en œuvre l’imagerie et la spectroscopie RMN, (ii) un volet moléculaire consacrée à l’étude de l’expression de gènes cibles des rétinoïdes impliqués dans le processus amyloïdogène. Les mesures ont été réalisées, d'une part, chez des animaux soumis à un régime dépourvu en vitamine A pendant 10 semaines et d'autre part, chez des animaux soumis à ce même régime pendant une durée de 13 ou 14 semaines. Une partie des animaux carencés a été traitée par de l'AR. Les résultats montrent que dès 10 semaines de carence, les animaux présentent une altération du métabolisme et de son action cellulaire de la vitamine A qui se traduit par (i) une diminution significative du taux de vitamine A sérique, (ii) une diminution du taux d'ARNm codant pour les récepteurs RAR, dans le cerveau entier, le striatum, l'hippocampe et de manière moins prononcée le cortex des animaux. Après 10 semaines de régime dépourvu en vitamine A, des modifications métaboliques ont été mises en évidence essentiellement dans le cortex. Elles se traduisent par une hausse du (i) NAA/Cr, marqueur de la densité neuronale corrigée par une administration d'AR, et (ii) du GSH/Cr, indicateur du potentiel antioxydant cellulaire dans cette structure. Au plan anatomique, un ralentissement de la croissance cérébrale a été observé dés la 7ème semaine de régime. Une diminution du volume hippocampique et une augmentation des espaces ventriculaires ont été observées à partir de 11 semaines de carence. Au plan moléculaire, aucune modification de l'expression du gène codant pour APP, ou du rapport APP770-751/APP695, considéré comme un indicateur précoce de la MA n'a été observée après 10 semaines de carence. Après 14 semaines de régime dépourvu en vitamine A, de profondes modifications métaboliques sont observées dans les trois structures à savoir le cortex, l’hippocampe et le striatum. Au plan moléculaire, les principaux résultats suggèrent un basculement du processus biochimique de dégradation de la protéine APP en faveur de la voie amyloïdogénique dans le cortex, et par voie de conséquence en faveur de la formation du peptide Aß. Cependant, aucune modification du taux protéique des peptides Aß n'a été mise en évidence dans le cortex et l'hippocampe des rats carencés. Le modèle d'hypothyroïdie que nous avons mis en oeuvre entraine bien une hypoactivité de la voie de signalisation de la T3, observée dans l'hippocampe des animaux et une diminution du taux d'ARNm codant pour RARß observée dans le cortex des rats hypothyroïdiens. Au plan moléculaire, l'augmentation du rapport APP770-751/APP695 a été observée chez les rats rendus hypothyroïdiens par rapport aux rats témoins. Comme chez les rats carencés en vitamine A, les indicateurs de la voie physiologique ne sont que très faiblement affectés chez les rats rendus hypothyroïdiens
Some data reveal that retinoid hyposignalling, presumably resulting from decreased bioavailability of retinoid ligands naturally, was shown to result in aging-related synaptic plasticity and long term potentiation (LTP) alterations as well as in aging-related decline of cognitive function. Moreover, genetic, metabolic and dietary evidence has been provided for a defective retinoid metabolism in Alzheimer disease (AD). Thus, key steps of the amyloid production process are under the control of proteins whose expression is positively regulated by RA in vitro. In this context, the aims of this work were to better understand neuro-anatomical and functionnal consequences of retinoid signaling brain hypoactivity. Our experimental method uses two animal models: a Vitamin A deficiency model which induce especially an hypoactivity of retinoid pathway, and an hypothyroid model which was also characterized by an hypoactivity of retinoid pathway. In the fisrt model, two main approch were used : (i) an NMR imaging and spectroscopy approach, (ii) a molecular approach to study expression of retinoid target genes implicated in amyloidogenic process. NMR results showed that VAD induces severe anatomic and metabolic disorders in particular a slowing of brain growth, hippocampus atrophy, and a decrease of NAA/Cr, marker of neuronal density which was observed in cortex, hippocampus and striatum. Molecular results reveal a vitamin A deficiency-related dysregulation of the amyloid pathway in the cortex of rats, which is known to be the first brain area altered by AD development. In this area, 14 weeks of deprived diet induces physiological dysregulation in the modulation of RA target genes leading to an increased amount of ADAM10, BACE and PS1, with some modifications in amyloidogenic pathway but without increased amount of Aß peptides. In hypothyroid model, molecular results suggests that adult onset-hypothyroidism may induce the amyloidogenic pathway of APP processing by increasing activity of ß and ?secretases and levels of amyloid peptides mainly in hippocampus. Together these data argue for the idea that hypoactivity of retinoid signalling which occurs naturally with aging could be a factor participating in accelerating aging and that hypothyroidism that become more prevalent with advancing age, could increase, via a hyposignaling of T3 pathway, the vulnerability of amyloidogenic pathway of APP processing as well as of other clinical symptoms of AD

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

Doutoramento em Biologia
A Doença de Alzheimer (DA) é uma das doenças neurodegenerativas mais comuns, e apresenta uma incidência mundial de 2-7% em indivíduos com mais de 65 anos e de cerca de 15% em indivíduos acima dos 85 anos de idade. Apesar da sua etiologia multifactorial, há uma correlação bem descrita entre esta patologia e um peptídeo neurotóxico denominado Abeta. Este peptídeo deriva fisiológica e proteoliticamente de uma glicoproteína transmembranar com características de receptor: a Proteína Percursora de Amilóide de Alzheimer (PPA). As possíveis funções fisiológicas da proteína PPA, o seu destino e vias de processamento celulares, conjuntamente com possíveis proteínas celulares que com ela interajam, são assim tópicos de interesse e objectos de investigação científica mundial. Neste contexto tem sido amplamente descrito o envolvimento do processo de fosforilação de proteínas, uma importante modificação pós-transducional que regula muitos e variados acontecimentos intracelulares, na regulação do processamento da PPA. Apesar do exposto, muito pouco é conhecido acerca da fosforilação directa da própria PPA. Esta proteína possui na sua estrutura primária sequências consenso para fosforilação, quer no seu ectodomínio quer no seu domínio intracelular, já descritas como sofrendo fosforilação “in vitro” e “in vivo”. O resíduo Serina 655 pertence a um motivo funcional da APP, 653YTSI656, que forma um sinal de internalização e/ou de “sorting” basolateral. Este domínio é também o local de ligação para a APPBP2, uma proteína que interage com os microtubulos da célula. Embora ainda mal elucidados, os mecanismos pelos quais a fosforilação proteica regula o processamento da PPA parecem incluir uma alteração no tráfego desta proteína, sugerindo que o domínio fosforilável 653YTSI656 desempenha um papel importante nesse processo. Esta dissertação visou assim contribuir para elucidar o papel da fosforilação directa da molécula de APP, mais especificamente no seu resíduo Serina 655, na regulação do direcionamento e tráfego subcelular da proteína, e nas suas possíveis clivagens proteolíticas. De forma a respondermos a essas questões desenvolvemos um modelo experimental para seguir o tráfego intracelular, que usa uma combinação de biologia molecular, técnicas de microscopia de epifluorescência e técnicas de cultura celular. Os resultados obtidos implicam este resíduo como um sinal de direcionamento subcelular da proteína APP, e revelam como o redireccionamento desta proteína por fosforilação favorece um tipo de processamento não amiloidogénico desta. Adicionalmente, a fosforilação do resíduo Serina 655 parece possuir um papel regulador da actividade da PPA como molécula de transdução de sinais. As implicações destas observações na DA e em novas aplicações terapêuticas para a doença são subsequentemente discutidas.
Alzheimer’s Disease (AD) is a common neurodegenerative disease affecting individuals worldwide with an incidence of 2-7% of post-65 and 15% of post-85 years old. This disease is multifactorial in its etiology but central to its pathology is a neurotoxic peptide termed Abeta. This peptide is physiologically derived by a proteolytic process on the transmembranar Alzheimer’s Amyloid Precursor Protein (APP). Protein phosphorylation-dependent APP processing has been widely described and although the mechanisms involved remain far from clarified, alterations in APP trafficking seem to occur as part of the answer. Furthermore, the occurrence of consensus phosphorylation sites in the APP intracellular domain has been known for long, but little was known regarding the direct phosphorylation of APP. Efforts in unravelling the role of these domains are finally being successful in placing them as key control points in APP targeting and processing. Among these consensus sequences, the less studied 653YTSI656 motif forms a characteristic internalisation and/or basolateral sorting signal sequence, and is known to be the binding site for a microtubuleinteracting protein (APPBP2). Phosphorylation of this motif was thus suggested to be involved in APP targeting regulation, hitherto all attempts failed to confirm it or even to reveal substantial evidences. In this project, the role of the 653YTSI656 idomain, and in particular the phosphorylatable serine 655, in APP trafficking and proteolytic processing was studied. In order to address this question a new experimental methodology was developed, which coupled molecular biology, fluorescence imaging, and cell culture techniques. APP point mutants, mimicking serine 655 phosphorylatedand dephosphorylated-status, and tagged with the green-fluorescent protein, were used to study protein trafficking dynamics and processing. Results obtained place serine 655 phosphorylation as a key signal in APP sorting and targeting to specific subcellular locations. Also of high relevance was the observed implication of serine 655 phosphorylation as a regulatory mechanism that maybe involved in controlling APP function as a signal transducer. The implications of these observations in AD pathogenesis and therapeutic approaches are discussed.
FCT - PRAXIS XXI/BD/16218/98
FCT - POCTI/BCI/34349/1999
Project DIADEM, QLK3-CT- 2001/02362
Fundação Calouste Gulbenkian
Fundação Astrazeneca

The misfolding and aggregation of the beta cell hormone islet amyloid polypeptide (IAPP) into amyloid fibrils is the main pathological finding in islets of Langerhans in type 2 diabetes. Pathological assemblies of IAPP are cytotoxic and believed to contribute to the loss of insulin-producing beta cells. Changes in the microenvironment that could trigger the aggregation of IAPP are largely unknown. So is the possibility that islet amyloid can spread within or between tissues. The present thesis have explored the roles of glycosaminoglycan heparan sulfate (HS) and the novel anti-amyloid chaperone Bri2 BRICHOS domain in the assembly of IAPP amyloid and cytotoxic IAPP aggregates. Furthermore, cross-seeding as a molecular interaction between the observed connection of type 2 diabetes and Alzheimer disease has been examined. The N-terminal region of IAPP was required for binding to HS structures and induction of binding promoted amyloid formation. Interference in the HS-IAPP interaction by heparanase degradation of HS or by introducing short, soluble HS-structure fragments reduced amyloid deposition in cultured islets. Cytotoxicity induced by extracellular, aggregating IAPP was mediated via interactions with cell-surface HS. This suggests that HS plays an important role in islet amyloid deposition and associated toxicity. BRICHOS domain containing protein Bri2 was highly expressed in human beta cells and colocalized with IAPP intracellularly and in islet amyloid deposits. The BRICHOS domain effectively attenuated both IAPP amyloid formation and IAPP-induced cytotoxicity. These results propose Bri2 BRICHOS as a novel chaperone preventing IAPP aggregation in beta cells. The intravenous injection of IAPP, proIAPP or amyloid-β (Aβ) fibrils enhanced islet amyloidosis in transgenic human IAPP mice, demonstrating that both homologous- and heterologous seeding of islet amyloid can occur in vivo. IAPP colocalized with Aβ in brain amyloid from AD patients, and AD patients diagnosed with T2D displayed increased proportions of neuritic plaques, the more pathogenic plaque subtype. In conclusion, both IAPP amyloid formation and the cytotoxic effects of IAPP is dependent on interactions with HS whereas interactions with Bri2 BRICHOS is protective. Cross-seeding between Aβ and IAPP can occur in vivo and the two peptides colocalize in brain amyloid in AD patients.

The amyloid-β precursor protein (APP) has been widely studied due to its role in Alzheimer´s disease (AD). When APP is sequentially cleaved by β- and γ-secretase, amyloid-β (Aβ) is formed. Aβ is prone to aggregate and is toxic to neurons. However, the main processing pathway for APP involves initial cleavage at the α-site, within the Aβ region, instead generating a neuroprotective soluble fragment, sAPPα. APP is a member of a protein family, also including the proteins APLP1 and APLP2, which are processed in a similar way as APP. In addition, K/O studies in mice have shown that the three proteins have overlapping functions where APLP2 play a key physiological role. The aim of this thesis was to study mechanisms underlying the α-secretase processing of APP and APLP2. We have used the human neuroblastoma cell-line SH-SY5Y as a model system and stimulated α-secretase processing with insulin-like growth factor-1 (IGF-1) or retinoic acid (RA). Our results show that the stimulated α-site cleavage of APP and APLP2 is regulated by different signaling pathways and that the cleavage is mediated by different enzymes. APP was shown to be cleaved by ADAM10 in a PI3K-dependent manner, whereas APLP2 was cleaved by TACE in a PKC-dependent manner. We further show that protein levels and maturation of ADAM10 and TACE is increased in response to RA, mediated by a PI3K- or PKC-dependent signaling pathway, respectively. Another focus of our research has been O-GlcNAcylation, a dynamic post-translational modification regulated by the enzymes O-GlcNAc transferase and O-GlcNAcase (OGA). We show that decreased OGA activity stimulates sAPPα secretion, without affecting APLP2 processing. We further show that ADAM10 is O-GlcNAcylated. Lastly, we show that APP can be manipulated to be cleaved in a similar way as APLP2 during IGF-1 stimulation by substituting the E1 domain in APP with the E1 domain in APLP2. Together our results show distinct α-site processing mechanisms of APP and APLP2.

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 4: Manuscript; Paper 5: Manuscript.

Doutoramento em Biomedicina
The Amyloid Precursor Protein (APP) is a type 1 membrane glycoprotein, mainly known as the precursor of the amyloid β-peptide, a central player in Alzheimer’s disease. Nevertheless, APP has been established as a neuromodulator of developing and mature nervous system. Alterations in the level or activity of APP and APP fragments seem to play a critical role in several neurodegenerative and neurodevelopment disorders. APP is a complex molecule due to the intricate relationships between its intracellular trafficking, posttranslational modifications, proteolytic cleavages, and multiple protein interactors. Various studies currently address the physiological roles of APP and its fragments, but there are contradictory results and missing pieces that need further work. The main objective of this thesis was to contribute to the characterization of the role of APP in neuronal differentiation. Particularly, we focused on mechanisms mediated by APP, its fragment sAPP, and APP phosphorylation at serine 655. First, we characterized the APP protein in Retinoic Acid (RA)-induced SHSY5Y cell differentiation. The comprehensive analysis of this model exposed a biphasic temporal response: a first early phase (D0-D4), where a sAPP/APP peak assists the emergence of new processes and their elongation into neurites; and a second phase (D4-D8) when increased holoAPP protein levels are necessary to sustain neuritic elongation and stabilization. In line with our main aim, we subsequently characterized the relationship between APP and the neurotrophic EGF-EGFR-ERK signaling pathway. We showed, for the first time, that APP interacts with proEGF, and confirmed the interaction with EGFR. Furthermore, we showed that combined APP and EGF have a synergistic effect on neuronal-like differentiation, related to enhanced ERK1/2 activation, and observed that APP modulates EGFR expression levels and trafficking. Both ERK1/2 activation and EGFR seem to be modulated by the APP S655 phosphorylation state, and phosphorylation at this residue favours dendritogenesis in mice cortical neurons. Finally, we focused on discovering APP protein interactors dependent on S655 phosphorylation and with a role in neuronal differentiation. SH-SY5Y differentiated cells, overexpressing APPWt or S655 phosphomutants, were used to immunoprecipitate the specific APP proteins and their respective interacting partners, later identified by mass spectrometry. The dephosphoS655 APP interactome was enriched in functions associated with cytoskeleton organization, and these cells were particularly associated with actin remodeling. The phosphoS655 APP interactome included proteins involved in the regulation of survival and differentiation, and in various signaling pathways, correlating well with an enhanced neurite outgrowth displayed by these cells. We hope that the knowledge here gathered can contribute to a better comprehension of APP-driven neurotrophic roles and underlying mechanisms.
A Proteína Precursora de Amilóide (APP) é uma proteína membranar mais conhecida por ser precursora do péptido Amilóide β, tendo por isso um papel central na doença de Alzheimer. Não obstante, a APP tem sido reconhecida como neuromodulador do sistema nervoso central. Alterações nos níveis ou na atividade da APP e seus fragmentos estão implicadas em diferentes doenças neurológicas. As relações entre o seu transporte intracelular, modificações pós-traducionais, corte proteolítico, e proteínas com as quais interage são complexas e multifacetadas. Talvez por isso, estudos focados no papel fisiológico da APP apresentem resultados contraditórios e muitas questões em aberto. O objetivo deste trabalho consistiu na caracterização do papel fisiológico da APP na diferenciação neuronal. Particularmente, focámo-nos nos mecanismos mediados pela APP e fragmento sAPP, e a fosforilação da APP no resíduo serina 655. Inicialmente, caracterizámos a proteína APP ao longo da diferenciação de células SH-SY5Y com ácido retinóico (RA). A análise sistemática deste modelo permitiu delimitar uma resposta bifásica: na primeira fase (D0-D4), um pico de sAPP/APP acompanha o aparecimento de novos processos e o crescimento a neurites; na segunda fase (D4-D8) o aumento nos níveis da APP suporta o crescimento e manutenção das neurites. Caracterizámos posteriormente a relação entre a APP e a via de sinalização EGF-EGFR-ERK na diferenciação neuronal. Demonstrámos, pela primeira vez, que a APP interage com o proEGF, e confirmámos a sua ligação ao EGFR. Adicionalmente, observámos que a APP e o EGF têm um efeito sinérgico na diferenciação tipo-neuronal e aumento da ativação da ERK1/2, e que a APP afeta os níveis e transporte do EGFR. Estes mecanismos são modulados pela fosforilação da APP na S655, que favorece a dendritogénese em neurónios corticais de ratinho. Por último, focámo-nos na identificação de proteínas interatoras da APP dependentes da fosforilação em S655 e com função na diferenciação neuronal. Usando células SH-SY5Y diferenciadas e a sobrexpressar a APPWt ou fosfomutantes da S655, imunoprecipitámos as diferentes APPs e seus interatores, posteriormente identificados por espectrometria de massa. O interatoma da APP desfosforilada é enriquecido em funções associadas à organização do citoesqueleto, levando a uma maior reorganização da actina. O interatoma da APP fosforilada incluí proteínas envolvidas na regulação de sobrevivência e diferenciação, e em várias vias de sinalização, o que se correlaciona com o favorecimento de neurites nestas células. Com este trabalho esperamos ter contribuído para uma melhor compreensão do papel neurotrófico da APP e dos mecanismos subjacentes a este.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by neurofibrillay tangles and deposition of amyloid-β (Aβ) peptides. Several mutations in the gene for amyloid precursor protein (APP) cause familial AD and affect APP processing leading to increased levels of Aβ42. However, the Arctic Alzheimer mutation (APP E693G) reduces Aβ levels. Instead, the increased tendency of Arctic Aβ peptides to form Aβ protofibrils is thought to contribute to the pathogenesis.

In this thesis, the pathogenic mechanisms of the Arctic mutation were further investigated, specifically addressing if and how the mutation affects APP processing. Evidence of a shift towards β-secretase cleavage of Arctic APP was demonstrated. Arctic APP did not appear to be an inferior substrate for α-secretase, but the availability of Arctic APP for α-secretase cleavage was reduced, with diminished levels of cell surface APP in Arctic cells. Interestingly, administration of the fatty acid docosahexaenoic acid (DHA) stimulated α-secretase cleavage and partly reversed the effects of the Arctic mutation on APP processing.

In contrast to previous findings, the Arctic mutation generated enhanced total Aβ levels suggesting increased Aβ production. Importantly, this thesis illustrates and explains why measures of both Arctic and wild type Aβ levels are highly dependent upon the Aβ assay used, with enzyme-linked immunosorbent assay (ELISA) and Western blot generating different results. It was shown that these differences were due to inefficient detection of Aβ oligomers by ELISA leading to an underestimation of total Aβ levels.

In conclusion, the Arctic APP mutation leads to AD by multiple mechanisms. It facilitates protofibril formation, but it also alters trafficking and processing of APP which leads to increased steady state levels of total Aβ, in particular at intracellular locations. Importantly, these studies highlight mechanisms, other than enhanced production of Aβ peptide monomers, which could be implicated in sporadic AD.

Neben aktivierten Astrozyten weisen zahlreiche Zytokine und Wachstumsfaktoren, die in der Nähe amyloidogener Ablagerungen im Hirn des Alzheimer-Patienten auftreten, auf eine Alzheimer-assoziierte Neuroinflammation hin, die an der Ausprägung der Neurodegeneration ursächlich beteiligt sein kann. Bisher ist die Bedeutung aktivierter Astrozyten für die Alzheimer-Pathogenese wenig untersucht worden. Von besonderem Interesse war in diesem Zusammenhang das interzelluläre Adhäsionsmolekül ICAM-1, da es mit amyloidogenen Ablagerungen und Astrozyten assoziiert vorliegt. Um einen ersten Hinweis auf eine mögliche pathophysiologische Rolle des ICAM-1 in der Alzheimer-assoziierten Neuroinflammation zu erhalten, wurde im ersten Teil der vorliegenden Arbeit die astrozytäre ICAM-1-Expression in Abhängigkeit unterschiedlicher Mitogene untersucht, die für die Alzheimer-Pathogenese von Bedeutung sind. Aus diesen Untersuchungen ging hervor, daß Astrozyten, die mit den Zytokinen IL1[beta], TNF[alpha] und TNF[alpha] mit IFN[gamma] behandelt wurden, ihre ICAM-1-Expression verstärkten. Es konnte erstmals gezeigt werden, daß eine Erhöhung der intrazellulären cAMP-Konzentration durch Forskolin, Rolipram und Prostglandine die Zytokin-verstärkte ICAM-1-Expression verminderte. Allerdings konnte eine schon bestehende astrozytäre Aktivität durch cAMP-erhöhende Substanzen nicht beeinflußt werden. Im Rahmen weiterer Untersuchungen wurde in dieser Arbeit gezeigt, daß die astrozytäre ICAM-1-Expression nicht durch das Amyloid[beta]-Protein ausgelöst oder verstärkt wird, dagegen konditionierte Medienüberstände A[beta]-aktivierter Mikroglia einen starken Einfluß auf die astrozytäre Aktivität ausüben. Im zweiten Teil dieser Arbeit ist die Untersuchung aktivierter Astrozyten in der Alzheimer-assoziierten Amyloidogenese beschrieben, die im Zusammenhang mit der amyloidogenen Prozessierung des Amyloid-Vorläuferproteins APP steht. Bisher standen Neurone als Hauptproduzenten des Amyloid [beta]-Proteins im Vordergrund der Alzheimer-Forschung. Da jedoch verschiedene Veröffentlichungen darauf hinweisen, daß Astrozyten ebenfalls eine direkte Rolle in der Entstehung der amyloidogenen Ablagerungen spielen können, wurde die amyloidogene APP-Prozessierung aktivierter Astrozyten untersucht. Neben vergleichenden Analysen der astrozytären und neuronalen APP-Prozessierung konnte im Rahmen dieser Arbeit erstmals gezeigt werden, daß sich die APP-Prozessierung aktivierter Astrozyten zugunsten des nicht-amyloidogenen Prozessierungsweges verlagert, d.h. aktivierte Astrozyten verringerten die Sekretion des A[beta]-Proteins. Ein charakteristisches Merkmal aktivierter Astrozyten ist eine verstärkte [alpha]-Sekretaseaktivität bei der APP-Prozessierung. Astrozyten, die mit den Zytokinen IL1[beta], TNF[alpha] und TNF[alpha] mit IFN[gamma] aktiviert wurden, akkumulierten nicht-amyloidogene C-terminale Fragmente, sekretierten verstärkt sekretorisches APP nach [alpha]-Sekretaseaktivität und zeigten eine verringerte A[beta]-Sekretion bei gleichzeitig verstärkter Sekretion des nicht-amyloidogenen p3-Fragmentes. Abschließend wurde die pathophysiologische Bedeutung der Astrozyten in der Alzheimer-Pathogenese im Zusammenhang mit den erzielten Ergebnissen aus dieser Arbeit und mit den Befunden anderer Forschungsgruppen diskutiert und ein Modell über die zellulären, neuroinflammatorischen Vorgänge in der Alzheimer-Pathogenese entwickelt.
Activated astrocytes, number of cytokines and growth factors are associated with amyloidogenic deposits in the brain of Alzheimer-patients, pointed to an Alzheimer-associated neuroinflammation, that could be involved in the progression of the neurodegeneration. So far the implication of activated astrocytes in the pathogenesis of Alzheimer's disease is not clear. In this connection the adhesion molecule ICAM-1 was of particularly interest, because it is associated with astrocytes and amyloidogenic deposits. To get insights in a possible pathophysiological role of ICAM-1 in the Alzheimer-associated neuroinflammation, in the first part of this thesis the astrocytic expression of ICAM-1 was investigated dependent on different mitogens, that are relevant to the pathogenesis of Alzheimer's disease. It was shown that the expression of ICAM-1 was enhanced in astrocytes treated with the cytokines IL1[beta], TNF[alpha] and the combination TNF[alpha] with IFN[gamma]. It was shown that an elevation of intracellular concentration of cAMP by forskolin, rolipram and prostaglandines results in an suppressed cytokine-stimulated ICAM-1 expression. But it was impossible to reduce an existing astrocytic activity by these cAMP-elevating agents. Within the scope of additional investigations it was shown that the expression of ICAM-1 was not induce by the amyloid [beta]-peptide, whereas the ICAM-1 expression was strongly enhanced by conditioned media of A[beta]-activated microglia. In the second part of this thesis the investigation of activated astrocytes in the Alzheimer-associated amyloidogenesis is described, the astrocytic processing of the amyloid precursor protein (APP). So far it is believed that the neurons are the main producers of the amyloid [beta]-peptide. But different publications pointed to the possibility that astrocytes could play a direct role in the generation of the amyloidogenic deposits. From this reason the amyloidogenic processing of APP was investigated in activated astrocytes. Besides comparative study of astrocytic and neuronal APP processing it was first shown that activated astrocytes practise more the non-amyloidogenic pathway of APP processing. A characteristic hallmark of activated astrocytes is an increased activity of [alpha]-secretase. Astrocytes that are activated by the cytokines IL1[beta], TNF[alpha] or TNF[alpha] with IFN[gamma] showed an accumulation of non-amyloidogenic C-terminal fragments, increased APP-secretion followed by [alpha]-secretase activity, decreased A[beta]-secretion and enhanced p3-secretion. In conclusion the pathophysiological role of astrocytes in the pathogenesis of Alzheimer's disease was discussed leading to a model of cellular neuroinflammatory events in the pathogenesis of Alzheimer's disease.

La Sostanza P (SP) è un neuropeptide di 11 aminoacidi, membro della famiglia delle tachichinine, ampiamente distribuita nel sistema nervoso centrale ed ha un ruolo funzionale sia in condizioni fisiologiche che patologiche, tra le quali le malattie neurodegenerative (Raffa, 1998; Severini et al., 2002). Alterati livelli di SP sono stati riscontrati nelle regioni corticali di tessuti cerebrali di pazienti malati di Alzheimer (AD) (Quigley and Kowall, 1991; Waters and Davis, 1997). Recentemente, è anche stata riportata una riduzione della SP nella corteccia cerebrale, nell’ippocampo, nei gangli basali e nel fluido cerebrospinale dei pazienti malati di AD, il che indica un possibile ruolo svolto dalla SP in questa patologia (Jiménez-Corral et al., 2006). Sulla base di numerose evidenze in vitro e in vivo che dimostrano il ruolo neuroprotettivo della SP, abbiamo utilizzato un modello di granuli cerebellari di ratto in vitro (CGCs). In condizioni di basse concentrazioni di K+ questi neuroni vanno incontro a morte neuronale per apoptosi con attivazione del processamento amiloidogenico dell’APP, mimando così i meccanismi molecolari che avvengono in vivo nella patologia di AD. Dati recenti hanno evidenziato che sostanze in grado di stimolare gli enzimi dell’α-secretasi (ADAM) e quindi la promozione della via non amiloidogenica dell’APP potrebbero avere una potenziale attività terapeutica nella patologia. Scopo del presente lavoro è stato quello di valutare il possibile effetto della SP sul processamento proteolitico dell’APP, con particolare interesse verso la via dell’α-secretasi. Abbiamo trovato che la SP, ad una concentrazione di 200nM, protegge i CGCs dalla morte per apoptosi, riduce la produzione di strutture β extracellulari e i livelli di Aβ1-42 intracellulari. Inoltre abbiamo dimostrato che la SP induce il processamento non amiloidogenico dell’APP, promuovendo la secrezione del frammento neuroprotettivo solubile APPα, aumenta l’attività enzimatica dell’α-secretasi, l’espressione genica e proteica di ADAM 9 e la maturazione di ADAM 10, senza influenzare l’espressione del precursore proteico dell’Aβ (APP) e di BACE 1, l’enzima coinvolto nel processamento amilodogenico dell’APP. In conclusione, il presente studio mette in evidenza che la SP, attivando la via non amilodogenica, potrebbe assumere un’importanza clinica come agente in grado di modificare la patologia di AD.
Substance P (SP) is an 11-aa neuropeptide, member of the tachykinins (TK) family, broadly distributed in the central nervous system and having a functional role both in physiological and pathological conditions, as in neurodegenerative diseases (Raffa, 1998, Severini et al., 2002). Altered levels of SP have been observed in the cortical regions of post-mortem brain tissues from patients with Alzheimer’s disease (AD) (Quigley and Kowall, 1991; Waters and Davis, 1997). Recently, a consistent SP reduction in the cerebral cortex, hippocampus, basal ganglia and cerebrospinal fluid of AD patients was reported, indicating a possible role of SP in the progression of this disease. On the basis of the in vitro and in vivo results demonstrating the involvement of SP in neuroprotection, we used CGCs in low K+ conditions. In this model, CGCs undergo to apoptotic cell death with activation of amyloidogenic processing of APP, mimicking molecular mechanisms that occur in vivo in AD. Recent data demonstrated that drugs that can regulate the processing of APP towards the non-amyloidogenic pathway (ADAM) may have a therapeutic potential in AD. Aim of the present work was to assess the possible effects of SP on proteolytic processing of APP, with particular interest towards the α-secretase pathway. Data of the present work demonstrate that SP, at a concentration of 200nM, protects CGCs against apoptotic cell death induced by low K+ conditions, significantly reduces the extracellular fibrils production and levels of intracellular Aβ1-42. In addiction, we demonstrate that SP increases α-secretase activity, through the secretion of the neuroprotective soluble fragment APPα, induces the activation of α-secretase enzymatic activity, ADAM 9 gene and protein expression, ADAM 10 maturation, without influencing the precursor protein expression of Aβ (APP) and of BACE 1, the enzyme involved in the amylodogenic processing of APP. In conclusion, this study demonstrates that SP, by activating the non-amyloidogenic processing of APP, may have a therapeutic potential as disease-modifying agent in AD.

Mestrado em Biomedicina Molecular
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide and the leading cause of dementia in the elderly. Abnormal processing of Alzheimer’s amyloid precursor protein (APP) and increased generation of its amyloid beta (Aβ) fragment are central events in the AD pathogenesis, propelling major studies on APP biology. APP is thought to be involved in important processes such as cell adhesion, survival, migration and differentiation. Therefore, because of the imperative need to study APP biological functions, the search for APP-binding partners has stand out. Recently in our laboratory, a plethora of putative APP-binding proteins was unraveled through the use of the Yeast Two Hybrid (YTH) system. Among them, the heparin-binding epidermal growth factor-like growth factor (HB-EGF) has emerged as an interesting target of study. HB-EGF is a heparin-binding member of the EGF family of growth factors that stimulates growth and differentiation. It has been purposed has an important trophic factor in the developing and adult central nervous system (CNS), being expressed at much higher levels than EGF in the CNS, which indicates that HB-EGF may serve as a major ligand for EGFR in neurons. HB-EGF is synthesized as a pre-pro-form of 208 amino acids in length and is expressed at the cell surface as a 20-30kDa type I transmembrane precursor, named proHB-EGF. This larger membrane-anchored precursor is then proteolytically processed, generating the mature soluble HB-EGF (sHB-EGF) that is released to the extracellular medium. Very interestingly, HB-EGF presents autocrine, paracrine and juxtacrine biological activities, with proHB-EGF evidencing unique biological characteristics distinct from sHB-EGF. In the work here described, we first validated the APP/HB-EGF interaction by yeast co-transformation, and unraveled that the interaction is not mediated by the APP intracellular domain (AICD). We further confirmed it as an in vivo interaction by GFP-Trap pull-down assays and accessed the physiological relevance of this novel interaction through co-localization and signaling studies in HeLa cells transfected with the APP-GFP and HB-EGF cDNAs. Although the functional role of the APP/HB-EGF complex was not determined, results suggest that these proteins physically and functionally interact, having potential value in regulating each other signal pathways, with a role for APP in inducing the activation of the MAPK signaling being evidenced. In addition, a putative novel interaction with a proEGF species was also detected and APP was shown to act synergistically with EGF to activate the MAPK signaling. These results deepen our understanding of the APP biology, a crucial protein in cerebral physiology and AD pathophysiology.
A Doença de Alzheimer (DA) é a doença neurodegenerativa mais prevalente a nível mundial, e a principal causa de demência na população sénior. O processamento anormal da Proteína Precursora de Amilóide de Alzheimer (PPA) e a produção aumentada do seu fragmento beta amilóide (Aβ) constituem eventos centrais na patogénese da DA, o que tem fomentado a investigação da PPA. Esta tem sido descrita como uma proteína envolvida em processos celulares determinantes, como adesão, migração, diferenciação, e sobrevivência celular. Como tal, devido à necessidade imperativa de caracterizar as suas funções biológicas, a investigação de proteínas que interagem com a PPA é de vital importância. Recentemente, diversas proteínas foram identificadas no nosso laboratório como putativos interatores da PPA, através da técnica de Yeast Two Hybrid (YTH). De entre estes, o fator de crescimento de ligação à heparina semelhante ao fator de crescimento epidermal (HB-EGF) revelou-se um interessante alvo de estudo. O HB-EGF é um membro da família do fator de crescimento epidermal (EGF) com capacidade de ligação à heparina, que se destaca pelas suas capacidades de estimular o crescimento e diferenciação celulares, tendo sido proposto como um relevante fator trófico para o desenvolvimento e manutenção do sistema nervoso central (SNC). Na verdade, o HB-EGF é mais abundante no SNC do que o próprio EGF, o que sugere que o HB-EGF é o principal ligando neuronal para o recetor do EGF (EGFR). O HB-EGF é sintetizado como um precursor de 208 aminoácidos (pre-proHB-EGF), sendo exposto na membrana celular como um precursor transmembranar de 20-30 kDa, o proHB-EGF. Esta forma é subsequentemente proteoliticamente processada, gerando um péptido solúvel que é libertado para o meio extracelular (sHB-EGF). Curiosamente, o HB-EGF apresenta atividades biológicas parácrinas, autócrinas e justácrinas. O presente trabalho teve como principal objetivo a validação da interação entre a PPA e o HB-EGF, primeiramente alcançado através da técnica de YTH, que revelou também que a interação entre as duas proteínas não é mediada pelo domínio intracelular da PPA. Esta interação foi subsequentemente confirmada por ensaios de GFP-Trap pull-down em cultura de células de mamífero, e a sua relevância fisiológica estudada através de estudos de co-localização e sinalização celulares, usando células HeLa transfectadas com os cDNAs da PPA e do HB-EGF. Apesar do papel funcional do complexo PPA/HB-EGF não ter sido determinado, os resultados obtidos sugerem que as duas proteínas interagem física e funcionalmente, influenciando a sinalização mediada por cada uma delas separadamente, como a ativação da via de sinalização MAPK que verificámos ser também induzida pela PPA. Adicionalmente, descrevemos uma nova interação proteica entre a PPA e uma forma precursora do EGF, e demonstrámos que a PPA atua de forma sinérgica com o EGF. Estes resultados levam a uma maior compreensão da biologia da PPA, uma proteína importante na fisiologia cerebral e na fisiopatologia da DA.

Mestrado em Biomedicina Molecula
Among different types of cell-cell interactions present in the body, the interaction between the oocyte and a sperm cell is of great importance for species perpetuation. Cellular adhesion is thus a crucial process in fertilization, which involves the oocyte’s zona pellucida (ZP), the first barrier found by sperm cells that mediates the first contact between gametes. ZP3 is a major ZP glycoprotein of the oocyte, and its ligand in sperm cells is still unknown. The APP glycoprotein was found by us to be in the sperm’s head, including its equatorial zone involved in sperm-oocyte interaction. This protein plays a role in the pathogenesis of the Alzheimer’s disease and its physiologic roles are still being unravelled. The present work aimed to characterize a putative APP-ZP3 interaction in cell-cell adhesion. Relevant functional motifs on the ZP3 amino acid sequence were analysed, and the role of ZP3 and APP in cell-cell adhesion was evaluated. Interference assays were performed with antibodies against epitopes of APP, ZP3 and β1-integrin, the latter an important protein in cellular adhesion known to bind APP. The subcellular co-localization of ZP3 and APP was analysed by immunocytochemistry assays, and a potential physical interaction between ZP3 and APP was evaluated by immunoprecipitation. Results indicate that, from the studied proteins, APP and β1-integrin are the most important in cell-cell adhesion, probably through a common pathway. ZP3 has only a minor effect in cell-cell adhesion, but is able to interfere with adhesion mediated by APP and β1-integrin, potentially sharing their common pathway. Surprisingly, ZP3 and APP only co-localize at low quantities in vesicles, mainly near the Golgi apparatus, while there is a slightly higher degree of co-localization between ZP3 and β1-integrin near the plasma membrane. Unexpectedly though, ZP3 and APP were found to co-localize in extracellular aggregates of secreted ZP3. Immunoprecipitation results so far further suggest that highly glycosylated ZP3 and APP forms physically interact, what may reflect an important interaction between these proteins in the oocyte’s ZP upon fertilization.
Entre os diversos tipos de interações entre células que ocorrem no organismo, a interação entre o oócito e o espermatozoide é de grande importância para a perpetuação da espécie. A adesão celular é então um processo fundamental para a fertilização, a qual envolve a zona pelúcida (ZP) dos oócitos, a primeira barreira encontrada pelos espermatozoides que medeia a primeira interação entre os gâmetas. A ZP3 é uma das principais proteínas da ZP, e ainda não se conhece qual o seu ligando no espermatozoide. A glicoproteína APP foi descrita por nós como estando presente na cabeça do espermatozoide, em particular na zona equatorial, envolvida na interação espermatozoide-oócito. Esta proteína é central à patogénese da Doença de Alzheimer e os seus papéis fisiológicos ainda não estão bem caracterizados. Este trabalho focou-se na caracterização de um possível interação entre a APP e a ZP3 na adesão célula-célula. Foi realizado um levantamento de motivos funcionais relevantes no ZP3, e avaliado o papel deste e da APP na adesão célula-célula. Para tal foram realizados ensaios de interferência com anticorpos contra epitopos da APP, da ZP3, e também da β1-integrina, um proteína importante na adesão celular e que se sabe ligar à APP. A co-localização subcelular entre a ZP3 e a APP foi analisada por imunocitoquímica, e uma possível interação física entre a ZP3 e APP monitorizada através de ensaios de imunoprecipitação. Os resultados indicam que, de entre as proteínas estudadas, a APP e a β1-integrina são as mais importantes na adesão celular, provavelmente através de uma via comum. A ZP3 parece ter apenas um pequeno efeito na adesão célula-célula, possivelmente através da mesma via da APP e da β1-integrina. Surpreendentemente, ZP3 e APP apresentam pouca co-localização, e especialmente em vesiculas perto do complexo de Golgi. Contudo, existe um maior grau de co-localização entre a ZP3 e a β1-integrina, particularmente perto da membrana plasmática. Inesperada foi também a presença de agregados extracelulares de ZP3 secretada contendo também APP. Os resultados preliminares dos ensaios de imunoprecipitação sugerem uma interacção física entre as formas altamente glicosiladas da APP e da ZP3, o que pode refletir uma importante interação destas duas proteínas na ZP do oócito aquando da fertilização.

La maladie d'Alzheimer (AD) est un désordre neurodégénératif progressif et la forme la plus commune de démence. A l’heure actuelle, il n'y a aucun remède et la maladie est toujours fatale. Une des caractéristiques histopathologiques de l'AD est la présence de dépôts protéiques, les plaques amyloïdes, dans le cerveau. Ces plaques sont formées par les peptides amyloïdes β (Aβ) de 40 et 42 résidus, qui sont les produits de clivage par des protéases de la protéine précurseur de l’amyloïde (l'APP). L'élucidation de certains des processus clés dans la cause et le développement de l'AD est une étape cruciale pour le développement de traitements nouveaux et efficaces.

Les propriétés conformationnelles du segment transmembranaire (TM) de l’APP peuvent affecter sa protéolyse par la γ-sécrétase. Ces propriétés ne sont pas encore clairement établies. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, des détails structurels des peptides APP_TM4K, chimiquement synthétisés, ont été étudiés dans la bicouche lipidique en utilisant la réflexion totale atténuée par spectroscopie infrarouge à transformée de Fourier (ATR-FTIR) et la résonance magnétique nucléaire à l’état solide (ssNMR). Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, une hétérogénéité conformationnelle et orientée a été observée pour le site de clivage γ et, dans une plus faible mesure, pour le site de clivage ζ. Ces variabilités conformationnelles autour des sites de clivage γ et ζ peuvent avoir des implications importantes dans le mécanisme de clivage et donc dans la production d’Aβ. Il a été aussi démontré que la dernière glycine dans le motif de dimérisation GxxxG est transmembranaire. Ceci peut impliquer que la dimérisation via ce motif pourrait servir d’ancrage et conférer une orientation transmembranaire stable au segment transmembranaire de l’APP.

Le peptide amyloïde β est directement lié à la maladie d’Alzheimer. Partant de sa forme monomérique, l’Aβ s'agrège pour produire en final des fibrilles et aussi de manière transitoire toute une gamme d'oligomères, ces derniers étant la plupart neurotoxiques. Une dérégulation de l’homéostasie du Ca2+ dans le cerveau vieillissant et dans des troubles neurodégénératifs joue un rôle crucial dans de nombreux processus et contribue au dysfonctionnement et à la mort cellulaire. Nous avons postulé que le calcium peut permettre ou accélérer l'accumulation d'Aβ. Le modèle d'accumulation d'Aβ (1-40) et celui d'Aβ (1-40) E22G, un peptide amyloïde portant la mutation arctique qui cause une apparition prématurée de la maladie, ont été comparé. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères semblables à ceux formés par l’Aβ (1-40) E22G avec ou sans Ca2+, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Les ressemblances morphologiques entre oligomères ont été confirmées par microscopie de force atomique. La distribution des oligomères et des fibrilles dans des échantillons différents a été détectée par électrophorèse sur gel suivie d’une analyse par Western blot, dont les résultats ont été confirmés par des expériences de fluorescence à la thioflavine T. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. En général, ces résultats nous ont ameré à conclure que les ions calcium stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans la pathogénèse d'AD.

Malgré les progrès énormes obtenus dans la compréhension de la maladie (AD), il reste un défi majeur, celui du développement de médicaments nouveaux et efficaces. Afin d’obtenir des éclaircissements sur le mécanisme d'action de deux nouveaux puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen dans la bicouche lipidique, la technique de RMN à l’état solide a été employée. Précédemment, les dérivés du carprofen ont été localisés dans des membranes de lipides par des expériences de diffusion (scattering) des neutrons. Les contraintes déterminées à partir des expériences de ssNMR ont permis d’affiner leurs positions et d’obtenir une orientation précise dans la double couche lipidique. Ces résultats combinés indiquent que le mécanisme probable de modulation du clivage par la γ-sécrétase est une interaction directe des carprofènes avec le domaine TM de l’APP. Une telle interaction, empêcherait à la formation de dimères d'APP, dimérisation nécessaire au clivage séquentiel par la γ-sécrétase, diminuant ou réduisant ainsi énormément la production d’Aβ, tout particulièrement d’Aβ42.

Les résultats de ce travail apporte de nouvelles informations sur les processus clés impliqués dans l'AD; Production de l'Aβ à partir de l'APP, formation des oligomères d'Aβ et mécanisme d'action potentiel de molécules thérapeutiques. Nous pensons que ces résultats pourront permettre une meilleure compréhension de la maladie et pourront aider dans la conception de nouveaux médicaments contre cette maladie.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. There is no cure and the disease is fatal. One of the characteristic histopathological markers of AD is the presence of proteinaceous deposits, amyloid plaques, in the brain. These plaques are formed by the amyloid β-peptides (Aβ) 40- and 42-residue-long, which are protease cleavage products of the amyloid precursor protein (APP). Elucidation of some of the key processes in the cause and the development of AD is crucial for the development of new and efficient treatments.

Conformational properties of the transmembrane (TM) segment of APP may affect its proteolytic processing by γ-secretase. These properties have not been definitely established. In addressing the role of structural variations of the TM sequence in APP processing, structural details of the chemically synthesized APP_TM4K peptides within the membrane bilayers were studied using Attenuated total reflection Fourier transform spectroscopy (ATR-FTIR) and solid-state nuclear magnetic resonance (ssNMR) techniques. While the overall secondary structure of the APP_TM4K peptide is an α-helix, conformational and orientational heterogeneity was observed for the γ-cleavage site and, to a smaller extent, for the ζ-cleavage site. Evidence for the conformational variability around γ- and ζ-cleavage sites may have important implications for the cleavage mechanism and hence for the Aβ production. It was also found that the last glycine within the sequence of GxxxG motifs is in the transmembrane orientation, implying that dimerization via these motifs may act as an anchor, confining the TM dimer to the stable transmembrane orientation.

Amyloid β-peptide is directly linked to AD. Starting from its monomeric form, Aβ aggregates into fibrils and / or oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. The aggregation pattern of Aβ(1-40) and of Aβ(1-40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease, were compared. We found that in the presence of Ca2+, Aβ(1-40) preferentially formed oligomers similar to those formed by Aβ(1-40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1-40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy (AFM) imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1-40), that have been implicated in the pathogenesis of AD.

Despite the tremendous progress in understanding AD, there remains the challenge of the development of new and efficient drugs. In order to shed light onto the mechanism of action of two new potent γ-secretase modulators -- benzyl-carprofen and sulfonyl-carprofen within lipid bilayers, ssNMR technique was employed. Using neutron scattering experiments it was previously found that sulfonyl-carprofen and benzyl-carprofen partition into the headgroup region of the lipid bilayer. The orientational constraints derived from the ssNMR experiments refined their position into precise orientation. Combined, these results indicate that carprofen-derivatives can directly interact with the region of APP that mediates dimerization. Such interaction, would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by γ-secretase, diminishing or greatly reducing Aβ42 production.

Results obtained during this work shed new light onto some of the key processes in AD: Aβ production from APP, formation of Aβ oligomers and insights into the mechanism of action of potential therapeutics. We believe that these results will promote a better understanding of the disease and will help in future drug design.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP)
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP).
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

En plus d'être clivée par les β- et les γ-sécrétases lors du processus amyloïdogénique de la maladie d'Alzheimer, l'APP (Amyloid Precursor Protein) peut également subir un clivage grâce à l'α-sécrétase qui conduit à la libération des fragments d'APP soluble α (sAPPα)(voie non-amyloïdogénique) prévenant ainsi l'accumulation des peptides β-amyloïdes pathogènes. Les études sur le clivage de l'APP par l'α-sécrétase ont montré que l'activité de cette enzyme était constitutive mais aussi régulée. Lors de mon travail de thèse, nous avons montré que l'expression du récepteur de la sérotonine de type 4 (R 5-HT4) favorise la coupure constitutive de l'APP par l'α sécrétase ADAM10 et la libération des fragments non-amyloïdogéniques, sAPPα, aussi bien dans les cellules HEK-293 que dans les neurones corticaux en culture primaire. Ce mécanisme est totalement indépendant de la production d'AMPc, mais reste dépendant d'une interaction entre le R 5-HT4, l'APP et la forme mature de l'ADAM10. Le R 5-HT4, contrairement à d'autres récepteurs couplés aux protéines G (RCPG) décrits pour promouvoir la libération de sAPPα, est en effet capable d'interagir physiquement avec l'α-sécrétase ADAM10 et son substrat, l'APP. Cette interaction, directe ou indirecte, favorise l'adressage de l'ADAM10 et de l'APP à la membrane plasmique, là où la coupure en α est majoritaire. Toute rétention du R 5-HT4 à l'intérieur de la cellule retient également l'APP et l'ADAM10 sous sa forme inactive et annule la production de sAPPα. La régulation de l'α-sécrétase est toujours dépendante de l'activation du R 5-HT4 par un agoniste. Cet effet étant cette fois dépendant de la voie de signalisation de l'AMPc et de la protéine Epac comme cela avait déjà été démontré. Ces résultats décrivent pour la première fois un mécanisme par lequel un RCPG stimule le clivage constitutif de l'APP par l'α sécrétase et fournit de nouvelles perspectives pour la régulation de l'APP et le contrôle de l'adressage de l'α-sécrétase ADAM10
In addition to the amyloidogenic pathway of Alzheimer's disease whereby Amyloid Precursor Protein (APP) is cleaved by β- et γ-secretases, the substrate can also be cleaved by α secretases, producing soluble APP alpha (sAPPα)(non-amyloidogenic pathway) and thus preventing the generation of pathogenic Amyloid-beta peptides. Despite, intensive research, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. In this study, we tried to elucidate how 5-HT4Rs stimulate the release of sAPPα. We show that expression of serotonin type 4 receptors (5-HT4Rs) constitutively induces APP cleavage by the α-secretase ADAM10 and release of non-amyloidogenic fragments, sAPPα, in HEK-293 cells and cortical neurons. This effect is fully independent of cAMP production and relies on the transport of the 5-HT4R/APP/mature ADAM10 complex to the plasma membrane. Indeed, 5-HT4Rs but not other G protein-coupled receptors (GPCRs) known to activate sAPPα release, physically interact, directly or indirectly, with ADAM10 and APP to promote their targeting to the plasma membrane. Stimulation of 5 HT4Rs by an agonist further increases sAPPα fragments release and this effect is mediated through cAMP/Epac signalling. These findings describe a new mechanism whereby a GPCR stimulates the cleavage of APP by α-secretases and provide novel insights into the regulation of APP and α-secretase sorting

L’élargissement des endosomes précoces (EP) a été amplement décrit dans la trisomie 21 (T21) et la maladie d’Alzheimer (MA). La présence d’EP élargis a été démontrée dans des neurones avant même le développement de la neuropathologie et des symptômes de la MA. L’élargissement des EP a également été identifié dans de nombreux modèles de MA et de T21. Il a été proposé que la surexpression d’APP induise l’élargissement des EP, mais certaines observations suggèrent une origine multifactorielle à l’élargissement des EP. Malgré la petite taille des EP, leur morphologie n’a jamais été analysée à haute résolution. Dans cette étude, nous caractérisons les EP et la voie endosomale dans la T21 afin de mieux en comprendre les phénotypes et les mécanismes moléculaires associés. Grâce à différentes méthodes de microscopie électronique et super-résolutive, nous observons dans de nombreux modèles de T21 une augmentation du nombre d’EP de taille normale ayant tendance à s’agréger plutôt que des EP élargis. Nous montrons que le recyclage du récepteur à la transferrine est augmenté alors que la dégradation du récepteur à l’EGF est ralentie. Nous montrons ensuite que les niveaux de Rab5 et de PI(3)P sont augmentés et le niveau d’EEA1 est diminué. Enfin, l’inhibition de la kinase PIKfyve permet de rétablir le niveau d’EEA1 et la production d’Aβ dans des fibroblastes T21. Notre étude redéfinit les phénotypes de la voie endo-lysosomale dans la T21 aux niveaux morphologiques, dynamiques et moléculaires. Ces résultats apportent de nouvelles hypothèses mécanistiques pour expliquer les anomalies endosomales dans la T21 et la MA
Abnormal early endosome (EE) enlargement was widely characterized in the brain of individuals with Down syndrome (DS) and Alzheimer’s disease (AD) patients. Neurons bearing enlarged EE have been described before the development of the neuropathological hallmarks and clinical symptoms of AD. EE enlargement was also detected in various models of AD and DS. It was proposed that APP overexpression would lead to EE enlargement, but several lines of evidence rather suggest multifactorial causes to EE enlargement. Despite the nanometric size range of EE, this phenotype was never quantified with high resolution microscopy. Here, we characterize EE and the endo-lysosomal pathway in DS in order to better understand endosomal phenotypes and their underlying molecular mechanisms. With electron microscopy technics and super-resolution, we observe an increased number of normal-sized EE with a tendency to clustering rather than enlarged EE in various DS models. We show that the transferrin receptor recycling is accelerated, while t573.84he degradation of the EGF receptor is delayed. Then, we show that the levels of Rab5 and PI(3)P are decreased but the level of EEA1 is increased. Finally, the inhibition of the PIKfyve kinase rescues the level of EEA1 and Aβ production in fibroblasts from individuals with DS. Our study redefines phenotypes of the endo-lysosomal pathway in DS, at morphological, dynamic and molecular levels. These results provide new hypotheses to explain endosomal abnormalities in DS and AD, and bring insights into the comprehension of the role the endo-lysosomal pathway in both pathologies

La maladie d'Alzheimer est une maladie neurodégénérative, elle représente 70% des formes de démences et affecte près de 860 000 personnes en France. Cette maladie est caractérisée par deux lésions neuropathologiques : les Dégénérescences neurofibrillaires et les Plaques séniles. Ces dernières sont principalement constituées de peptides amyloïdes (Aβ) résultant du clivage d'une protéine membranaire appelée Précurseur du peptide amyloïde (APP). L'étude des formes familiales monogéniques a montré que des mutations des gènes de l'APP et des Présénilines 1 et 2 conduisaient systématiquement à une augmentation de la production d'Aβ. Cette observation a permis l'élaboration de la cascade amyloïde plaçant le métabolisme de l'APP au centre du processus physiopathologique. Même si aujourd'hui ce métabolisme commence à être relativement bien connu, plusieurs zones d'ombres subsistent encore. Dans l'optique de caractériser de nouveaux acteurs intervenant dans ce métabolisme, nous avons émis une hypothèse qui repose sur deux constatations : (i) les protéines impliquées dans l'étiologie de la maladie sont différentiellement exprimées entre les cerveaux des patients et ceux des témoins (ii) dans le cerveau, de nombreuses métalloprotéases participent aux même mécanismes que l'APP (adhésion cellulaire, neuroinflammation, plasticité neuronale...), certaines sont aussi directement actrices du métabolisme de l'APP en tant qu'α-sécrétase (ADAM9, ADAM10 et ADAM17) ou en dégradant l'Aβ (NEP, IDE, MMP2, MMP3 et MMP9). Nous avons donc supposé que les métalloprotéases présentant une différence d'expression entre le tissu cérébral des malades et celui des témoins soient des candidates intéressantes pour moduler le métabolisme et le trafic de l'APP. Une première analyse transcriptomique par biopuce, à partir d'ARN totaux issus des cerveaux de 12 malades et de 12 témoins, nous a permis d'identifier quatre métalloprotéases présentant une différence d'expression significative (p<10-5) : ADAMTS16, ADAM17, ADAM30 et ADAM33. Nous avons cherché à confirmer ce résultat par une autre technologie sur un plus grand nombre d'échantillons (malades n=52 et témoins n=42). Seules ADAM30 et ADAM33 ont pu être validées. Nous avons également pu observer que l'expression d'ADAM30 dans le tissu cérébral des malades est inversement proportionnelle à la quantité d'Aβ42 déposée dans la parenchyme (Aβ42 la forme d'Aβ la plus neurotoxique). De plus, au niveau cérébral, l'expression d'ADAM30 est restreinte aux neurones, cellules sièges du métabolisme de l'APP. Nous avons donc sélectionné ADAM30 comme intervenante potentielle dans le métabolisme de l'APP. Pour tester notre hypothèse, nous avons sous- et sur-exprimé ADAM30 dans deux modèles cellulaires différents. Nous avons mis en évidence que la sur-expression d'ADAM30 entraîne une diminution de l'ensemble des produits du métabolisme de l'APP. En mutant le site catalytique de cette protéase, nous avons remarqué que cette action sur le métabolisme de l'APP est dépendante de cette activité catalytique. De manière cohérente, une sous-expression d'ADAM30 entraîne une augmentation de l'ensemble des produits du métabolisme de l'APP. En utilisant les inhibiteurs alcalisant, nous avons démontré que l'effet d'ADAM30 sur le métabolisme de l'APP met en jeu la dégradation par le lysosome. Des expériences d'immunofluorescence ont attesté qu'ADAM30 est localisée dans le réticulum endoplasmique et l'appareil de Golgi et qu'elle co-localise fortement avec l'APP dans ces organites. Au vu des résultats obtenus durant ces quatre années, nous pensons qu'ADAM30 pourrait être une protéine clé de la régulation de l'APP en inhibant son acheminement jusqu'à la membrane plasmique et en favorisant sa dégradation par le lysosome. [...]

L’accumulation cérébrale progressive de peptides amyloïdes générés à partir du clivage du précurseur du peptide amyloïde (APP) par les sécrétases est un mécanisme central de la maladie d’Alzheimer. C’est pourquoi, améliorer la compréhension de la régulation et de l’homéostasie du métabolisme de l’APP est devenu primordial. Partant de ce constat, nous avons supposé qu’une partie de la réponse pourrait être apportée par la caractérisation de nouveaux acteurs du métabolisme de l’APP. De part leurs rôles cruciaux dans le cerveau (développement, plasticité et réparations) et dans le métabolisme de l’APP (α-sécrétases), les ADAMs sont des protéines d’intérêt dont certaines fonctions ou rôles restent à déterminer. Précédemment, par une approche transcriptomique ciblant la famille des ADAMs dans des cerveaux de patients et de contrôles, ADAM30 a été retrouvée sous-exprimée dans le cerveau des patients atteints de la pathologie. Dans deux modèles cellulaires nous avions constaté que la sous-expression d’ADAM30 entraînait une augmentation de tous les produits du métabolisme de l’APP comme chez les patients. Le résultat opposé a été obtenu lors de la sur-expression d’ADAM30 dans ces cellules. Pour tenter de répliquer ces résultats dans un modèle plus proche de la physiopathologie humaine, nous avons développé un modèle de souris triples transgéniques surexprimant l’APPSweInd et ADAM30 de manière conditionnelle. Dans ce modèle nous avons observé et mesuré une diminution des dépôts amyloïdes dans le cerveau des souris exprimant ADAM30. Dans un second temps puisqu’il avait été montré au laboratoire qu’ADAM30 ne module pas l’activité des sécrétases et ne clive pas directement l’APP, nous avons cherché à déterminer les substrats d’ADAM30 dans le cadre du métabolisme de l’APP. Par une approche systématique nous avons pu déterminer que la Cathepsine D (CTSD) et l’Insuline Receptor Substrat 4 (IRS4) sont deux substrats potentiels d’ADAM30. Dans nos modèles cellulaires et de souris, nous avons pu constater qu’ADAM30 est capable de cliver et d’activer la CTSD. L’activité de la CTSD semble nécessaire pour l’action d’ADAM30 sur le métabolisme de l’APP. Nous avons pu déterminer que l’action spécifique d’ADAM30 pour la CTSD est dépendante de la séquence d’adressage au lysosome située dans l’extrémité C-terminale de l’APP. Comme la CTSD est une protéine Lysosomale, ADAM30 pourrait favoriser spécifiquement l’activation de la CTSD augmentant ainsi la dégradation de l’APP au sein de la voie endosome/lysosome. Ce mécanisme limiterait l’entrée de l’APP dans son métabolisme et donc la production de peptides amyloïdes. Afin de mieux comprendre la spécificité d’action d’ADAM30 pour la CTSD et l’APP, nous avons commencé à travailler sur le rôle potentiel d’IRS4 et la relation entre la voie de signalisation de l’Insuline et le métabolisme de l’APP. Nos travaux nous ont donc permis de mettre en évidence un nouvel acteur du métabolisme de l’APP, ADAM30, intervenant dans la régulation et la dégradation de ce dernier et ainsi d’améliorer notre compréhension des mécanismes de régulations fins impliqués dans le processus physiopathologique de la maladie d’Alzheimer
Progressive intra-cerebral accumulation of amyloid peptides formed after sequential cleavage of the amyloid peptide precursor (APP) by secretases , is a central mecanism for Alzheimer’s disease. Therefore, a better understanding of APP regulation and homeostasy is now crucial. With this background, we postulate that the characterization of new actors in the APP metabolism could provide a more subtle understanding of this APP metabolism and trafficking. From their obvious implication in brain (development, plasticity and repair) and in APP metabolism (α-secretases), ADAMs (A Disintegrin And Metalloprotease) are an important protein proteins family which still have some undetermined function or role. Previously, a transcriptomic approach targeting ADAMs family bas been done at the laboratory on Alzheimer’s patient or control brains and found ADAM30 as under-expressed in Alzheimer’s patient brains. On cellular models, we confirmed that ADAM30 under-expression was associate with an increase in production/secretion of all the APP metabolim byproducts. Opposite results were found with ADAM30 over-expression. To replicate those results in another model closest to human pathophysiology, we have developed a triple transgenic mice model over-expressing APPSweInd and conditionally over-expressing ADAM30. In this model, we have observed and measured a decrease in amyloid deposits in mice brains over-expressing ADAM30. Secondly, because ADAM30 did not modulate secretase activities and did not cleave APP directly, we decided to determine ADAM30 substrats in the APP metabolism context. With a systematic approach, we have determined that Cathepsin D (CTSD) and Insulin Receptor Substrat 4 (IRS4) are two ADAM30 potential substrats. In our cellular models, we have found that ADAM30 is able to cleave and activate CTSD. This CTSD activity is required for ADAM30 action on APP metabolism. We have determined that ADAM30 specific action for CTSD is dependent on lysosome adressing sequence localised in APP C-terminal part. CTSD is a lysosomal protein and so ADAM30 would make CTSD specific activation easier. This mecanism would be able to increase APP degradation in endosome/lysosome pathway and reduce APP entry in its metabolism. To better understand ADAM30 specific action on CTSD and APP, we begin to investigate the potential role of IRS4 and the relation between insulin signaling pathway ans APP metabolism. Combined together, those data suggest that ADAM30 is a new APP metabolism actor, involved in an early APP regulation and degradation pathway dependent on lysosome activation. This study participate in a better understanding of the fine mecanism regulations involved in Alzheimer’s disease pathophysiological process

Die Alzheimer-Krankheit (AD) ist gekennzeichnet durch ein massives Absterben von Neuronen in bestimmten Gehirnregionen. Die zwei charakteristischen histopathologischen Hauptmerkmale sind extrazelluläre Amyloidplaques bestehend aus dem APP-Peptidfragment Abeta und intrazelluläre Fibrillen hyperphosphorylierten Tau-Proteins. Familiäre Formen von AD (FAD) werden verursacht durch Mutationen in den beiden sehr homologen Presenilin-Genen 1 und 2 oder dem APP-Gen. Verschiedene Studien zeigen, dass ein Zusammenhang zwischen Presenilin Mutationen, Abeta-Generierung und Tau-Phosphorylierung beim Auslösen des Neuronentods vorliegt. Immer noch ungeklärt ist, inwiefern Abeta und Presenilin die Tau-abhängige Degeneration beeinflussen. In dieser Arbeit wird gezeigt, dass eine HSV-1-vermittelte Expression von fluoreszenzmarkiertem Tau in kortikalen Primärkulturen einen neurotoxischen Effekt ausübt. Dieser ist drastisch erhöht bei Verwendung eines Konstruktes, welches die pathologische Hyperphosphorylierung von Tau simuliert (pseudohyperphosphoryliertes Tau (PHP-Tau)). Die durch PHP-Tau induzierte Neurodegeneration ist assoziiert mit einer Induktion apoptotischer Mechanismen. Die transgene Expression von wildtyp (wt), aber nicht von FAD-mutiertem PS1 (M146L), unterdrückt PHP-Tau-induzierte Neurodegeneration. Dagegen erhöht die transgene Expression mutierten APPs (SDL) die Degeneration und Phosphorylierung in der Gegenwart von wt, aber nicht von PHP-Tau. Die Daten weisen darauf hin, dass wt und FAD-mutiertes PS1 sowie Abeta die Neurodegeneration durch differentielle Mechanismen modulieren, wobei die Hyperphosphorylierung von Tau entscheidend beteiligt ist. Abeta amplifiziert die Tau-induzierte Neurodegeneration durch die erhöhte Modifikation von Tau. Während PS1 wt der Neurodegeneration durch modifiziertes Tau entgegenwirkt, besitzt FAD-mutiertes PS1 diese Funktion nicht mehr. Demnach könnte die Unterdrückung der Tau-Phosphorylierung eine effektive Therapiemöglichkeit darstellen.

The present thesis focuses on deciphering the biochemical sequelae responsible for the precipitation of Alzheimer's disease (AD). The hallmark occurrence in AD is the assembly of Abeta (a normally soluble component of cerebrospinal fluid) into insoluble, extracellular plaques. The genetics of AD and recent observations of partial AD phenotype (i.e. Abeta plaque deposits) in transgenic mice overproducing Abeta indicate that Abeta amyloid deposition in the neocortex is intimately involved in the pathophysiology of the disorder. Hence, neurochemical candidates, which may impact the solubility of Abeta, were investigated. Recent studies conducted by our laboratory show that Abeta is a physiological metalloprotein, saturably binding zinc and copper. In fact, the interaction of Abeta with physiological concentrations of zinc, copper, and iron generate amyloid plaques in vitro, which can be resolubilized by the extraction of metal ions via chelation. Extending these findings, a vertically innovative approach was used to determine whether our in vitro findings are germane to AD pathology First, we investigated whether plaque deposited preferentially within cerebral regions enriched in zinc and copper. It was observed that brain regions targeted by AD (e.g. the hippocampal formation) experience drastic fluxes in metal ion concentrations, which may rise as much as 300muM in the case of zinc. In particular, it was noted that subdivisions of the inferior pulvinar that receive putative zinc-enriched projections from the primary visual cortex contain Abeta amyloid plaques. Interestingly, the region most affected in the thalamus is known to mediate visual attention which is compromised in AD Based on our above in vitro findings, the ability of metal chelators to resolubilize Abeta aggregates from the brains of transgenic mice overexpressing the human amyloid precursor protein (APP) and bearing cerebral amyloid was assessed. We noted that homogenization of APP transgenic mouse brains in the presence of metal chelators highly specific for zinc and copper significantly increased extractable Abeta (p < 0.01) as compared to homogenization in the presence of phosphate buffered saline alone. These findings suggest that the assembly of plaques in APP transgenic mouse brains appeared to parallel that in human, providing an exploitable model of AD amyloidosis Finally, United States Pharmacopeia (USP) drugs (i.e. clioquinol and triene), having zinc and copper chelating abilities, were administered daily to APP transgenic mice at 12 months of age. After 3 months of drug therapy, it was observed that animals receiving clioquinol, a chelator highly specific for zinc, were healthy and had more than a 50% reduction in Abeta load (p < 0.05) and a 60% decrease in sedimentable Abeta (p < 0.01). There was also a corresponding decrease (p < 0.05) in the progenitor pool of Abeta species, namely the APP beta-carboxyl terminal fragment concentration as measured by Western blot analysis. These findings suggest that chelation therapy may alter APP processing, thereby arresting Abeta production. Thus, metal chelators are one class of compounds that may be highly effective for the treatment of AD
acase@tulane.edu

Indiana University-Purdue University Indianapolis (IUPUI)
Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ-generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD.

Alzheimer's disease is the most common type of dementia in the elderly; it is characterized by early deficits in learning and memory formation and ultimately leads to a generalised loss of higher cognitive functions. While amyloid beta (Aβ) and tau are traditionally associated with the development of Alzheimer disease, recent studies suggest that other factors, like the intracellular domain (APP-ICD) of the amyloid precursor protein (APP), could play a role. In this study, we investigated whether APP-ICD could affect synaptic transmission and synaptic plasticity in the hippocampus, which is involved in learning and memory processes. Our results indicated that overexpression of APP-ICD in hippocampal CA1 neurons leads to a decrease in evoked AMPA-receptor and NMDA-receptor dependent synaptic transmission. Our study demonstrated that this effect is specific for APP-ICD since its closest homologue APLP2-ICD did not reproduce this effect. In addition, APP-ICD blocks the induction of long term potentiation (LTP) and leads to increased of expression and facilitated induction of long term depression (LTD), while APLP2-ICD shows neither of these effects. Our study showed that this difference observed in synaptic transmission and plasticity between the two intracellular domains resides in the difference of one alanine in the APP-ICD versus a proline in the APLP2-ICD. Exchanging this critical amino-acid through point-mutation, we observed that APP(PAV)-ICD had no longer an effect on synaptic plasticity. We also demonstrated that APLP2(AAV)-ICD mimic the effect of APP-ICD in regards of facilitated LTD. Next we showed that the full length APP-APLP2-APP (APP with a substitution of the Aβ component for its homologous APLP2 part) had no effect on synaptic transmission or synaptic plasticity when compared to the APP-ICD. However, by activating caspase cleavage prior to induction of LTD or LTP, we observed an LTD facilitation and a block of LTP with APP-APLP2-APP, effects that were not seen with the full length APLP2 protein. APP is phosphorylated at threonine 668 (Thr668), which is localized directly after the aforementioned critical alanine and the caspase cleavage site in APP-APLP2-APP. Mutating this Thr668 for an alanine abolishes the effects on LTD and restores LTP induction. Finally, we showed that the facilitation of LTD with APP-APLP2-APP involves ryanodine receptor dependent calcium release from intracellular stores. Taken together, we propose the emergence of a new APP intracellular domain, which plays a critical role in the regulation of synaptic plasticity and by extension, could play a role in the development of memory loss in Alzheimer’s disease.
La maladie d’Alzheimer est la forme la plus commune de démence liée au vieillissement ; elle est caractérisée par des déficits précoces d’apprentissage et de mémorisation et entraîne à terme une perte généralisée des fonctions cognitives supérieures. Alors que l’amyloïde-bêta (Aβ) et la protéine tau sont traditionnellement associées au développement de la maladie d’Alzheimer, des études récentes suggèrent que d’autres facteurs, tels que le domaine intracellulaire (APP-ICD) du précurseur de la protéine amyloïde (APP), pourraient jouer un rôle. Dans notre étude, nous avons testé si l’APP-ICD pourrait affecter les mécanismes de transmission ou de plasticité synaptique dans l’hippocampe, qui sous-tendent les processus d’apprentissage et de mémorisation. Nos résultats ont indiqué que la surexpression de l’APP-ICD dans des neurones du CA1 de l’hippocampe entraînait une diminution de la transmission synaptique dépendante des récepteurs AMPA et NMDA. Notre étude a montré que cet effet était spécifique de l’APP-ICD puisque son plus proche homologue l’APLP2-ICD n’a pas eu cet effet. De plus, l’APP-ICD entraînait un blocage de la potentialisation à long terme (LTP), une augmentation de l’expression et une facilitation de l’induction de la dépression à long terme (LTD), mais l’APLP2-ICD n’a eu aucun de ces effets. Notre étude a montré que cette différence observée en transmission et en plasticité synaptique entre les deux peptides réside dans le changement d’une seule alanine dans l’APP-ICD pour une proline dans l’APLP2-ICD, et que l’APP(PAV)-ICD n’avait pas d’effet sur la plasticité synaptique. Nous avons aussi démontré que l’APLP2(AAV)-ICD mimait l’effet de l’APP-ICD pour la facilitation de la LTD. Ensuite nous avons montré que la longue forme APP-APLP2-APP (APP avec un changement de la séquence de l’Aβ pour celle homologue de l’APLP2) ne montrait pas d’effet en comparaison avec l’APP-ICD, ni sur la transmission synaptique ni sur la plasticité synaptique. Cependant, en activant le clivage par les caspases préalablement à l’induction de la LTD ou la LTP, nous avons observé une facilitation de la LTD et un iii blocage de la LTP avec l’APP-APLP2-APP, des effets que nous n’avons pas reproduit avec la longue forme APLP2. La thréonine 668 phosphorylable se trouve immédiatement après l’alanine et le site de clivage par les caspases dans l’APP-APLP2-APP. La mutation de la Thr668 pour une alanine a aboli son effet sur la LTD et restauré la LTP. Finalement, nous avons montré que la facilitation de la LTD par l’APP-APLP2-APP dépendait de la libération de calcium intracellulaire par les récepteurs ryanodines. En conséquence, nous proposons l’émergence d’un nouveau domaine de l’APP jouant un rôle critique, en plus de l’Aβ, dans les processus à la base de l’apprentissage et qui en conséquence pourrait jouer un rôle dans le développement de la maladie d’Alzheimer.

Die Bildung von Amyloidablagerungen ist ein Kennzeichen der Alzheimerschen Erkrankung. Hauptbestandteil dieser senilen Plaques sind sogenannte A beta Peptide, die durch proteolytische Prozessierung aus einem Vorläufermolekül (APP) gebildet werden. Die vorliegende Arbeit beschreibt die Klonierung des Meerschweinchen - APP. Diese cDNA-Sequenz zeigt auf DNA-Ebene eine Homologie zum Human-APP von ca. 90%, auf Proteinebene beträgt die Identität ca. 97 %. Damit wird ein weiterer experimenteller Beweis für die evolutionäre Konservierung des Amyloidvorläuferproteins in Säugetieren erbracht. APP mRNA wird in Meerschweinchen-Geweben ubiquitär exprimiert. Durch alternatives Spleißen wird ein zum Human-APP im wesentlichen ähnliches Isoformenmuster gebildet: Isoformen, welche eine Proteaseinhibitordomäne enthalten, werden dominierend in peripheren Organen exprimiert, dagegen ist im Zentralnervensystem das APP 695 mit über 60 % der Gesamttranskripte die bevorzugt exprimierte Isoform. Die klonierte cDNA des Meerschweinchen-APP wurde in prokaryontischen wie auch eukaryontischen Zellsystemen exprimiert. Dabei wurde die Eignung einer Anzahl von gegen Human-APP gewonnenen Antikörpern zur Detektion des Meerschweinchen-APP und seiner Prozessierungsprodukte gezeigt. Die Expression der neuronal dominierend exprimierten Isoform APP 695 des Meerschweinchen-APP in humanen Neuroblastom-Zellen zeigte keine Unterschiede hinsichtlich der APP-Prozessierung und A beta-Bildung im direkten Vergleich zu Human-APP 695. Die proteolytische Prozessierung des Proteins wurde durch Detektion der typischen Spaltprodukte in vivo (im Liquor) als auch in einem neu etablierten in vitro-Modell primär kultivierter neuronaler Zellen untersucht. Diese Zellkulturen wurden zunächst immunhistochemisch und biochemisch charakterisiert und als "mixed brain"-Typ mit einem hohen neuronalen Anteil beschrieben. Die Prozessierung des endogenen Meerschweinchen-APP in kultivierten Zellen führt dabei zur Bildung und Akkumulation aggregationsfähiger A beta - Peptide. Zur Detektion dieser Peptide wurde ein sensitiver Nachweis durch Western-Blot etabliert. Es wird damit ein Modellsystem für in vitro-Untersuchungen vorgeschlagen, welches ein Studium der Expression und Prozessierung des Amyloidvorläuferproteins unter angenähert physiologischen Bedingungen ermöglicht.
A beta peptides, the major component of neuritic plaques found in the brains of patients with Alzheimer’s disease, are derived by proteolytic processing from a larger precursor molecule (amyloid precursor protein - APP). A combination of PCR methods was used to clone and sequence APP cDNA from guinea pig (Cavia porcellus). Guinea pig APP exhibits extensive similarities to human APP in terms of primary structure, mRNA expression of differentially spliced isoforms as shown by Northern blot and RT-PCR analysis as well as proteolytic processing to amyloidogenic A beta peptides. In contrast to rat and mouse APP, guinea pig APP - recombinantly expressed in human neuroblastoma-cells - was processed indistinguishable from human APP thus excluding intrinsic sequence-specific factors influencing processing. Further studies were performed using newly established primary cell cultures of guinea pig neurons. Refined methods have been used to detect and characterize major proteolytic processing products of APP in vitro and in vivo. In conclusion, guinea pigs provide a model to study expression and processing of APP that closely resembles the physiological situation in humans and should, therefore, be important in elucidating potential strategies to prevent amyloid formation in Alzheimers Disease.