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Das Amyloid-Vorläufer-Protein (APP) ist ein integrales Membranprotein, welches in neuronalen und nicht neuronalen Geweben des Menschen exprimiert wird. Neben seiner bekannten Relevanz in der Pathogenese der Alzheimer-Erkrankung nimmt es eine weitere physiologische Rolle ein: APP ist ein Wachstumsfaktor. Diese Funktion machen sich auch Karzinomzellen zunutze. APP wird in Kolon-, Pankreas- und weiteren Karzinomzelllinien überexprimiert. Durch den HDAC-Inhibitor Valproat kann die Proliferation von Tumorzellen verringert werden. Dabei führt die Behandlung der Zellen mit Valproat zu einer Hyperacetylierung des Histons H4 und damit verbunden auch zu einer erhöhten Konzentration des Chaperon GRP78. Dieses hemmt die APP-Reifung, indem es das unreife APP bindet. Die intrazelluläre APP-Konzentration sinkt und die extrazellulären wachstumsfördernden Spaltprodukte wie sAPPα werden vermindert sezerniert. In der Folge wird die Proliferation der Tumorzellen signifikant inhibiert. Die Identifikation neuer und potenter HDAC-Inhibitoren hat eine große Relevanz, um neue Therapiestrategien in der Krebstherapie zu ermöglichen und die Überlebenschancen von Tumorpatienten zu verbessern. In der vorliegenden Arbeit wurde die Wirkung der Histon-Deacetylase-Inhibitoren Apicidin, Scriptaid, Sirtinol sowie APHA-Compound 8 auf die Tumorzelllinien Sw480 (Kolonkarzinom) und BxPc3 (Pankreaskarzinom) getestet. In der vorliegenden Arbeit konnte erstmals gezeigt werden, dass alle vier getesteten HDAC-Inhibitoren die Proliferation der untersuchten Tumorzellen hemmen. Die getesteten Medikamente wirkten dabei ohne Ausnahme in geringeren Konzentrationen auf Sw480 und BxPc3 als Valproat. Apicidin, Scriptaid und Sirtinol inhibierten zudem die Reifung von APP. In der Western-Blot-Analyse zeigte sich, dass die Konzentration des Amyloid-Vorläufer-Proteins nach Zugabe der Medikamente sank. Die Immunfluoreszenz bestätigte, dass Apicidin die APP-Konzentration in Sw480-Zellen konzentrationsabhängig vermindert. Dieses Ergebnis stützt die These, dass der Wachstumsfaktor APP auch durch die getesteten HDAC-Inhibitoren gehemmt wird. Zudem konnte in der Western-Blot-Analyse der molekulare Mechanismus der Medikamente, welcher mit dem von Valproat identisch ist, verifiziert werden. Histon H4 zeigte sich erwartungsgemäß hyperacetyliert, GRP78 wurde hochreguliert und APLP2 als APP-homologes Protein blieb unverändert. Eine Ausnahme stellt der HDAC-Inhibitor APHA-Compound 8 dar. Dieser inhibierte zwar die Proliferation der Zellen und in der Western-Blot-Analyse konnte eine starke Hyperacetylierung von Histon H4 dargestellt werden. Dennoch konnte in der Western-Blot-Analyse weder eine Inhibition des reifen noch des unreifen APP nachgewiesen werden. Zusammenfassend zeigte sich, dass Apicidin und Scriptaid das Amyloid-Vorläufer-Protein in niedriger Dosierung inhibieren können. Somit könnten die Medikamente in Zukunft in der Therapie von APP-positiven Tumoren und besonders im Fall der getesteten Karzinome neue Behandlungsoptionen bieten und das Spektrum der Behandlung erweitern.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Heat shock proteins function as molecular chaperones which help protein folding and prevent protein aggregation. My study shows that celastrol, a pharmacological compound capable of up-regulating the levels of heat shock proteins, inhibits cell death and protein aggregation caused by expanded polyglutamine containing protein, and the protective effects of celastrol are dependent on heat shock factor 1. These results suggest the potential of celastrol as a therapeutic agent in the treatment of polyglutamine diseases. Sumoylation is a protein modification which plays diverse roles in regulating the target proteins. My study shows that lamin A is a target of protein sumoylation, and two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. My results also indicate that sumoylation is important for the normal localization of lamin A, and support a role for altered sumoylation in the underlying molecular mechanism of cardiomyopathies associated with the E203G/E203K lamin A mutations. In the third project, my results show that amyloid precursor protein is another target of SUMO modification, and sumoylation of amyloid precursor protein reduces the levels of amyloid β aggregates, which are the primary causative factor for Alzheimer’s disease. My results provide a new mechanism for the generation of amyloid β, and indicate the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer’s disease. My results also provide the first demonstration that SUMO E2 enzyme exists in the lumen of the endoplasmic reticulum, extending the sub-cellular reach of sumoylation to include the regulation of proteins in secretory pathways.

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 (A&#946;) 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’A&#946;. 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’&#945;-sécrétase (ADAM9, ADAM10 et ADAM17) ou en dégradant l’A&#946; (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’A&#946;42 déposée dans la parenchyme (A&#946;42 la forme d’A&#946; 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. [...] / Alzheimer’s disease (AD) is the most common neurodegenerative disorder of the old age, characterized by the presence of two major neuropathological features : neurofibrillary tangles and senile plaques. These plaques are composed of the A&#946; peptides cleavage product of the amyloid precursor protein (APP). Proteolytic processing of APP is modulated by the action of enzymes &#945;-, &#946;- and &#947;-secretases with the latter two mediating the amyloidogenic pathway. Suggesting that processing of APP is a key step in the pathology of AD. However, even if extensively studied, this APP metabolism is still not fully characterized. With this background, we postulate that the characterization of new actors of the APP metabolism might help for a more subtle understanding of this APP metabolism and trafficking. We focused on the ADAMs and related proteins with the hypothesis that ADAMs and related proteins, under- or over-expressed in the brain of AD cases compared with the one of controls, may be of particular interest. Beyond the obvious implication of several ADAMs as &#945;-secretases, this hypothesis was also driven by several observations : (i) ADAMs have been involved in numerous biological processes including brain development, plasticity and repair as APP; (ii) several metalloproteases (MMP-2, -3 and -9) have been described to degrade A&#946; peptides. Using microarray to screen the expression of 117 ADAMs and MMPs was analyzed using total RNA extracted from cerebral tissue of 12 AD cases and 12 controls. We observed that 4 ADAMs were differentially expressed. We first confirmed that the ADAM30 expression was decreased in AD brains and we observed that ADAM30 under-expression was correlated with an increase in A&#946;42 deposition in AD brains. Consistently, over-expression of ADAM30 led to decrease APP metabolism and as a consequence, A&#946; secretion in two different cell lines (Moreover, under-expressed ADAM30 increases APP processing and A&#946; generation). This modification of the APP metabolism was directly linked to the ADAM30 catalytic properties. Our data suggest that catalytic activity of ADAM30 takes an important place in APP processing in a lysosome dependent manner and AD pathophysiological process.

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

The present study evaluated olfactory and cognitive abilities of the Tg6799 (also called 5xFAD) strain of Alzheimer’s disease (AD) model mice of two different age groups (2-3 and 8-10 months of age), and one group of healthy control mice (9-10 months). Employment of an operant conditioning paradigm using an automated olfactometer, an olfactory habituation/dishabituation test and a spatial learning test with non olfactory cues resulted in data showing that the 5xFAD mice develop olfactory impairments already at 2-3 months of age. The impairments consisted in a robust impairment in olfactory sensitivity, decreased responsiveness to novel odors and an inability to discriminate between enantiomeric odor molecules in the 5xFAD mice compared to control mice. Spatial learning deficits were also detected at this age, suggesting that cognitive functions were also affected. No differences in magnitude of the olfactory or spatial learning impairments could be detected between the age groups of model mice tested. Histological examination of development and presence of amyloid β (Aβ) plaques in the brains showed that plaques develop mainly between the ages of 3 and 8 months. This indicates that soluble Aβ rather than the formation of plaques might be responsible for the olfactory impairment and spatial learning impairments found. By 10 months of age plaque load of the 5xFAD mice was massive. The results of the present study clearly show that the 5xFAD strain might be suitable for research on human AD with regard to the early onset of olfactory impairments.

Lead (Pb) is one of the most well known toxic heavy metals in human beings and animals, which leads to toxic neurological disorders, cognitive problems, learning and memory disabilities. Epidemiological studies revealed that chronic lead exposure is one of the environmental risk factors which may cause Alzheimer¡¦s Disease, which were speculated for the observation of cellular necrosis, apoptosis, and £]-amyloid deposition frequently occuring altogether after chronic lead exposure. Recent studies have shown that the £]-amyloid formed during autophagic turnover of APP-rich organelles supplied by both autophagy and endocytosis. Therefore, we will conduct the new perspective for studying the possible role of autophagy on amyloidogensis disorders after lead exposure. SH-SY5Y human neuroblastoma cells, used in this study, were differentiated to a neuronal phenotype by retinoic acid (RA) to the culture medium at 10 £gM for 1, 2, 3 and 4 days. Doses of lead acetate with of lead acetate were 5 £gM and applied to the neuronal culture and then cell viability measurement by MTT assay. The apoptotic effect of non-differentiation and differentiation neuroblastoma cells after lead exposure was determined by cleaved DNA fragments. Furthermore, APP, intracellular A£]1-40 and A£]1-42 expression were quantified by Real-time PCR and ELISA, respectively. The autophagy process and variation of total and phosphorylated mammalian target of rapamycin (mTOR) forms were determined after lead exposure in non-differentiation and differentiation neuroblastoma cells by western blot. The results indicate that lead exposure enhances autophagy response in both non-differentiation and differentiation SH-SY5Y cells, which might cause neuronal apoptosis associated with £]-amyloidgenesis. Otherwise, lead exposure resulted in the inhibition of mTOR signaling, which correlated with the autophagic process. Besides, in our studies, non-differentiated cells exhibited more toxic vulnerability than RA induced differentiated neuron is congruous to previous finding that lead exposure during fetal development might be a potential risk factor for AD in the adulthood.

Despite intense interest in the proteolysis of Amyloid Precursor Protein (APP) in Alzheimer’s disease (AD), how the normal processing and function of this type I receptor-like glycoprotein is regulated remains ill-defined. APP is reported to function in neurodevelopment, including migration of neuronal precursor cells into the cortical plate. In recent years, several candidate ligands for APP, including F-spondin, Reelin, \(\beta1\) Integrin, Contactins, and Lingo-1 have been reported. However, a cognate ligand for APP that regulates its function or processing has yet to be widely confirmed in multiple laboratories. First, in an unbiased approach to reveal novel ligands, Pancortin was identified by a mass spectrometry-based screen for factors that bind to the APP ectodomain in rodent brain. Each of the Pancortin isoforms was confirmed to interact with APP. However, only specific Pancortin isoforms reduced \(\beta\)-secretase but not \(\alpha\)-secretase cleavage of endogenous APP. Using in utero electroporation to overexpress or knockdown Pancortin isoforms in rodent cortex, a previously unidentified role for Pancortin in cortical cell migration with evidence for a functional interaction with APP was discovered. Next, I developed new assays in an effort to confirm a role for one or more of the published candidate ligands in regulating APP ectodomain shedding in a biologically relevant context. A comprehensive quantification of APPs\(\alpha\) and APPs\(\beta\), the immediate products of secretase processing, in both non-neuronal cell lines and primary neuronal cultures expressing endogenous APP yielded no evidence that any of these published candidate ligands stimulate ectodomain shedding. Rather, Reelin, Lingo-1 and Pancortin emerged as the most consistent ligands for significantly inhibiting ectodomain shedding. These studies clarify mechanisms regulating the function and processing of APP, which is needed to understand consequences of chronically altering APP proteolysis to treat AD and to develop new potential drug targets.

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