Oxidativer Stress und β-Amyloidspiegel bei APP/PS1-transgenen Mäusen

Projektdetails:

Thematik:	Ursachenforschung
Förderstatus:	abgeschlossen
Art der Förderung:	Research
Institution:	Universität Frankfurt, Biocenter
Projektleitung:	Prof. Dr. Anne Eckert
Laufzeit:	01. November 2001 - 31. Oktober 2003
Fördersumme:	76.000,00 Euro

Projektbeschreibung

Es häufen sich die Beweise, dass Störungen im Energiestoffwechsel zur Pathogenese von Alzheimer beitragen. Alzheimer-Patienten weisen bestimmte neuropathologische Schädigungen wie Plaques und Neurofibrillenbündel auf. Die Amyloidplaques bestehen aus dem Beta-Amyloid (Aβ) Peptid, das aus Amyloid Precursor Protein (APP) entsteht. In vitro-Studien konnten nun zeigen, dass Aβ eine vermehrte Produktion reaktiver Sauerstoffformen auslöst, was darauf hindeutet, dass die durch freie Radikale entstandene Zytotoxizität von Aβ schließlich zu Störungen im Abwehrmechanismus der Zellen führen könnte. Trotz dieser Beobachtungen konnte bisher noch kein eindeutiger Zusammenhang zwischen den durch oxidativen Stress verursachten Schäden und den Aβ-Spiegel in vivo hergestellt werden.

Dr. Eckert verwendet transgene Mäuse zur Erforschung der Rolle von oxidativem Stress bei Alzheimer, um Veränderungen bei der Entstehung von Oxidantien, Abwehrmechanismen gegen Oxidantien und (Dys)Funktionen der Mitochondrien im Mäusehirn zu untersuchen. Die Mäuse tragen zwei verschiedene menschliche Gene, APP und/oder Presenilin 1 (PS1), mit den entsprechenden Mutationen, die bei den erblich auftretenden Alzheimerformen (FAD) bei Patienten vorkommen. Dr. Eckert konnte bereits nachweisen, dass sich Schädigungen durch oxidativen Stress mit zunehmendem Alter häufen. Interessanterweise ist das Alter der zentrale Risikofaktor für Alzheimer. Im zweiten Schritt will sie daher prüfen, ob in den transgenen Tiere durch den Alterungsprozess oxidative Schäden vermehrt auftreten und ob ein Zusammenhang mit der altersabhängigen Ansammlung intraneuronaler Aβ und/oder Aβ-Plaquebildung im Gehirn besteht.

Die Grundlagenforschung am Gehirn transgener Mäuse mit FAD-Mutationen im menschlichen APP und/oder PS1 wird zur Aufklärung des zugrunde liegenden Mechanismus der Neurodegeneration beitragen. Langfristiges Ziel der Studie sind neue Perspektiven für die Entwicklung vorbeugender Medikamente.

Abschlussbericht

Despite a lot of research in the last decades, the exact causes of neurodegeneration in Alzheimer's disease (AD) are still not definitely known. One hypothesis is that a phenomenon called oxidative stress leads to the development of the disease. What is oxidative stress? We all breathe oxygen to fuel our metabolism. Although indispensable to life, a very small part of inhaled oxygen is converted to so-called "reactive oxygen species".

These are toxic by-products of our metabolism and can damage the cells of our body. However, cells are usually equipped with good defence against these toxic products: vitamins like vitamin C and E and other antioxidants can directly inactivate reactive oxygen species and a system of several antioxidant enzymes can convert reactive oxygen species to non-toxic metabolites. These enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR), which detoxify reactive oxygen species in a cascade of successive reactions. If the balance between formation of and defence against reactive oxygen species is perturbed, reactive oxygen species can accumulate - a state that is termed oxidative stress.

Oxidative stress has been shown to increase with advancing age. Since Alzheimer's disease is more common in aged people, it seems likely that oxidative stress, accumulating over life, contributes to the neurodegeneration in the disease. A part from aging - which is the most important risk factor for Alzheimer's disease- other factors that increase the risk of developing the disease have been explored in epidemiological studies, among these are genetic susceptibility factors and gender: females have been shown to suffer from the disease more frequently than males at the same age.

In our present study, we investigated the enzymatic defence against reactive oxygen species in autopsy brain samples from AD patients and control persons of the same age. The activities of the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were measured. These enzymes catalyse subsequent reactions in detoxification of reactive oxygen species. Our results show that the defence mechanisms are increased in Alzheimer's disease patients, which indicates that more reactive oxygen species have to be detoxified requiring a higher activity of enzymes. Furthermore, we have analysed our data with respect to a possible gender difference. Importantly, female Alzheimer patients exhibited higher activities of antioxidant enzymes (SOD, GPx) than male patients indicating higher levels of oxidative stress in brains from female patients. This was also confirmed with the measurement of toxic products of reactive oxygen species: female patients had higher levels of hydroxynonenal, an indicator of oxidative damage.

Based on these results we have extended our research to an animal model of Alzheimer's disease. The animal model is based on a genetic mutation in a gene called Amyloid Precursor Protein (APP) gene. APP mutations are found in very few families worldwide. In these families carriers of the APP mutations develop the so called "familial" form of Alzheimer's disease. Patients show the same symptoms and neuroptahological features as in the sporadic form of the disease, but the onset of disease is slightly earlier at an age between 40 and 65 years. The APP genes can be used to generate transgenic mice that exhibit pathological structures in their brains resembling the pathology in human Alzheimer's disease brains. Interestingly, female mice carrying APP mutations develop pathological lesions earlier than male mice. Although there is no evidence in humans so far that female patients develop pathological lesions earlier than male patients, the situation in transgenic mice reminds one of epidemiological findings that Alzheimer's disease is more frequent in women than in men. We have conducted the same experiments as with brains from Alzheimer's disease patients on brains of APP transgenic mice to investigate whether there is a similar gender difference in antioxidant enzymatic defence. We have analysed mice at an age of 3 months, which corresponds to young adulthood, and at an age of 12 months, which is half the average lifespan of the mice and corresponding to aged adulthood. In these mice we have found that - unlike in Alzheimer's disease patients – antioxidant defence is not higher but instead lower (activity of SOD-enzyme) or unaltered (activities of GPx- and GR-enzymes). It has been reported by other researchers that the mutated APP leads to a loss of trace elements from the brains of these transgenic mice, and this could explain why the antioxidant defence is impaired in the transgenic mice, because the SOD enzyme specifically requires trace elements to operate properly. But with respect to gender differences, the situation was similar to human brains: female mice exhibit higher activity of GPx. This leads us to speculate that the levels of reactive oxygen species are higher in female brains - and thus, females would be more susceptible to oxidative stress than males

The next step (in the second part of project) will be to investigate markers of oxidative stress in the brains of transgenic mice at different ages. Since our experiments using cell lines expressing human APP strongly indicate that mitochondria are an early cellular target within cells for damage by ß-amyloid, studies investigating the function of mitochondria in transgenic mice will be pursued.

Wissenschaftliche Publikationen auf Basis des geförderten Projekts

Schuessel, K., Schäfer, S., Bayer, T. A., Czech, C., Pradier, L., Müller-Spahn, F., Müller, W. E., and Eckert, A. (2005). Impaired cu/zn-sod activity contributes to increased oxidative damage in app transgenic mice. Neurobiology of Disease, 18(1):89-99.

Keil, U., Bonert, A., Marques, C.A., Schwerping, I., Weyermann, J., Strosznajer, J.B., Müller-Spahn, F., Haass, C., Czech, C., Pradier, L. Müller, W.E., Eckert, A. (2004). Amyloid-beta induced changes in nitric oxide production and mitochondrial activity leads to apoptosis. J. Biol. Cem., 279(48):50310-50320.

Keil, U., Bonert, A., Scherping, I. Marques, C.A., Müller-Spahn, F., Strosznajer, J.B., Müller, W.E., Eckert, A. (2004). Elevated nitric oxide production mediates beta-amyloid-induced mitochondria failure. Pol J Pharmacol.,56(5):631-634.

Schüssel, K., Leutner, S., Cairns, N.J., Müller, W.E., Eckert, A. (2004). Impact of gender on upregulation of antioxidant defence in Alzheimer’s disease brain. J Neural Trans, 111:1167-1182.

Eckert, A., Keil, U., Marques, C.A., Keil, U., Schüssel, K., Müller, W.E. (2003). Mitochondrial dysfunction, apoptotic cell death and Alzheimer’s disease. Biochem. Pharmacol, 66(8):1627-34.

Marques, C.A., Keil, U., Steiner, B. Haass, C., Müller, W.E. Eckert, A. (2003). Neurotoxic mechanisms caused by the Alzheimer’s disease-linked Swedish APP mutation: Oxidative stress, caspases and JNk pathway. J. Biol.Chem., 278:28294-28302.

Schindowski, K., Kratzsch, T., Steiner, B., Leutner, S., Touchet, N., Czech, C., Pradier, L., Maurer, K., Frölich, L., Müller, W.E:, Eckert, A. (2003). Impact of aging, sporadic and genetic Alzheimer’s risk factors on vulnerability to cell death, NeuroMolecular Medicine, 4:161-177.

Eckert, A., Marques, C.A., Keil, U., Schüssel, K., Müller, W.E. (2003). Increased apoptotic cell death in sporadic and genetic Alzheimer’s disease. Ann. NY Acad. Sci.1010:1-6.

Eckert A., Steiner B., Marques C., Leutz S., Romig H., Haass C., & Müller W. (2001). Elevated Vulnerability to Oxidative Stress-Induced Cell Death and Activation of Caspase-3 by the Swedish Amyloid Precursor Protein Mutation. J Neurscience Research 64:183-192.