Mammalian validation of Drosophila melanogaster-identified y-secretase modulators

• Applicant: Dr. Tina Katrin Wahle-Schuldt
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2007 to 2010
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 53121166

Several pathological features define Alzheimer’s disease, the most common form of dementia, striking amongst them the presence of cumuli in the brain parenchyma of the amyloidogenic peptide Aβ, the product of the sequential proteolysis of the Amyloid precursor protein (APP) by two proteases, named β- and γ-secretase. Genetic analysis of familial forms of Alzheimer’s disease revealed that mutations in APP or in the γ-secretase complex component Presenilin result in an increase in the generation of Aβ. Given this, the adoption of pharmaceutical approaches in order to reduce the activity of the γ-secretase complex has become a therapeutic goal against this disease. However, the fact, that γ-secretase activity is essential for physiological events, required for proper functioning of a number of metabolic pathways, including the Notch pathway, has stimulated the scientific community to search for modulators of γ-secretase acitvity on APP without affecting other substrates. With this idea in mind, an in vivo genetic screen using Drosophila melanogaster was performed in the host laboratory, resulting in the identification of a number of genes which selectively interfere with the Notch and/or APP cleavage by γ-secretase.In the proposed project it is planned to determine to which extent the insect identified γ-secretase modifiers participate in γ-secretase activity in mammalian cells, with special emphasis in hippocampal neurons, both in vitro and in situ. In a first step, the Drosphilaacting genes whose mutations result in enhancement or suppression of γ-activity, and for which mammalian ortologues were found, will be analyzed by siRNA knock-down in mammalian cells of non-neuronal origin, expressing constitutively APP or Notch. In a second step, the genes whose down-regulation had an effect in the above assay will be tested in hippocampal neurons in primary culture. For this, siRNA knock-down will be performed via lentivirus-mediated gene transfer, via a collaboration already existing between the hostlaboratory and the Gene Therapy Unit at the same University of the host laboratory. In the third step, genes with an effect in hippocampal neurons in vitro, will be investigated in mice models of Alzheimer’s disease, through the use of local injection of Adeno-associated virus, also through the existing collaboration with the Gene Therapy Unit. The selected γ-secretase modifiers will be functionally characterized and their role in the development of the disease determined.

• DFG Programme: Research Fellowships
• International Connection: Belgium
• Host: Professor Dr. Carlos G. Dotti