Investigation of the role of PICALM on amyloid metabolism in a transgenic mouse model of Alzheimer’s disease

Recently, two large independent genome-wide association studies have provided compelling evidence that polymorphisms at the phosphatidylinositol-binding clathrin assembly protein (PICALM) locus are linked to Alzheimer’s disease (AD). At the cellular level, PICALM is involved in clathrin-mediated endocytosis, a fundamental process that is essential for both membrane receptor signalling and membrane homeostasis. In the context of AD, this phenomenon has been notably involved in the clearance of amyloid-beta peptides, the main constituent of amyloid plaques that accumulate in the brain of AD patients. In this master thesis, we hypothesized that the polymorphisms identified in the PICALM gene might lead to a decrease of clathrin-mediated endocytosis and might impair the degradation of amyloid-beta peptides by glial cells, fostering its accumulation in the extracellular space. To test this hypothesis, we examined the effect of PICALM brain-specific silencing in a transgenic mouse model of AD by using recombinant Adeno-Associated-Viral (AAV) vectors. We first generated several molecular constructions overexpressing different shRNAs targeting PICALM and selected them in vitro for their efficacy by performing transient transfection experiments in HEK cells. The best constructs were next subcloned in an AAV shuttle plasmid carrying a reporter gene in order to produce the corresponding recombinant AAVs. Finally, AAV vectors were injected in the brain of transgenic mice and amyloid accumulation was evaluated by using histochemical methods 4 months after injections. Preliminary results indicate that mice, which have received the AAV-shRNA targeting PICALM, present an increase in amyloid plaque load in the cortex compared to non-injected animals, suggesting that PICALM is indeed involved in the regulation of the amyloid metabolism in vivo. Further experiments will be performed to decipher the molecular mechanisms linking PICALM function to the amyloid-beta peptide metabolism.