Tau sensor, an approach based on FRET to study Tau protein in Alzheimer's disease: imaging and molecular characterization

Alzheimer’s disease is a neurodegenerative disorder which is very diffuse during the last two centuries. It’s characterized by progressive dementia and two main lesions lead to the beginning and development of the disease: neuritic plaques and neurofibrillary tangles formed due to aggregation of APP and Tau protein respectively. Tau protein is a microtubule-associated protein (MAP) expressed mainly in neurons whose main function is to stabilize cytoskeleton by binding tubulin. During Alzheimer’s disease Tau undergoes many post-translational modifications and it’s not able to perform its function. In this condition Tau aggregates forming toxic tangles which cause neurodegeneration. Because of the importance of this protein in Alzheimer’s disease, many groups are working to elucidate the physiological and pathological role of Tau and to develop new therapies for this severe disorder. In order to study the role of Tau in Alzheimer’s disease in the laboratory has been developed a chimeric protein (Tau sensor) made of a Tau protein linked to a Cfp and a Yfp at the N- and C-terminals. The aim of my thesis is to characterize Tau sensor by imaging employing FRET and FRAP techniques and by molecular analysis using Western blot technique. As a previous study the cellular system employed has been HeLa cells. The study has revealed that Tau sensor binds microtubules and it’s in part cleaved and secreted in physiological conditions. The response to microtubules-destabilizing agents is the same of wild type Tau with the sensor diffusing into the cytoplasm. The effect of taxol, a microtubules-stabilizing agent, causes the displacement of Tau sensor from the microtubules and remarkably it determines the formation of aggregates. Staurosporine, a kinase inhibitor, stabilizes Tau sensor on the cytoskeleton. These data suggest that the sensor shares the same function of Tau protein and allow to employ it in neuronal cells, a system nearer to that in which Alzheimer’s disease develops