Development of reversible chemogenetic reporters for studying organelle contact sites and autophagy in Alzheimer’s disease

In the last years, several defects in the autophagy pathway have been reported in different Alzheimer’s disease (AD) models, suggesting a possible key role for them in disease onset and progression (1). Previous work in our laboratory has shown that familial AD (FAD)-linked Presenilin 2 mutants induce a block in the late stages of the autophagy flux, likely as consequence of a defective recruitment at autophagosomes of RAB7, a small GTPase essential for autophagosome-lysosome fusion (2). Importantly, evidence has been provided suggesting that key steps of autophagy are regulated at organelle membrane contact sites (MCSs) (3), although the possible link between perturbations of MCSs and autophagy defects in the AD context remains unclear. This gap is partly due to the lack of tools to monitor MCS dynamics with sufficient spatiotemporal resolution. To overcome these limitations, in this thesis we developed a series of chemogenetic, reversible splitFAST-based reporters (4) to investigate MCSs between different organelles involved in autophagy, including endoplasmic reticulum (ER), mitochondria, autophagosomes and lysosomes. We envisage these reporters will be helpful to dissect the mechanisms underlying autophagy dysfunction in AD.