The lysosomal exonuclease PLD3 as an endolysosomal flux regulator: impact on neuronal homeostasis and relevance in neurodegeneration.

 Late-onset Alzheimer’s disease (LOAD) is characterized by an unbalanced proteostasis. Dysfunctions in endolysomal transport and degradation kinetics arise at early preclinical stages and progressively worsen. Among the known LOAD risk factors, I focus on phospholipase D (PLD)-3. PLD3 is selectively expressed in neurons where it resides in late endosomes and lysosomes. It is an aspecific member of the PLD family, being an exonuclease that degrades ssDNA. I will investigate the functional link between PLD3 and lysosomal homeostasis focusing on three major aims. (1) A crosslink-IP sequencing approach will allow me to identify PLD3specific DNA substrates. (2) Identified substrates will be linked to cellular organelles and/or mechanisms, using functional assays combined with advanced imaging including super-resolution and electron microscopy. (3) This will be corroborated with unbiased proteome/lipidome analyses of magnetically isolated lysosomes (wild-type vs PLD3 SNP/knockout-derived). Prioritized hits will identify dysregulated pathways that will be validated in control and patient-derived human neurons and in vivo, using an APP knock-in mouse model wherein PLD3 is deficient. Linking lysosomal exonuclease dysfunction to neurodegeneration will provide fundamental new insights on the physiological relevance of lysosomes in degrading ssDNA, how this affects neuronal homeostasis and add support to endolysosomal dysfunction as a common denominator in neurodegeneration