- Vivi Padova
- Il BO
Mutations in LRRK2 comprise the most common cause for autosomal-dominant familial Parkinson´s disease (PD), and variations increase risk for disease, indicating that LRRK2 is key to the entire spectrum of PD pathogenesis (1-4). The protein displays kinase activity, and most pathogenic mutations increase this activity (5-7), making LRRK2 a prime druggable target for disease-modifying strategies. A range of specific and even brain-permeant kinase inhibitors have been developed (8-10). However, there is a lack of understanding of the precise cellular deficits associated with pathogenic LRRK2, and a lack of understanding of the cell type-specific mechanisms which may explain the observed safety liability issues associated with genetic or pharmacological LRRK2 inhibition in vivo (10-13). Pathogenic LRRK2 has been consistently shown to cause deficits in endocytic and autophagic trafficking paralleled by structural endolysosomal alterations (14-18). Our studies indicate that mutant LRRK2 deregulates endolysosomal trafficking at the step of the late endosome, impacting upon both endocytic and autophagic turnover (18,19). The endolysosomal deficit is reverted by distinct LRRK2 kinase inhibitors, correlates with a decrease in Rab7 GTPase activity, and can be rescued by overexpression of active Rab7 (18). The GTPase activity of Rab7 is key for a variety of vesicular trafficking steps in and out of the late endosome, and alterations are reflected by changes in the size and subcellular distribution of endolysosomal structures (14,20). We propose to determine whether changes in endolysosomal number, size and subcellular distribution, and altered autophagosome numbers, are a robust cellular readout for pathogenic LRRK2 in neuronal as well as non-neuronal and patient-derived cells. Apart from Rab7, LRRK2 has been linked to other Rab proteins, some of them with restricted expression patterns and implicated in distinct vesicular trafficking pathways involving the late endosome (17,21-26). We will determine whether these Rab proteins rescue endolysosomal and autophagic alterations caused by pathogenic LRRK2, whether they alter Rab7 GTPase activity, and whether Rab7 activity modulates their localization. We will further decipher whether pathogenic LRRK2 directly phosphorylates those Rab proteins, or modifies their GTP binding and/or GTPase activities. LRRK2 kinase inhibitors will be employed to address possible reversal of the observed cellular changes in neuronal as well as non-neuronal cells. Our studies may provide a robust pharmacodynamic readout for compounds targeting LRRK2 activity in a pathopysiological neuronal as well as non-neuronal context. In addition, insight into the LRRK2-mediated regulation of other Rab proteins with restricted tissue distribution may warrant further studies aimed at targeting the Rab proteins expressed in tissues which display kinase inhibitor-mediated safety liability issues (10).