AXONAL TRAFFICKING DEFICITS CAUSED BY PARKINSON’S-ASSOCIATED MUTATIONS IN LRRK2

Parkinson’s disease (PD) is a devasting neurodegenerative disease that causes cardinal motor symptoms as well as debilitating non-motor and cognitive manifestations. Neurons face logistical and bioenergetic challenges for maintaining homeostasis in complex axonal arbors. Therefore, disruptions to axonal transport may contribute to PD pathogenesis. Autosomal dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of PD, resulting in hyperactivity of LRRK2 kinase activity and increased phosphorylation of downstream RAB GTPases (RABs). LRRK2-phosphorylated RABs have a modified set of effector binding partners. Thus, pathogenic LRRK2 has the potential to alter intracellular trafficking, but these specific pathways have not been fully explored. During my thesis work, I investigated the mechanisms by which hyperactive LRRK2 disrupts the axonal transport of two crucial cargoes: autophagosomes and synaptic vesicle precursors (SVPs). In work described in this document, I employed an array of live-imaging and immunocytochemistry techniques to explore the effects of hyperactive LRRK2 on cargo transport. In addition, I performed biochemical assays to interrogate the binding properties of phosphoRABs. In gene-edited iPSC-derived human neurons, I contributed to work that showed that the most common pathogenic LRRK2 mutation impairs processive retrograde autophagosome transport. In later work, I showed that these autophagosome transport deficits scale with the magnitude of hyperactivity. Furthermore, I demonstrated that regulation of autophagosome trafficking relies on a tightly-controlled interplay between LRRK2, its opposing phosphatase PPM1H, and the small GTPase ARF6. In addition to autophagosomes, I went on to show in another study that hyperactive LRRK2 disrupts axonal transport of SVPs. My work characterized the mechanism by which this disruption arises, showing that LRRK2-phosphorylated RAB3A has impaired binding to MADD, a motor adapter. As a consequence, I found that the compartmental distribution of synaptic proteins is altered, with decreased delivery to pre-synaptic sites. Together, these studies provide important insights into how mutant LRRK2 disrupts multiple essential pathways for neuronal homeostasis by phosphorylating distinct RAB substrates. The mechanisms explored in this work will aid efforts to identify how regulation of LRRK2 or other therapeutic targets can be leveraged for management of PD motor and non-motor disease progression.