The LRRK2 variant E193K affects the readily releasable pool of synaptic vesicles via modulation of LRRK2 interactome

The Leucine-Rich Repeat Kinase 2 (LRRK2) is a complex protein, expressed in neurons and implicated in Parkinson disease (PD). LRRK2 contains a dual enzymatic activity and several structural domains that constitute a versatile platform for multiple protein interactions at the synapses. In this study, we characterize the functional role of the N-terminal Armadillo repeats domain of LRRK2 and the impact on synaptic vesicle (SV) dynamics of a novel variant, E193K, harboured within this domain and identified in an Italian family affected by PD. Using a genetically encoded sensor of recycling, synaptopHluorine, and total internal reflection fluorescence microscopy, we visualized SV trafficking in the N2A neuroblastoma cells expressing the wild type LRRK2 protein, a mutant lacking the Armadillo domain (ΔN LRRK2) or the E193K variant. We found that expression of the ΔN construct increased the frequency and the amplitude of spontaneous synaptic events. A similar phenotype was detected in the presence of the E193K variant, suggesting that this mutation behaves as a loss-of-function mutation. A domain-based pulldown approach demonstrated that the LRRK2 N-terminus binds to cytoskeletal (β-actin and α-tubulin) and SV (synapsin I) proteins and the E193K substitution alters strength and quality of LRRK2 interactions. The results support a role of the Armadillo domain in interaction with synaptic proteins and suggest that the E193K mutation affects LRRK2 function via perturbation of its physiological network of interactors, resulting in impaired vesicular trafficking. These findings may have important implications for understanding the role of LRRK2 at the synapses and the pathophysiological mechanism for LRRK2-linked diseases.