The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions

Abstract

Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson’s disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely β3-Cav2.1 at N-terminus part and β-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.

The LRRK2 N-terminal domain influences vesicle trafficking: impact of the E193K variant

“…We observed that the E193K variant increases SV fusion. Interestingly, we and other have reported a similar phenotype upon LRRK2 silencing 4,5,31 . Furthermore, we were able to rescue the SV phenotype correlated with E193K upon co-expression of the isolated N-terminal domain.…”

“…Our data are consistent with such model and suggest that the N-terminal and C-terminal domains act together to shape the interacting site for SV and SV-associated proteins. www.nature.com/scientificreports www.nature.com/scientificreports/ However, we reported that the isolated LRRK2-WD40 domain prevents SV fusion by itself 4,6 . In contrast, the isolated N-terminal fragment did not elicit an overt effect on SV dynamics.…”

“…The N-terminal domain influences LRRK2 interactions. We have brought evidence that LRRK2 modulates SV trafficking via interaction with synapsin I, α-tubulin and β-actin [4][5][6] . To appreciate the role of the LRRK2 N-terminal domain in such interactions, we expressed Strep-FLAG full-length LRRK2 and Strep-FLAG LRRK2ΔN-term in N2A cells.…”

“…LRRK2 is a complex protein that consists of multiple functional domains, allowing a dual enzymatic activity and multiple protein-protein interactions 3 . We have contributed to describe LRRK2 as a synaptic protein that influences the synaptic vesicle (SV) cycle [4][5][6][7] . Noteworthy, LRRK2 rodent models show age-related neurotransmission defects [8][9][10] and LRRK2 binds and phosphorylates several presynaptic proteins [11][12][13][14] .…”

Thein situstructure of Parkinson’s disease-linked LRRK2

“…These data support our model that the WD40:WD40 protomer interface is important for the formation of LRRK2 filaments. Given the correlation between breaking the dimerization interface, loss of filament formation, and change in kinase activity seen in the G2385 LRRK2 mutant (9,11,(30)(31)(32), supporting that oligomerization plays a role in regulating LRRK2 kinase activity. Here, we have shown that recent advances in cryo-ET can be used to determine novel structures of proteins in situ, making it possible to generate structural hypotheses in a cellular context.…”