Total Internal Reflection Fluorescence Microscopy to unravel the impact of LRRK2/Park8 and its pathogenic mutants on neurotransmitter vesicle trafficking

LRRK2/Park8 is a leucine-rich repeat kinase expressed in neurons. Mutations in LRRK2 were identified in familial Parkinson’s disease (PD), a neurodegenerative motor disorder caused by degeneration of midbrain dopaminergic neurons. The protein comprises several functional domains, including a C-terminal WD40 module involved in interactions with synaptic vesicles (SV) proteins. The physiological role of LRRK2 and the specific mechanisms by which its mutants cause neuronal toxicity in PD are still elusive. Here we focus on the role of LRRK2 on SV dynamics. We co-transfected the SH-SY5Y neuroblastoma cell line with wt/mutants LRRK2 and a pH-sensitive GFP selectively targeted to SV and we visualized SV dynamics by means of Total internal reflection fluorescence microscopy (TIRFM) under resting conditions. We confirmed that overexpression of the wt WD40 domain perturbed vesicle dynamics determining a significant decrease in both the frequency and the amplitude of spontaneous synaptic events (80% reduction). Overexpression of the WD40 G2385R, a genetic variant that acts as PD risk factor, attenuated the phenotype. Viceversa, overexpression of a LRRK2 lacking the WD40 domain increased the frequency of synaptic events. Interestingly, a significant increase in the amplitude of the fluorescent signal associated with vesicle fusion was detected, thus suggesting changes in vesicle cluster organization. Our data confirm a key role of the LRRK2 WD40 domain in the control of SV fusion/endocytosis and propose the presynaptic site as a pathological target of PD.