Abstract
The LRRK2 protein consists of multiple functional domains, including protein-binding domains at its N and C-terminus. Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) have been linked to familial and sporadic Parkinson’s disease (PD). We have recently described a novel variant falling within the N-terminal armadillo repeats, E193K. Herein, our aim is to investigate the functional impact of LRRK2 N-terminal domain and the E193K variant on vesicle trafficking. By combining Total Internal Reflection Fluorescence (TIRF) microscopy and a synaptopHluorin assay, we found that expression of a construct lacking the N-terminal domain increases the frequency and amplitude of spontaneous synaptic events. Complementary biochemical approaches showed that the E193K variant alters the binding properties of LRRK2, decreases LRRK2 binding to synaptic vesicles, and promotes vesicle fusion. Our results confirm the physiological and pathological relevance of the nature of the LRRK2-associated macro-molecular complex solidifying the idea that different pathological mutations critically alter the scaffolding function of LRRK2 resulting in a perturbation of the vesicular trafficking as a common denominator.
Highlights
The Leucine-rich repeat kinase 2 gene (LRRK2) protein consists of multiple functional domains, including protein-binding domains at its N and C-terminus
Since the N-terminal Armadillo domain is involved in LRRK2 supra-molecular organization[19], we further investigate the functional role of LRRK2 N-terminal domain by means of Total Internal Reflection Fluorescence (TIRF) microscopy (TIRFM) coupled with a synaptopHluorin assay as previously described[20]
We over-expressed in N2A neuronal line the sypHy reporter together with a panel of RFP-tagged LRRK2 derived constructs: LRRK2 full-length, LRRK2 lacking the first 913 amino acids as well as a complementary a deletion construct LRRK2 aa 1–983, containing the N-terminal Armadillo and Ankyrin domains (Fig. 1A)
Summary
The LRRK2 protein consists of multiple functional domains, including protein-binding domains at its N and C-terminus. Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) have been linked to familial and sporadic Parkinson’s disease (PD). We have recently described a novel variant falling within the N-terminal armadillo repeats, E193K. Our aim is to investigate the functional impact of LRRK2 N-terminal domain and the E193K variant on vesicle trafficking. By combining Total Internal Reflection Fluorescence (TIRF) microscopy and a synaptopHluorin assay, we found that expression of a construct lacking the N-terminal domain increases the frequency and amplitude of spontaneous synaptic events. The majority of PD cases do not correlate with clear genetic causes, late-onset autosomal dominant PD have been robustly linked to mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene (PARK8; OMIM 609007). We investigated the functional impact of the E193K variant identified in an Italian family with 3 siblings affected by PD18. We combined TIRF microscopy and a synaptopHluorin assay and found that LRRK2-E193K expression increases the frequency and time length of SV fusion events
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