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

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.

Figure 1

Domain-wise dissection of LRRK2 impact on vesicle trafficking. (A) Schematic representation of RFP-LRRK2 derived constructs. The distinct LRRK2 domains are indicated. Protein-protein domains: ARM, armadillo repeats; ANK, ankyrin repeats; LRRs, leucine-rich repeats; WD40, WD40 repeats; Roc, Ras of complex proteins; COR, C-terminal of ROC; Kin, kinase domain. (B) Western blotting analysis of cells expressing synaptopHluorin reporter (sypHy) together with RFP-LRRK2 derived constructs. (C) Time course analysis of fusion events occurring in N2A cells transfected with the different LRRK2 derived constructs. N2A cells were co-transfected with sypHy reporter and empty vector (E.V.) or the indicated RFP-LRRK2 derived constructs. TIRFM imaging was performed 48 h after transfection. Peaks of fluorescence intensity correspond to single fusion events. Fluorescence data are expressed as F/F0. The graphs show the total number of fusion events (D) and the resulting fluorescence changes (E) expressed as Area Under Curve (AUC) for each construct. Data are normalized for the cell area and are expressed as mean ± SE; n = 20 cells per construct, in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus empty vector, ANOVA.

Figure 2

LRRK2 interacts with proteins involved in vesicle trafficking. (A) We isolated on streptavidin resin full-length Strep-FLAG-LRRK2 and Strep-FLAG-LRRK2ΔN-terminus proteins from N2A over-expressing cells. Interacting proteins were resolved by western-blotting. (B) We evaluated the extent of Synapsin I, α-tubulin and β-Actin bound to the different LRRK2 variants. Data are expressed as optical density and normalized versus amount of precipitated LRRK2 protein. Graphs report mean ± SE, *p < 0.05, **p < 0.01 Student’s T-test, n = 4 independent experiments.

Figure 3

E193K variant does not influence neuritic tree. (A) We expressed in DIV4 cortical neurons GFP together with empty vector or RFP-LRRK2 wild type or RFP-LRRK2 E193K variant. We processed culture at DIV14 for imaging purposes. Scale bar = 50 μm. (B) The graph reports neurite number, expressed as mean ± SE, n = 12 cells. (C) The graph reports total neurite length, expressed as mean ± SE, n = 12 cells.

Figure 4

E193K variant affects vesicle trafficking. (A) Schematic representation of LRRK2 wild type, E193K variant and N-terminus domain. The distinct LRRK2 domains are indicated. (B) Western-blotting analysis of WT and E193K N2A clones expressing sypHy reporter together with empty vector or RFP-LRRK2 N-terminus constructs. Arrowhead indicates the specific RFP positive band detected by the anti-RFP antibody. (C) Vesicle density in the TIRFM zone after NH₄Cl treatment. The cells were incubated with the membrane permeant NH₄Cl solution for 5 minutes, to label all sypHy positive clusters. Then, cells were fixed and imaged by epifluorescence or TIRFM to visualize vesicles docked to the plasma membrane. Each spot corresponds to a sypHy positive cluster. Scale bar = 10 μm. (D) The graph reports the number of sypHy positive clusters visualized in the same cell under epifluorescence or TIRFM. Data are normalized for the cell area and are expressed as mean ± SE; n = 15 cells for construct. ***p < 0.001, Student’s T-test. (E) Time course analysis of synaptic events occurring in N2A stable clones expressing LRRK2 wild type (WT) or E193K LRRK2 and transfected with the empty-vector or with the isolated N-terminal domain together with the sypHy reporter. Transfected cells were imaged by TIRFM 48 h later. Peaks of variable fluorescence intensity correspond to single fusion events. Fluorescence data are expressed as F/F0. The graphs show the total number of fusion events (F) and the resulting fluorescence changes (G) expressed as Area Under Curve (AUC) for each construct. Data are normalized for the cell area and are expressed as mean ± SE of up to 20 cells per construct, in three independent experiments. *p < 0.05, **p < 0.01 compared to LRRK2 wild type, ^###p < 0.001 compared to empty vector transfected clones.

Figure 5

E193K mutation affects LRRK2 binding properties. (A) We measured the extent of LRRK2 and SV binding by ultracentrifugation sedimentation assay. We incubated purified RFP-LRRK2 wild type and E193K variant with isolated SV (10 μg protein/sample). Bound RFP-LRRK2 was separated from free RFP-LRRK2 by high-speed centrifugation. We appreciated SV-bound LRRK2 by immunoblotting with anti-RFP antibody. The recovery of SV in the pellet was evaluated based on synaptophysin immunoreactivity. (B) The binding of RFP-LRRK2 wild type and E193K to SV was calculated as the ratio of total RFP-LRRK2 and expressed as mean ± SE, n = 6, ***p < 0.001, Student’s t-test. (C) We isolated on streptavidin resin full-length FLAG-LRRK2 wild-type and FLAG-LRRK2 E193K proteins from N2A over-expressing cells. Interacting proteins were resolved by western-blotting. (D) We evaluated the extent of synapsin I, α-tubulin, β-Actin bound to the different LRRK2 variant. Data are expressed as the ratio over LRRK2 wild-type. Graphs report mean ± SE; n = 4. **p < 0.01, Student’s T-test.