Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain

Abstract

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.

FIG 1

LRRK2 binds presynaptic proteins. (A) Distribution of LRRK2, synapsin I, and synaptophysin immunoreactivities in subcellular fractions of rat forebrain. LRRK2 is present in highly purified vesicle fractions (US), and its association with the vesicle membrane is not affected by salt treatment (SSV). H, homogenate. (B) LRRK2 binds to SV. Full-length FLAG-LRRK2, purified from transfected HEK293T cells, was incubated with unstripped SV (US) or salt-stripped SV (SSV) before high-speed sedimentation. Representative Western blots stained with anti-FLAG antibody show the initial amount of FLAG-LRRK2 protein (total), the yield of protein precipitated by US or SSV (bound), and the amount of endogenous synapsin I associated to SV. Fusion proteins were incubated with equal amounts of SV (monitored by antisynaptophysin staining). (C) The graph reports the yield of FLAG-LRRK2 precipitated by US and SSV, expressed as a fraction of FLAG-LRRK2 total protein and normalized against the SV total protein amount. (D) The graph reports the amount of endogenous synapsin I associated to US or SSV. Data are expressed as optical density (OD) in arbitrary units. *, P < 0.05, n = 4 (Student's t test). (E) Immunoprecipitation of endogenous LRRK2 from adult brain lysate confirms the interaction between LRRK2 and selected presynaptic and cytoskeletal proteins.

FIG 2

The C-terminal WD40 domain of LRRK2 represents a hub for protein-protein interactions. Western blots are shown, confirming the specific interaction of proteins following a domain-based pulldown assay for the LRRK2 WD40 domain. s-physin, synaptophysin.

FIG 3

LRRK2 interacts with presynaptic proteins. (A) The network of LRRK2 interactors was modeled from STRING annotation on the Cytoscape representation. annotated, interactions annotated on STRING; experimental, interactions described in the manuscript. (B) Gene symbols, names, and gene ontology (GO) terms for the proteins included in the network.

FIG 4

Expression of an LRRK2 WD40 domain construct is sufficient to induce neurotoxicity in primary neurons. Neurons were transfected at DIV3 and imaged at DIV16. Long-lasting overexpression of RFP-LRRK2 WD40 and full-length RFP-LRRK2 significantly reduced the number of processes and increased the amount of swollen or fragmented neurites compared to levels in DsRed-, RFP LRRK2 1–2141-, or RFP-RACK1-transfected neurons. Images show signals acquired for GFP (channel 1, CH1), DsRed or RFP (channel 2, CH2), superimposed channel signals (merge), and tracings. Arrow heads indicate an RFP-LRRK2 WD40-positive cell. Numbers in the tracing panel indicate total process numbers and are expressed as means ± SE. **, P < 0.01 versus DsRed; #, P < 0.01 versus full-length RFP-LRRK2 (ANOVA; Tukey's post hoc test, n = 3; 8 neurons were analyzed for each experimental case). Panel size shown is 200 by 200 μm.

FIG 5

Expression of an LRRK2 WD40 domain construct induces neurotoxicity in primary neurons. (A) Neurons were transfected at DIV10 and imaged at DIV16. Long-lasting overexpression of RFP-LRRK2 WD40 significantly reduced the number of processes and increased the amount of swollen or fragmented neurites compared to levels in DsRed-, LRRK2 1–2141-, or RFP-RACK1-transfected neurons. Images show signals acquired for GFP (channel 1, CH1), DsRed or RFP (channel 2, CH2), superimposed channel signals (merge), and tracings. Graphs report the number of total processes (B) and number of fragmented processes (C) (means ± SE). *, P < 0.05 versus DsRed; **, P < 0.01 versus DsRed; #, P < 0.05 versus LRRK2 WD40 (ANOVA; Tukey's post hoc test, n = 4; 7 neurons were analyzed for each experimental case). Panel size shown is 200 by 200 μm.

FIG 6

The LRRK2 WD40 domain sequesters cycling synaptic vesicles. The exoendocytotic assay was performed at DIV12 on cortical neurons transfected at DIV10. (A) Cycling SV appear as synaptotagmin (s-tagmin)-positive clusters along neuron processes. Images show signals acquired for synaptotagmin (channel 1, CH1) and DsRed or RFP or FLAG (channel 2, CH2) and their superimposition plus GFP (merge). (B) The total SV pool was revealed by staining with anti-SV2A antibodies upon permeabilization. Images show signals acquired for SV2A (channel 1, CH1) and DsRed or RFP (channel 2, CH2) and their superimposition plus GFP (merge). (C) SV cycling is strongly reduced upon either LRRK2 WD40 or RACK1 overexpression. The graph reports the number of synaptotagmin-positive clusters per 10 μm of GFP-positive process. (D) The total number of SV pools was not altered by LRRK2 WD40, RACK1, or LRRK2 1–2141 overexpression. (E and F) Ectopically expressed LRRK2 WD40 confines cycling SV within a perisomatic region, while total SV pool distribution is not affected. The graph reports the percentage of synaptotagmin- or SV2A-positive clusters distributed within the proximal half of the process. Data are expressed as means ± SE. *, P < 0.01 versus DsRed (ANOVA; Tukey's post hoc test, n = 4; 7 neurons were analyzed for each experimental case). Panel length is 10 μm.

FIG 7

The G2385R substitution impacts the local structure of LRRK2 WD40. (A) Combined stick-and-ribbon representation showing a structural homology model (based on RACK1 from Arabidopsis thaliana, PDB 3DM0) for the LRRK2 WD40 domain. The N terminus, C terminus, and the position of G2385 (red sphere) are indicated. (B) Transmission EM images of negatively stained (1% uranyl acetate) LRRK2 WD40, LRRK2 WD40 G2385R, and RACK1 proteins show doughnut-shaped particles consistent with the characteristic structure of WD40 folds. Four representative averaged single-particle two-dimensional projections are shown for each protein (scale bar, 5 nm). (C) LRRK2 WD40 binds SV. Increasing nanomolar amounts of LRRK2 WD40, LRRK2 WD40 G2385R domains, and RACK1 protein were incubated with unstripped SV (US) or salt-stripped SV (SSV) before high-speed sedimentation. Representative Western blots show initial amount of fusion protein (total) and the yield of GST fusion proteins precipitated by US or SSV (bound). Fusion proteins were incubated with equal amounts of SV (monitored by antisynaptophysin staining). (D) The table reports the dissociation constant (K_D) describing the binding between the indicated fusion protein and US or SSV. Data are expressed as means ± SE. *, P < 0.05 versus LRRK2 WD40 G2385R binding to US; °, P < 0.01 versus LRRK2 WD40 binding to SSV (ANOVA; Tukey's post hoc test, n = 4). (E) The graph reports the yield of precipitated GST fusion protein normalized versus the SV total protein amount (average data plus fitting) on the y axis and the initial amount of GST fusion protein on the x axis.