The WD40 domain is required for LRRK2 neurotoxicity

Abstract

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD). LRRK2 contains an "enzymatic core" composed of GTPase and kinase domains that is flanked by leucine-rich repeat (LRR) and WD40 protein-protein interaction domains. While kinase activity and GTP-binding have both been implicated in LRRK2 neurotoxicity, the potential role of other LRRK2 domains has not been as extensively explored.

Principal findings: We demonstrate that LRRK2 normally exists in a dimeric complex, and that removing the WD40 domain prevents complex formation and autophosphorylation. Moreover, loss of the WD40 domain completely blocks the neurotoxicity of multiple LRRK2 PD mutations.

Conclusion: These findings suggest that LRRK2 dimerization and autophosphorylation may be required for the neurotoxicity of LRRK2 PD mutations and highlight a potential role for the WD40 domain in the mechanism of LRRK2-mediated cell death.

Figure 1. LRRK2 C-terminus forms distinct beta-propeller configuration.

Molecular surface of the homology model of the WD40 domain of LRRK2 (left) using the structure of the BUB3 mitotic checkpoint protein (PDB code 1yfq) as a template. The coloring of the surface is determined by the electrostatic potential at each point on the surface (red = acidic, white = neutral and blue = basic). The prominent basic cleft shown at the center of the molecule was consistently present in other models of the WD40 domain of LRRK2 based on other templates. The basic character of this cleft was due to a set of basic residues that were consistently placed in each of the models we examined (K2367, R2413, K2415, R2456, R2477 and K2478). A ribbon diagram of the same model is shown at the right, highlighting these residues in sphere representation. These residues were consistently clustered together in all of the models examined. Similar modeling of LRRK1 C-terminus failed, highlighting previously suggested divergence between LRRK1 and LRRK2 in this region.

Figure 2. The WD40 domain is critical for LRRK2 neurotoxicity and autophosphorylation.

(A) Schematic of LRRK2 and ΔWD40-LRRK2 showing major domains. The WD40 domain was removed by terminating LRRK2 at amino acid 2146. (B) A LRRK2-transfected apoptotic neuron. The arrow indicates a non-apoptotic nucleus, and the arrowhead indicates an apoptotic cell co-transfected with GFP and RC-LRRK2. (C) Removal of the WD40 domain abolishes the neurotoxicity of PD-mutant forms of LRRK2. Wild-type LRRK2 (WT), R1441C LRRK2 (RC), and G2019S LRRK2 (GS) with and without the WD40 domain (ΔWD40) were assessed for neurotoxicity. Murine cortical neurons were co-transfected with LRRK2 constructs and GFP, and the percentage of apoptotic nuclei were assessed 48 hours post-transfection. Data represent the mean±seven individual experiments. Data was assessed using ANOVA followed by Duncan's Multiple Range analysis p<0.05. (D) Removal of the WD40 domain has a differential effect on LRRK2 autophosphorylation and trans-phosphorylation. The different forms of LRRK2 were immunoprecipitated from 293T cells and assessed for their ability to autophosphorylate and to trans-phosphorylate MBP. Top panel is an autoradiogram and bottom panel is silver-stained gel demonstrating the presence of the different forms of LRRK2 and MBP in similar amounts between different conditions.

Figure 3. LRRK2 is found in a dimeric and a high molecular weight complex.

(A) Native gel electrophoresis of wild type and PD mutant forms of LRRK2. GFP-tagged wild-type, R1441C- and G2019S-LRRK2 were separately transfected into 293T cells and whole cell lysates prepared from these cells were separated by a non-denaturing blue native gel and immunoblotted using anti-GFP antibody. (B) Size-exclusion gel filtration chromatography of wild type and PD mutant forms of LRRK2. Lysates prepared from LRRK2-transfected cells as in (A) were separated by gel filtration chromatography show a similar pattern of two complexes as seen using blue native electrophoresis. (C) The α-complex is a LRRK2 dimer. 293T cells were co-transfected with FLAG- and V5-tagged LRRK2 and separated by size exclusion gel filtration as in (B). Fractions 20–23 (α-complex) and 12–15 (β-complex) were pooled and immunoprecipitated with the anti-FLAG antibody and immunoblotted with anti-FLAG (upper panel) or anti-V5 (lower panel). The amount of V5-LRRK2 that co-immunoprecipitates with the anti-FLAG antibody indicates that the majority, if not all, of the α-complex is a LRRK2 dimer, while the majority of LRRK2 in the β-complex is monomeric. (D) V5-LRRK2 singly transfected and processed as in (C) demonstrates that there is no cross reactivity between anti-FLAG and V5-LRRK2.

Figure 4. The WD40 domain is necessary for the formation of the dimeric LRRK2 α-complex.

(A) Native gel electrophoresis demonstrates absence of the LRRK2 dimer for ΔWD40-LRRK2, and an increase in the high molecular weight LRRK2 immunoreactivity (B) Size-exclusion gel filtration chromatography of ΔWD40-LRRK2 similarly demonstrates a decrease in the α-complex and increase in β-complex.