Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

Keywords: BAG5; GAK; autophagy; trans-Golgi.

Fig. 1.

A LRRK2 protein complex. (A) Ideograms of LRRK2 and candidate protein interactors. LRRK2 shows the location of the mutations used in this study, including pathogenic mutations (red) and T1348N or K1906M hypothesis-testing mutants (black). Domains include leucine-rich repeat (LRR), Ras of complex protein (ROC), and COR as well as kinase and WD40 domains. The protoarray baits used in each experiment are boxed. BAG5 has five BAG domains. GAK has PTEN-like domain (PTEN), Clathrin binding domain (CBD), and J domain. Rab7L1 has a single Ras-like domain. (B–E) Single-protein interactions. Coimmunoprecipitations using antibodies to (B and C) Flag or (D) GFP from cell lysates expressing (B) BAG5, (C) Rab7L1, or (D) GAK with (B) mock-transfected cells, (C) Flag-β-glucoronidase (GUS), or (D) GFP as negative controls probed for endogenous LRRK2 (arrows), Hsc70 (closed arrowhead), and tagged proteins (open arrowheads). IB, immunoblotting; IP, immunoprecipitation. (E) IP of BAG5 probed for endogenous GAK (arrowheads). (F and G) Cocomplex formation between LRRK2, GAK, BAG5, and Rab7L1. (F) Lysates were immunoprecipitated first with Flag for BAG5 and then with GFP for GAK, and they were probed for LRRK2 and interacting partners. (G) Flag-tagged Rab7L1 complexes probed for endogenous LRRK2, GAK, Hsc70, and Bag5. For all panels, markers on the right of the blots are in kilodaltons, and results are representative of at least three independent experiments.

Fig. 2.

(A) LRRK2 complex and LRRK2 mutations promote clearance of trans-Golgi–derived vesicles. Clustering and clearance of trans-Golgi by LRRK2 and interacting partners. Primary neurons were transfected with the indicated constructs and stained for transfection markers (green) and trans-Golgi (GLG1; blue). Right shows higher magnification of the circled neurons, whereas adjacent cells (asterisks) show normal Golgi morphology, including clustered Golgi in Rab7L1 (Lower Right) transfections and a LRRK2-transfected neuron with minimal residual Golgi staining (Center Right). (B) Blinded counts of cells with normal Golgi (blue), clustered Golgi (orange), or minimal or no apparent Golgi staining (white) as a proportion of all counted cells (n > 50 cells in three independent cultures per construct, P values are by Fisher exact test for proportions against GUS-transfected cultures corrected for the number of tests applied). (C and D) Pathogenic mutations in LRRK2 increase clearance of the trans-Golgi network. HEK cells were transfected with indicated LRRK2 variants or GUS as a negative control and stained with antibodies to TGN46. Counting of different Golgi phenotypes after transfections with different LRRK2 variants (C) without or (D) with cotransfection of Rab7L1 and statistical analysis were performed as in B. (E) Minimal functional complex includes LRRK2, BAG5 GAK, and Rab7L1. Cells were transfected with GUS (negative control for transfection), BAG5, LRRK2, GAK, or Rab7L1 and simultaneously treated with siRNA against the other components. Counting of different Golgi phenotypes after transfections with different combinations was performed as in C and D.

Fig. 3.

Acute expression of mutant LRRK2 in adult mouse brain results in diminished staining of TGN markers in vivo. (A) Mouse striatum sections from animals 2 wk after injection with lentiviral vectors expressing either (Left) eGFP or (Right) G2019S mutant eGFP-LRRK2 stained for GFP (green), GLG1 (red), and TOPRO3 (blue in Bottom). (B) Counting of cells with minimal GLG1 staining in animals transduced with the vectors, with untransduced cells in the same sections also counted as controls. Error bars show SEM. ***P < 0.001 (Tukey posthoc test from one-way ANOVA compared with eGFP control).

Fig. 4.

Mechanism of turnover of Golgi-derived vesicles involves the autophagy–lysosomal system and is consistent with altered gene expression in the human brain. (A–C) Selective Golgi clearance involves the autophagy–lysosome system. (A) Cells coexpressing LRRK2 (red) and GUS (blue) stained for TGN46 (green) after treatment for 24 h with DMSO or 10 nM bafilomycin A1. A cell with only residual TGN46 is shown in Right. (B and C) Blinded counts of the proportions of Golgi phenotypes in cells treated with (B) bafilomycin A1 or (C) knockdown of Atg7. (D and E) Gene expression in the human brain. (D) Direct assay of rs708723 in Rab7L1 from the brain of an individual who was heterozygous at this marker using RNA-Seq. (E) Density plots for the proportion of reads with the C allele for RNA-Seq (red) and exome data for DNA (blue) in brains from individuals heterozygous for rs708723. There is departure from the expected proportion of 0.5 (black vertical line) for RNA-Seq data.