Pleiotropic effects for Parkin and LRRK2 in leprosy type-1 reactions and Parkinson's disease

Abstract

Type-1 reactions (T1R) are pathological inflammatory episodes and main contributors to nerve damage in leprosy. Here, we evaluate the genewise enrichment of rare protein-altering variants in 7 genes where common variants were previously associated with T1R. We selected 474 Vietnamese leprosy patients of which 237 were T1R-affected and 237 were T1R-free matched controls. Genewise enrichment of nonsynonymous variants was tested with both kernel-based (sequence kernel association test [SKAT]) and burden methods. Of the 7 genes tested 2 showed statistical evidence of association with T1R. For the LRRK2 gene an enrichment of nonsynonymous variants was observed in T1R-free controls (P_SKAT-O = 1.6 × 10^-4). This genewise association was driven almost entirely by the gain-of-function variant R1628P (P = 0.004; odds ratio = 0.29). The second genewise association was found for the Parkin coding gene PRKN (formerly PARK2) where 7 rare variants were enriched in T1R-affected cases (P_SKAT-O = 7.4 × 10^-5). Mutations in both PRKN and LRRK2 are known causes of Parkinson's disease (PD). Hence, we evaluated to what extent such rare amino acid changes observed in T1R are shared with PD. We observed that amino acids in Parkin targeted by nonsynonymous T1R-risk mutations were also enriched for mutations implicated in PD (P = 1.5 × 10^-4). Hence, neuroinflammation in PD and peripheral nerve damage due to inflammation in T1R share overlapping genetic control of pathogenicity.

Keywords: LRRK2; Parkin; Parkinson’s disease; inflammation; leprosy type-1 reaction.

Fig. 1.

Overview of the deep resequencing for the LRRK2 and PRKN genes. At the top, the population mean depth of coverage is plotted according to the exons encoding the LRRK2 gene (A) and PRKN (B). Blue circles indicate the depth of coverage per base pair with the light blue shade representing the SD of the mean. A red horizontal line marks the average depth of coverage for the 2 genes in the studied population. The protein functional domains are shown in the center and linked to their respective coding exons. The location of known coding mutation of LRRK2 and Parkin reported by either dbSNP or gnomAD, mutation associated with Parkinson’s disease in PDmutDB and LOVD and mutations associated with T1R are shown as yellow with blue bars denoting variants that overlap between PD and T1R. At the bottom, the allele count or the frequency of LRRK2 and PRKN mutations is shown for the T1R-affected and T1R-free samples.

Fig. 2.

The LRRK2 R1628P rare variant displays an increased respiratory burst in response to challenge with both live bacillus Calmette–Guérin and M. leprae. A and B show the relative abundance of LRRK2 for wild-type (WT) RAW cells and 3 CRISPR/Cas-edited mutant cell lines in both resting state and under bacillus Calmette–Guérin stimulated conditions in a representative Western blot (A) and as triplicates with SDs (B). C–F show the kinetics of total reactive oxygen species (ROS) produced by RAW cells upon challenge with bacillus Calmette–Guérin (C and D) or M. leprae (E and F). LRRK2 knockout (KO) cells and the nonsynonymous mutations (1628P/P, 1628R/P, and 755L/L) were compared with WT using two-way ANOVA. *P < 0.05; **P < 0.01 and ***P < 0.001. The experiments were repeated at least 3 times with similar results. Two independent clones for the 1628P/P were tested with similar results. Compared with WT, LRRK2 KO cells consistently produced less ROS upon bacterial challenge with bacillus Calmette–Guérin or M. leprae (C–F). In contrast, there was no significant difference in the kinetics of ROS production following exposure to bacillus Calmette–Guérin between WT and LRRK2 755L/L-expressing cells (D). Compared with cells expressing LRRK2 WT, cells expressing the LRRK2 1628P/P variant consistently showed increased ROS production following exposure to both bacillus Calmette–Guérin and M. leprae (C and E). Compared with LRRK2 WT, cells heterozygotic for LRRK2 1628R/P also displayed higher ROS production upon M. leprae stimulation which was not significantly different from the response of homozygous LRRK2 1628P/P carriers.

Fig. 3.

The LRRK2 R1628P mutation abrogates apoptosis induced by live bacillus Calmette–Guérin. Fluorescence-activated cell sorting (FACS) was used to evaluate the impact of LRRK2 nonsynonymous variants on apoptotic cell death upon bacillus Calmette–Guérin challenge. (A) Cellular staining as revealed by FACS analyses for RAW cells expressing LRRK2 WT and genome-edited proteins after 24 h of coincubation with live bacillus Calmette–Guérin and unchallenged baselines. (B and C) Proportion of cells undergoing apoptosis. The bar plots display the mean estimate for 2 experiments done in triplicate with their respective SEs. There were no significant differences in the proportions of live or apoptotic cells under unstimulated condition. Infection with bacillus Calmette–Guérin triggered a significant (P < 0.001) increase of apoptosis in wild-type, 755L LRRK2, and LRRK2-depleted (KO) cells with a corresponding decline of live cells. In contrast, 2 independent clones expressing 1628P/P LRRK2 protein did not respond with a significant increase of cell death or apoptosis. When stimulated with bacillus Calmette–Guérin, cells depleted for LRRK2 (KO) expressed significantly higher apoptosis compared with mutant clones or wild-type cells (P < 0.001). The levels of total apoptosis showed a significantly dose-dependent trend with the 1628R/P line situated between wild-type and homozygous 1628P/P lines (P = 0.003 including LRRK2 KO and P = 0.04 without LRRK2 KO). Statistical tests were performed using one-way ANOVA with Tukey correction and a linear mixed model for the trend test.

Fig. 4.

Characterization of Parkin mutations shared by PD and T1R cases. (A) Melting temperatures (T_m) of recombinant human Parkin mutants, measured using differential scanning fluorescence. (B) Mass spectrometry analysis of Parkin phosphorylation by PINK1. Human Parkin (20 μM) is phosphorylated by PINK1 (1 μM) for 15 min in the presence of ATP (5 mM) and ubiquitin (20 μM). The arrows indicate a 79.9-amu (atomic mass unit) shift resulting from phosphorylation. (C) The fraction (percent) of Parkin and ubiquitin phosphorylation was calculated from the MS peak intensities. (D) SDS/PAGE analysis of Parkin ubiquitination reactions following PINK1 phosphorylation. In step 1, human Parkin (2 μM) is phosphorylated by PINK1 (0.1 μM) for 15 min in the presence of ATP (5 mM) and ubiquitin (50 μM). In step 2, E1 (50 nM) and UbcH7 (2 μM) are added to allow ubiquitination for another 15 min at 37 °C. Parkin auto-ubiquitinates to form smears and also ubiquitinates UbcH7. (E) Ubiquitination activity was quantified using loss of the unmodified UbcH7 band, compared with the no ATP control. (F) Mitophagy was examined using a FACS-based analysis of mitochondrially targeted Keima (mt-Keima). Representative FACS data of mt-Keima expressing WT or PRKN mutants untreated (control) or treated with CCCP (20 μM) for 4 h from at least 3 independent experiments. (G) Bars showing mitophagy percentage for either untreated (control) or after 4 h of CCCP treatment quantified from mt-Keima signal in U2OS cells expressing GFP-Parkin variants normalized to wild-type (WT) Parkin. (H) Bars showing GFP intensity of untreated cells expressing GFP-Parkin variants normalized to WT Parkin. The quantification of average percentage for the mitophagy was a combination of at least 3 independent experiments. P values for all of the plots were determined by one-way ANOVA with Dunnett’s post hoc tests. *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant.