LRRK2 activation controls the repair of damaged endomembranes in macrophages

Abstract

Cells respond to endolysosome damage by either repairing the damage or targeting damaged endolysosomes for degradation via lysophagy. However, the signals regulating the decision for repair or lysophagy are poorly characterised. Here, we show that the Parkinson's disease (PD)-related kinase LRRK2 is activated in macrophages by pathogen- or sterile-induced endomembrane damage. LRRK2 recruits the Rab GTPase Rab8A to damaged endolysosomes as well as the ESCRT-III component CHMP4B, thereby favouring ESCRT-mediated repair. Conversely, in the absence of LRRK2 and Rab8A, damaged endolysosomes are targeted to lysophagy. These observations are recapitulated in macrophages from PD patients where pathogenic LRRK2 gain-of-function mutations result in the accumulation of endolysosomes which are positive for the membrane damage marker Galectin-3. Altogether, this work indicates that LRRK2 regulates endolysosomal homeostasis by controlling the balance between membrane repair and organelle replacement, uncovering an unexpected function for LRRK2, and providing a new link between membrane damage and PD.

Keywords: LRRK2; Parkinson's disease; endolysosomal damage; lysosomes; tuberculosis.

Figure EV1. Infection triggers LRRK2‐dependent Rab8A phosphorylation in macrophages

1. A–C

   RAW264.7 macrophages were left uninfected or infected with (A) Mycobacterium tuberculosis (Mtb), (B) C. albicans (Ca) or (C) L. monocytogenes (Lm) for the indicated time, and Rab8A pT72, Rab10 pT73 and LRRK2 pS935 levels were analysed by Western blot.

2. D–F

   WT and LRRK2 KO macrophages were left uninfected or infected with (D) Mtb for 24 h, (E) Ca or (F) Lm for 120 min. Rab8A pT72 and LRRK2 pS935 phosphorylation was analysed by Western blot.

3. G–I

   RAW264.7 macrophages were pre‐treated with 1 μM GSK2578215A (GSK inh) or 0.1 μM MLi‐2 and left uninfected or infected with (G) Mtb for 24 h, (H) Ca or (I) Lm for 120 min. Rab8A pT72 and LRRK2 pS935 phosphorylation was analysed by Western blot and quantified by densitometry. Beta‐actin was used as loading control. Data represent the mean + SEM of three independent biological replicates. One‐way ANOVA followed by Dunnett's test against DMSO control. ns = non‐significant; *P < 0.05; ***P < 0.001

Source data are available online for this figure.

Figure EV2. Galectin‐3 recruitment to the pathogens used in this study

RAW264.7 macrophages were infected with the indicated strains of Mtb, Ca or Lm, and Galectin‐3 recruitment was visualised by immunofluorescence. Data represent mean + SEM of three to four independent biological experiments.

1. Infection with Mtb WT or Mtb ΔRD1 for 24 h. Scale bar = 10 μm.

2. Infection with Lm WT or Lm Δhly for 60 min. Scale bar = 5 μm.

3. Infection with Ca WT or Ca YL for 60 min. Scale bar = 10 μm.

Figure 1. LRRK2 is activated by pathogens that damage phagosomal membranes

1. A–D

   RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One‐way ANOVA followed by Dunnett's test compared to WT strains. ns = non‐significant; *P ≤ 0.05; **P ≤ 0.01. (B) Recruitment of endogenous LRRK2 and EGFP‐Rab8A to Mycobacterium tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm. (C, D) Endogenous LRRK2 and Rab8A recruitment to (C) Candida albicans (Ca) and (D) Listeria monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represent the mean ± SEM of two to four independent biological replicates.

Source data are available online for this figure.

Figure EV3. LRRK2 mediates Rab8A recruitment to pathogen‐containing phagosomes

1. A

   WT and LRRK2 KO macrophages were transfected with EGFP‐Rab8A and infected with Mtb for 24 h. Scale bar = 10 μm.

2. B, C

   (B) WT and LRRK2 KO macrophages were infected with Ca and (C) Lm for 60 min. Rab8A recruitment was visualised by immunofluorescence. Scale bar = 5 μm.

3. D

   Macrophages were transfected with EGFP‐Rab8A, treated with 1 μM GSK2578215A (GSK inh) and infected with Mtb for 24 h. Scale bar = 10 μm.

4. E, F

   (E) Macrophages were treated with the GSK LRRK2 kinase inhibitor (1 μM) and infected with Ca or (F) Lm for 60 min. Rab8A recruitment was visualised by immunofluorescence. Scale bar = 5 μm.

5. G–I

   Representative images of macrophages transfected with EGFP‐Rab8A‐WT or EGFP‐Rab8A‐T72A and infected with (G) Mtb for 24 h, (H) Ca for 60 min and (I) Lm for 30 min. Scale bar = 5 μm.

Data information: Right panels show the quantification of the percentage of Rab8A‐positive phagosomes. Data represent the mean + SEM of three independent biological replicates. Student's t‐test; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Endolysosomal damage is sufficient for LRRK2 activation

1. A

   RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time, and Rab8A and Rab8A pT72 levels were analysed by Western blot.

2. B

   WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot.

3. C

   Macrophages were pre‐treated with either 1 μM GSK inh or 0.1 μM MLi‐2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.

4. D–F

   RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP‐1‐positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of (D, E). Data represent the mean ± SEM of three independent biological experiments.

5. G, H

   LRRK2 WT or LRRK2 KO macrophages were pre‐treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of (G) LRRK2‐ or (I) Rab8A‐positive vesicles per cell was monitored by immunofluorescence and high‐content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One‐way ANOVA followed by Dunnett's test against the untreated WT control. ns = non‐significant; **P ≤ 0.01.

Source data are available online for this figure.

Figure 3. Rab8A is recruited to damaged lysosomes containing Galectin‐3 and CHMP4B in response to calcium signalling

1. A

   RAW264.7 macrophages were electroporated with EGFP‐Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin‐3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.

2. B

   Macrophages were electroporated with EGFP‐Rab8A and RFP‐Galectin‐3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.

3. C

   RAW264.7 macrophages were treated with 1 mM of LLOMe, and LRRK2, Rab8A, CHMP4B, Galectin‐3 and LC3B foci over time were analysed by high‐content imaging. Data show the mean ± SEM of five biological replicates.

4. D–H

   RAW264.7 macrophages were pre‐treated with 10 μM BAPTA‐AM for 1 h, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high‐content imaging. Scale bar = 10 μm. (E) Rab8A pT72 phosphorylation was analysed by Western blot. (F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high‐content imaging. Scale bar = 10 μm. (H) Quantification of E and F. (D and H) Data represent the mean ± SEM of two to three independent biological experiments. One‐way ANOVA followed by Sidak's multiple comparisons test. ns = non‐significant; **P ≤ 0.01, *P ≤ 0.05.

Source data are available online for this figure.

Figure EV4. Characterisation of Rab GTPase behaviour after sterile damage

1. RAW264.7 macrophages were electroporated with EGFP‐Rab3A, EGFP‐Rab8A, EGFP‐Rab10 and EGFP‐Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high‐content immunofluorescence imaging. Scale bar = 10 μm.

2. WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot.

3. WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot.

4. RAW264.7 macrophages were electroporated with EGFP‐Rab8A‐WT, EGFP‐Rab8A‐Q67L, EGFP‐Rab8A‐T22N and EGFP‐Rab8A‐T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 μm.

5. CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean.

Source data are available online for this figure.

Figure 4. LRRK2 and Rab8A coordinate the recruitment of CHMP4B to damaged endolysosomes

1. A, B

   RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre‐treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. (A) CHMP4B and (B) Galectin‐3 vesicle numbers were analysed by immunofluorescence and high‐content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B‐ or Galectin‐3‐positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns = non‐significant, *P ≤ 0.05, **P ≤ 0.01 by one‐way ANOVA followed by Sidak's multiple comparisons test.

2. C, D

   Live cell imaging of LysoTracker‐positive spots in WT, LRRK2 KO or Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash‐out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.

Figure EV5. LRRK2 does not affect sensing of endolysosomal membrane damage by Galectin‐8

1. RAW264.7 cells were transfected with EGFP‐Rab8A and treated with 1 mM LLOMe for 30 min. Co‐localisation with Galectin‐8 was visualised by immunofluorescence. Scale bar = 5 μm.

2. RAW264.7 WT and LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min. Galectin‐8 vesicle numbers were analysed by immunofluorescence and high‐content imaging.

3. RAW264.7 macrophages treated or not with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin‐8 vesicle numbers were analysed by immunofluorescence and high‐content imaging.

4. RAW264.7 WT or Rab8A KO macrophages were treated with 1 mM LLOMe for 30 min. Galectin‐8 vesicle numbers were analysed by immunofluorescence and high‐content imaging.

Data information: Data represent number of vesicles per cell. Data show the mean ± SEM of one representative experiment out of three biological replicates shown. ns = non‐significant, *P < 0.05, ***P < 0.001 by one‐way ANOVA followed by Sidak's multiple comparisons test.

Figure 5. Damaged endolysosomes are targeted to lysophagy in the absence of LRRK2 and Rab8A signalling

1. A, B

   WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre‐treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (A) LC3B and (B) ubiquitin K63‐positive vesicle numbers were analysed by immunofluorescence and high‐content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns = non‐significant, *P ≤ 0.05, **P ≤ 0.01 by one‐way ANOVA followed by Sidak's multiple comparisons test.

2. C

   Representative images of LC3B/LAMP‐1 double‐positive endolysosomes in control and LLOMe‐treated macrophages. Scale bar = 10 μm.

3. D

   Analysis of the % of LC3B/LAMP‐1 double‐positive endolysosomes in LLOMe‐treated macrophages. Data show mean ± SEM of three independent biological experiments. *P ≤ 0.05, ***P ≤ 0.001 by Student's t‐test.

Figure 6. LRRK2 regulates membrane damage‐induced bacterial pathogen intracellular growth

1. A–C

   WT and LRRK2 KO RAW264.7 macrophages were infected with (A) Mtb for 24 h, (B) Ca for 60 min and (C) Lm for 60 min. Galectin‐3 and LC3B recruitment was analysed by immunofluorescence and high‐content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns = non‐significant, *P ≤ 0.05 by Student's t‐test.

2. D–F

   WT and LRRK2 KO RAW264.7 macrophages were infected with (D) Mtb WT or Mtb ΔRD1, (E) Ca WT or Ca YL and (F) Lm WT or Lm Δhly. Growth measured for (D) 72 h and (E, F) 2 h. Data show the mean ± SEM of three independent biological experiments. ns = non‐significant, *P ≤ 0.05, **P ≤ 0.01 by one‐way ANOVA followed by Sidak's multiple comparisons test.

Figure 7. Macrophages from PD patients accumulate damaged endolysosomes

Monocyte‐derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2–3) or R1441C (Donor 1) LRRK2 mutation were pre‐treated with 0.1 μM MLi‐2 and treated with 1 mM LLOMe for 30 min.

1. Rab8A and Rab8A pT72 levels were analysed by Western blot.

2. Rab8A‐ and Galectin‐3‐positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns = non‐significant, **P ≤ 0.01, by one‐way ANOVA followed by Sidak's multiple comparisons test.

Source data are available online for this figure.