PPM1M, a LRRK2-counteracting, phosphoRab12-preferring phosphatase with potential link to Parkinson's disease

Abstract

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of Rab GTPases that regulate receptor trafficking; activating mutations in LRRK2 are linked to Parkinson's disease. Rab phosphorylation is a transient event that can be reversed by phosphatases, including PPM1H, that acts on phosphoRab8A and phosphoRab10. Here we report a phosphatome-wide siRNA screen that identified PPM1M as a phosphoRab12-preferring phosphatase that also acts on phosphoRab8A and phosphoRab10. Upon knockout from cells or mice, PPM1M displays selectivity for phosphoRab12. As shown previously for mice harboring LRRK2 pathway mutations, knockout of Ppm1m leads to primary cilia loss in striatal cholinergic interneurons. We have also identified a rare PPM1M mutation in patients with Parkinson's disease that is catalytically inactive when tested in vitro and in cells. These findings identify PPM1M as a key player in the LRRK2 signaling pathway and provide a new therapeutic target for the possible benefit of patients with Parkinson's disease.

Figure 1.. PPM1H knockout does not influence the rate of phosphoRab turnover.

(A) Immunoblot analysis of wild-type (WT) and PPM1H knockout A549 cells, treated with 100 nM MLi-2 for the times indicated. Mass is shown at left in kDa here and in all subsequent figures; antigens are indicated at right. (B) Quantitation of pRab10/total Rab10 and pRab12/total Rab12 levels from immunoblots in (A), normalized to 1.0 for respective pRab10 or pRab12 WT 0 min conditions, as indicated. (C) Quantitation of pRab10/total Rab10 and pRab12/total Rab12 levels from immunoblots in (A), normalized to 1.0 for 0 min of each respective (WT or KO) condition to permit direct comparison. Error bars represent SEM from 3 independent experiments carried out in duplicate.

Figure 2.. Phosphatome-wide siRNA screen in 3T3 cells reveals PPM1M as a phosphoRab12-preferring phosphatase.

(A) Schematic describing screen workflow. (B, C) Summary plot of (B) pRab12/total Rab12 or (C) pRab10/total Rab10 levels after 72h siRNA and 20 min MLi-2 treatment, normalized to non-targeting control. Top 5 hits for each pRab as indicated. (D, E) Repeat immunoblots of the lysates of the top 10 hits from (B) for pRab12 and (C) for pRab10.

Figure 3.. PPM1M overexpression preferentially decreases phosphoRab12 compared with phosphoRab10.

(A) Immunoblot analysis of HEK293 cells overexpressing Flag-LRRK2 R1441C and HA-empty or HA-tagged PPM1H, PPM1H 153D, PPM1H D288A, PPM1M, PPM1M H127D, or PPM1M D235A. Cells were treated with 200 nM MLi-2 for 2h where indicated. (B) Quantitation of pRab12/total Rab12 and (C) pRab10/total Rab10 levels from immunoblots in (A), normalized to 1.0 for HA-empty. Error bars indicate SEM from two independent experiments analyzed in duplicate. Statistical significance determined by one way ANOVA, relative to HA-empty. For pRab10, ***p=0.0002 for PPM1H WT, **p=0.0097 for PPM1H H153D, ****p<0.0001 for PPM1H D288A, **p=0.0044 for PPM1M WT, **p=0.0014 for PPM1M D235A. For pRab12, ****p<0.0001 for PPM1H WT, PPM1H H153D, PPM1M WT, ***p=0.0002 for PPM1H D288A.

Figure 4.. Knockout of PPM1 subfamily phosphatases in MEF cells and tissues confirms PPM1M substrate preferences.

(A) Immunoblot analysis of parental (wild-type) and PPM1H, PPM1M, and PPM1J pooled knockouts in MEF cells. (B) Quantitation of pRab12/total Rab12 and pRab10/total Rab10 levels from immunoblots in (A), normalized to 1.0 for parental. Error bars indicate SEM from two independent experiments analyzed in duplicate. Statistical significance determined by one way ANOVA, respective to parental. For pRab10, *p=0.0122 for PPM1H knockout. For pRab12, *p=0.0117 for PPM1M knockout. (C) Immunoblot analysis of mouse embryonic fibroblasts (MEFs) derived from Ppm1m wild-type (Ppm1m^+/+), heterozygous knockout (Ppm1m^+/−), or homozygous knockout (Ppm1m^−/−) mice. (D) Quantitation of pRab12/total Rab12 and pRab10/total Rab10 levels from immunoblots in (C), normalized to 1.0 for the highest value. Each dot represents the average of two independent replicates from one mouse. Statistical significance determined by one way ANOVA. For pRab10, **p=0.0036, for pRab12 *p=0.0399 and **p=0.0029. (E) Immunoblot analysis of lung lysates from mice as in (C). (F) Quantitation of immunoblots in (E), normalized as in D. Each dot represents the average of three independent replicates from one mouse. For pRab12, *p=0.0335. (G) Immunoblot analysis of whole brain lysates from mice as in (C). (H) Quantitation from immunoblots in (G), as in F. Statistical significance determined by Kruskal-Wallis test for pRab10 and one way ANOVA for pRab12. For pRab10, *p=0.0349.

Figure 5.. PPM1M prefers phosphoRab12 over phosphoRab10 in vitro.

(A) Immunoblot analysis of pRab12 and pRab10 levels from in vitro biochemical reactions using 1.5μM pRab12 or pRab10 substrate and 50 or 100nM PPM1M enzyme, as indicated. (B) Quantitation of pRab12 and pRab10 levels from immunoblots in (A), normalized to 1.0 for 0 min. Error bars represent SEM from five independent experiments. (C) Phos-tag gel analysis of pRab8A dephosphorylation after in vitro biochemical reactions containing 2.5 μg pRab8A and indicated amounts of PPM1M. (D) Phos-tag gel analysis of pRab8A dephosphorylation after in vitro biochemical reactions containing 2.5 μg pRab8A and 200 ng of the indicated phosphatases.

Figure 6.. PPM1H and PPM1M flap domains are necessary for proper substrate recognition.

(A) Alphafold modeling of PPM1M (blue) and pRab12 (magenta). The PPM1H flap domain is shown in navy; phosphoserine 106 of the pRab12 substrate is indicated at the metal-containing PPM1M active site. (B) Diagram of PPM1H and PPM1M swapped flap domain constructs. (C) Immunoblot analysis of HEK293 cells overexpressing Flag-LRRK2 R1441C and HA-empty or HA-tagged PPM1H, PPM1M, PPM1H with PPM1M flap domain (PPM1H_M flap), or PPM1M with PPM1H flap domain (PPM1M_H flap). (D) Quantitation of pRab12/total Rab12 and pRab10/total Rab10 levels from immunoblots in (C), normalized to 1.0 for HA-empty. Error bars indicate SEM from four independent experiments analyzed in duplicate. Statistical significance determined by one way ANOVA, respective to HA-empty, ****p<0.0001.

Figure 7.. PPM1M knockout phenocopies LRRK2 hyperactive ciliation phenotype.

(A) Example confocal immunofluorescence micrographs of sections of the dorsal striatum from 3-month-old wild-type or Ppm1m^−/− mice; scale bar, 10 μm. Cholinergic interneurons were labeled using anti-choline acetyltransferase (ChAT) antibody (green); primary cilia were labeled using anti-AC3 (adenylate cyclase 3) antibody (magenta; yellow arrowhead). Nuclei were labeled using DAPI (blue). (B) Quantitation of ChAT⁺ neurons and (C) surrounding (mostly medium spiny) neurons containing a cilium. Error bars represent SEM from six individual brains per group, two to three sections per mouse. >36 ChAT⁺ neurons and >500 ChAT⁻ cells were scored per mouse. Statistical significance was determined using an unpaired t-test. ***p=0.0001 for cholinergic neurons; ns p=0.3835 for medium spiny neurons.

Figure 8.. Parkinson’s disease patient linked to PPM1M D440N mutation.

(A) Individuals genotyped for the c.1318G>A p.D440N mutation are indicated as D440N^+/−. In the case of II-4, the mutation was inferred from its presence in both of his children, III-3 and III-4, and is shown in brackets (D440N^+/−). A second-degree cousin of II-3 and II-4 also had PD but did not carry the D440N variant. The shared common ancestors of this individual with II-2 and II-4 are their great-grandparents (the grandparents of I-2, not shown in the pedigree). No clinical phenotype information for any of the members of generation III was available. (B) Alphafold modeling of D440 in the PPM1M (blue) active site shown with pRab12 (magenta, residues 38–222). Inset shows enlarged view of PPM1M D440 and Rab12 pS106. (C) Immunoblot analysis of HEK293 cells overexpressing Flag-LRRK2 R1441G and HA-empty, HA-PPM1M WT, HA-PPM1M H127D, HA-PPM1M D440N, with untransfected (UT) control. MLi-2 (200 nM) treatment was for 90 min as indicated. (D) Quantitation of pRab12/total Rab12 and pRab10/total Rab10 levels from immunoblots in (C), normalized to HA-empty. Error bars indicate SD from three independent experiments carried out in duplicate. Statistical significance determined by Welch’s t-test, followed by Benjamini-Hochberg correction for multiple comparisons, respective to HA-empty. ***p=0.00011 for pRab12 - PPM1M D440N, ***p=0.00078 for pRab10 - PPM1M H127D, otherwise ****p<0.0001.