Ayan Adhikari

Parkinson’s disease is characterized by loss of dopamine neurons that project to the dorsal striatum, and mutations in LRRK2 and GBA1 are the most common genetic causes of familial Parkinson’s disease. Previously, we showed that pathogenic LRRK2 mutations inhibit primary cilia formation in rare interneurons and astrocytes of the mouse and human dorsal striatum. This blocks Hedgehog signaling and reduces synthesis of neuroprotective GDNF and NRTN, which normally support dopamine neurons vulnerable in PD. Here, we show that GBA1 mutations also impair Hedgehog signaling and Hedgehog-dependent neuroprotective factor production by a distinct mechanism. Loss of GBA1 activity increases lysosomal accessible cholesterol and thus decreases accessible cholesterol in primary cilia of cultured cells; this change in lipid composition blocks ciliary Hedgehog signaling that depends on accessible cholesterol. Consistent with defects in Hedgehog signaling in the mouse dorsal striatum, GBA1 mutant mice show reduced Hedgehog-induced Gdnf RNA expression in striatal cholinergic interneurons, with no detectable impact on cilia formation. Also, both LRRK2 and GBA1 mutations suppress Hedgehog-induced Bdnf expression in striatal astrocytes. These findings underscore the role of Hedgehog signaling in the nigrostriatal circuit and reveal a convergent mechanism by which distinct LRRK2 and GBA1 mutations may contribute to PD pathogenesis.

PPM1H phosphatase reverses Parkinson’s disease-associated, Leucine Rich Repeat Kinase 2-mediated, Rab GTPase phosphorylation. We showed previously that PPM1H relies on an N-terminal amphipathic helix for Golgi membrane localization and this helix enables PPM1H to associate with liposomes in vitro ; binding to highly curved liposomes activates PPM1H’s phosphatase activity. We show here that PPM1H also contains an allosteric binding site for its non-phosphorylated reaction products, Rab8A and Rab10. Microscale thermophoresis revealed that PPM1H binds thio-phosphorylated Rab8A at the active site with a KD of ∼1µM; binding of Rab8A and Rab10 to an alternative site is of similar affinity and is not detected for another LRRK2 substrate, Rab12. Non-phosphorylated Rab8A or Rab10 inhibit PPM1H phosphatase reactions at concentrations consistent with their measured binding affinities and fail to inhibit PPM1H L66R phosphatase reactions. Independent confirmation of non-phosphorylated Rab binding to PPM1H was obtained by sucrose gradient co-flotation of non-phosphorylated Rabs with liposome-bound PPM1H. Finally, Rab8A or Rab10 binding also requires PPM1H’s amphipathic helix, without which the interaction affinity is decreased about 6-fold. These experiments indicate that Golgi associated Rab proteins contribute to the localization of PPM1H and non-phosphorylated Rabs regulate PPM1H phosphatase activity via an allosteric site. Targeting this site could represent a strategy to enhance PPM1H-mediated dephosphorylation of LRRK2 substrates, offering a potential therapeutic approach to counteract LRRK2-driven Parkinson’s disease.

This protocol is used to purify full length PPM1H enzyme in good yield and purity after expression in bacteria. We generally obtain 2mg from 4L bacterial culture and express the protein at 18°C to avoid aggregation due to overexpression.

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. Teaser: Parkinson’s linked Rab phosphorylation is reversed by PPM1M; the inactive D440N variant is implicated in rare patient cases.

This protocol describes our method to measure phosphoRab GTPase dephosphorylation using purified phosphoRab GTPases and PPM family phosphatases. We include our protocol for generation of phosphatase substrates by phosphorylation of Rab GTPases by MST3 kinase.

SUMMARY: Mutations in LRRK2 and GBA1 are the most common genetic causes of familial Parkinson’s disease. Previously, we showed that pathogenic LRRK2 mutations inhibit primary cilia formation in rare interneurons and astrocytes of the mouse and human dorsal striatum. This blocks Hedgehog signaling and reduces synthesis of neuroprotective GDNF and NRTN, which support neurons vulnerable in PD. Here we show that GBA1 mutations also impair Hedgehog signaling through a distinct mechanism. Loss of GBA1 activity decreases accessible cholesterol in primary cilia of cultured cells, thereby disrupting Hedgehog signaling. In the mouse striatum, Gba1 mutations result in reduced Hedgehog-induced Gdnf RNA expression in cholinergic interneurons, despite having no detectable impact on cilia formation. Also, both Lrrk2 and Gba1 mutations suppress Hedgehog-induced Bdnf expression in striatal astrocytes. These findings underscore the role of Hedgehog signaling in the nigrostriatal circuit and reveal a convergent mechanism by which distinct mutations may contribute to PD pathogenesis.

There exist at least three different pools of cholesterol in the plasma membrane: the essential pool, the sphingomyelin-sequestered pool, and the accessible pool (Radhakrishnan et al, 2020). Ciliary Hedgehog signaling is regulated by the accessible pool of cholesterol (Kinnebrew et al, 2019), and His-mNeon-FLAG-ALOD4, a toxin-based probe, can be used to visualize this accessible pool. Here, we present a method for staining and measuring the amount of accessible cholesterol on cilia in 3T3 fibroblasts stably expressing Somatostatin Receptor (SSTR3)-mApple, a ciliary marker. The protocol described here is based upon previously established methods (Kinnebrew et al., 2019; Johnson and Radhakrishnan, 2021) and has also been used successfully to label cilia in RPE cells.

We present here a protocol for preparing liposomes that can be used to monitor binding of proteins to vesicles of various membrane curvatures. We used this to study the association of the PPM1H phosphatase with highly curved membranes due to its N-terminal amphipathic helix.

PPM1H phosphatase reverses Parkinson’s disease-associated, LRRK2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor co-localization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H maintains active Rab GTPases on the Golgi to carry out their non-ciliogenesis-related functions in membrane trafficking. Significance Statement: Pathogenic, hyperactive LRRK2 kinase is strongly linked to Parkinson’s disease and LRRK2 phosphorylates a subset of Rab GTPases that are master regulators of membrane trafficking. PPM1H phosphatase specifically dephosphorylates Rab8A and Rab10, the major LRRK2 substrates. Here we provide novel cell biological and biochemical insight related to the localization and activation of PPM1H phosphatase. Understanding how PPM1H modulates LRRK2 activity is of fundamental interest and also important, as activators of PPM1H may eventually benefit Parkinson’s disease patients.