Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson's disease.

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.

Lysosomal glucocerebrosidase is needed for ciliary Hedgehog signaling: A convergent pathway contributing to Parkinson's disease.

Parkinson's disease is characterized by loss of dopamine neurons that project to the dorsal striatum, and mutations in and are the most common genetic causes of familial Parkinson's disease. Previously, we showed that pathogenic 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.

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

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of Rab GTPases that regulate receptor trafficking, and LRRK2-activating mutations are linked to Parkinson's disease. Rab phosphorylation is a transient event that can be reversed by phosphatases, including protein phosphatase, Mg2/Mn2 dependent 1H (PPM1H), which acts on phosphorylated Rab 8A (phosphoRab8A) and phosphoRab10. Here, we report a phosphatome-wide small interfering RNA (siRNA) screen that identified PPM1M as a phosphoRab12-preferring phosphatase that also acts on phosphoRab8A and phosphoRab10.

Restoration of striatal neuroprotective pathways by kinase inhibitor treatment of Parkinson's disease-linked -mutant mice.

Parkinson's disease is associated with activating mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), which suppresses primary cilia formation in cholinergic and parvalbumin interneurons and astrocytes in the striatum. As a result, there is a decrease in the production of neuroprotective glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), which normally support the viability of dopaminergic neurons. MLi-2 is a brain-penetrant, selective, and now experimental inhibitor of LRRK2.

Leucine-Rich Repeat Kinase 2: Pathways to Parkinson's Disease.

The past 10 years have seen tremendous progress in our understanding of leucine-rich repeat kinase 2 (LRRK2) and how mutations activate the kinase and trigger downstream pathology, contributing to Parkinson's disease. A breakthrough came from the identification of key LRRK2 substrates-a subset of small guanosine triphosphatases (GTPases) called Rab proteins. Cryoelectron microscopy has revealed structures of LRRK2 and showed how inhibitors engage and inhibit the kinase.

End-product inhibition of the LRRK2-counteracting PPM1H phosphatase.

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 ; 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.

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

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of Rab GTPases that regulate receptor trafficking; activating mutations in 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.

Endogenous LRRK2 and PINK1 function in a convergent neuroprotective ciliogenesis pathway in the brain.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) and PTEN-induced kinase 1 (PINK1) are associated with familial Parkinson's disease (PD). LRRK2 phosphorylates Rab guanosine triphosphatase (GTPases) within the Switch II domain while PINK1 directly phosphorylates Parkin and ubiquitin (Ub) and indirectly induces phosphorylation of a subset of Rab GTPases. Herein we have crossed LRRK2 [R1441C] mutant knock-in mice with PINK1 knock-out (KO) mice and report that loss of PINK1 does not impact endogenous LRRK2-mediated Rab phosphorylation nor do we see significant effect of mutant LRRK2 on PINK1-mediated Rab and Ub phosphorylation.

Pathogenic mutations cause loss of primary cilia and Neurturin in striatal parvalbumin interneurons.

Parkinson's disease-associated, activating mutations in the LRRK2 kinase block primary cilium formation in cell culture and in specific cell types in the brain. In the striatum that is important for movement control, about half of astrocytes and cholinergic interneurons, but not the predominant medium spiny neurons, lose their primary cilia. Here, we show that mouse and human striatal parvalbumin interneurons that are inhibitory regulators of movement also lose primary cilia.

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's.

Leucine-Rich Repeat Kinases.

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65 human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors.

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Activating LRRK2 mutations cause Parkinson's disease. Previously, we showed that cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2 mutant mice. Single nucleus RNA sequencing shows that cilia loss in cholinergic interneurons correlates with higher LRRK2 expression and decreased glial derived neurotrophic factor transcription.

Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B.

We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding.

Localization of PPM1H phosphatase tunes Parkinson's disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis.

PPM1H phosphatase reverses Parkinson's disease-associated, Leucine Rich Repeat Kinase 2-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.

Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase.

Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022).

A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation.

Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease, and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation.

LRRK2 phosphorylation of Rab GTPases in Parkinson's disease.

Rab GTPases comprise a large family of conserved GTPases that are critical regulators of the secretory and endocytic pathways. The human genome encodes ~ 65 Rabs that localize to discrete membrane compartments and, when in their GTP-bound state, bind to effector proteins to carry out diverse functions. Activating mutations in LRRK2 kinase cause Parkinson's disease, and subsets of Rab GTPases are important LRRK2 substrates.

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export.

We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER-lysosome membrane contact sites.

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain.

Activating LRRK2 mutations cause Parkinson's disease, and pathogenic LRRK2 kinase interferes with ciliogenesis. Previously, we showed that cholinergic interneurons of the dorsal striatum lose their cilia in R1441C LRRK2 mutant mice (Dhekne et al., 2018).

Rab29 Fast Exchange Mutants: Characterization of a Challenging Rab GTPase.

Rab29 has been implicated in multiple membrane trafficking processes with no described effectors or regulating proteins. Its fast nucleotide exchange rate and inability to bind GDI in cytosol make it a unique and poorly understood Rab. Because the conventional, "GTP-locked" Rab mutation does not have the desired effect in Rab29, we present here the use of a fluorescence-based assay to characterize novel Rab29 mutants (I64T and V156G) that display faster nucleotide exchange rates, allowing for GEF-independent Rab29 activation.

LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade.

Activating mutations in LRRK2 kinase causes Parkinson's disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis.

Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins.

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1, and together, these proteins block cilia formation in a variety of cell types, including patient derived iPS cells. We have used live-cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and the loss of cilia seen upon serum readdition.

Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins.

Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1's N-terminal domain need not release from the rest of the protein for efficient cholesterol export.

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins.

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins.

Membrane association but not identity is required for LRRK2 activation and phosphorylation of Rab GTPases.

LRRK2 kinase mutations cause familial Parkinson's disease and increased phosphorylation of a subset of Rab GTPases. Rab29 recruits LRRK2 to the trans-Golgi and activates it there, yet some of LRRK2's major Rab substrates are not on the Golgi. We sought to characterize the cell biology of LRRK2 activation.