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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. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's. [ABSTRACT FROM AUTHOR]

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. 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 colocalization 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 may maintain active Rab GTPases on the Golgi to carry out their nonciliogenesis-related functions in membrane trafficking. [ABSTRACT FROM AUTHOR]

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. Here, we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360–450 and show that this domain, termed ‘site #1,’ can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher-affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed ‘site #2’ that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

LRRK2 serine/threonine kinase is associated with inherited Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal‐dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here, we have identified the molecular determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110‐residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3‐D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimaera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab‐specific interaction domain within a conserved phosphatase fold. Synopsis: PPM1H phosphatase belongs to the PPM (PP2C) family of enzymes that have a conserved Mg2+/Mn2+‐dependent catalytic domain. PPM1H counters the LRRK2 kinase pathway by dephosphorylating a subset of Rab GTPases. Through X‐ray, biochemical and cellular studies, the molecular basis for specificity can be attributed to a novel flap domain that has evolved to recognize Rab GTPases. Mutations in the flap domain reduce the ability of PPM1H to hydrolyze a phosphorylated threonine in the switch 2 helix of Rab8a and Rab10Grafting of the PPM1H flap domain onto PPM1J phosphatase is sufficient to enable PPM1J to dephosphorylate Rab8a and Rab10 both in vitro and in cellular assaysA hydrophobic N‐terminal anchor that is conserved in PPM1H, PPM1J and PPM1M contributes to folding of their catalytic domains [ABSTRACT FROM AUTHOR]

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). Here, we show that cilia loss is seen as early as 10 weeks of age in these mice and also in two other mouse strains carrying the most common human G2019S LRRK2 mutation. Loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases yields the same cilia loss phenotype seen in mice expressing pathogenic LRRK2 kinase, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. Moreover, astrocytes throughout the striatum show a ciliation defect in all LRRK2 and PPM1H mutant models examined. Hedgehog signaling requires cilia, and loss of cilia in LRRK2 mutant rodents correlates with dysregulation of Hedgehog signaling as monitored by in situ hybridization of Gli1 and Gdnf transcripts. Dopaminergic neurons of the substantia nigra secrete a Hedgehog signal that is sensed in the striatum to trigger neuroprotection; our data support a model in which LRRK2 and PPM1H mutant mice show altered responses to critical Hedgehog signals in the nigrostriatal pathway.

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. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition. [ABSTRACT FROM AUTHOR]

This patent describes the novel pyrroloppyrimidine compounds as LRRK2 kinase inhibitors. The patent includes the synthesis of compounds, compositions containing them and their use in the treatment of or prevention of diseases associated with LRRK2 kinase activity, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS)., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Three-dimensional quantitative structure–activity relationship (3D-QSAR) modelling was conducted on a series of leucine-rich repeat kinase 2 (LRRK2) antagonists using CoMFA and CoMSIA methods. The data set, which consisted of 37 molecules, was divided into training and test subsets by using a hierarchical clustering method. Both CoMFA and CoMSIA models were derived using a training set on the basis of the common substructure-based alignment. The optimum PLS model built by CoMFA and CoMSIA provided satisfactory statistical results (q2= 0.589 andr2= 0.927 andq2= 0.473 andr2= 0.802, respectively). The external predictive ability of the models was evaluated by using seven compounds. Moreover, an external evaluation set with known experimental data was used to evaluate the external predictive ability of the porposed models. The statistical parameters indicated that CoMFA (after region focusing) has high predictive ability in comparison with standard CoMFA and CoMSIA models. Molecular docking was also performed on the most active compound to investigate the existence of interactions between the most active inhibitor and the LRRK2 receptor. Based on the obtained results and CoMFA contour maps, some features were introduced to provide useful insights for designing novel and potent LRRK2 inhibitors. [ABSTRACT FROM AUTHOR]

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. Here, we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'site #1,' can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher-affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'site #2' that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity., Competing Interests: EV, AA, CC, PL, EP, CL, JS, ES, HD, DA No competing interests declared, SP Senior editor, eLife, (© 2022, Vides et al.)

Abstract: LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. A combination of steady-state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady-state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in a 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S. [Copyright &y& Elsevier]

Abstract: Mutations in the alpha synuclein gene (SNCA) are the most potent cause of autosomal dominant Parkinson disease (PD) while mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause. We hypothesized that a direct interaction may exist between their protein products. Here we show that full-length Lrrk2 or fragments containing its kinase domain have a significant capacity to phosphorylate recombinant alpha synuclein (Asyn) at serine 129. Such phosphorylated Asyn is the major component of pathological deposits in PD. We further show that the G2019S mutation in Lrrk2, which is the most common genetic determinant of PD, has a significantly greater capacity than wild-type Lrrk2 to phosphorylate Asyn. This suggests that the G2019S mutant protein may cause PD by generating pathological levels of phosphorylated Asyn. Controlling Lrrk2 Asyn phosphokinase activity may be an approach to disease modifying therapy for PD and other synucleinopathies. [Copyright &y& Elsevier]

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). Here, we show that cilia loss is seen as early as 10 weeks of age in these mice and also in two other mouse strains carrying the most common human G2019S LRRK2 mutation. Loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases yields the same cilia loss phenotype seen in mice expressing pathogenic LRRK2 kinase, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. Moreover, astrocytes throughout the striatum show a ciliation defect in all LRRK2 and PPM1H mutant models examined. Hedgehog signaling requires cilia, and loss of cilia in LRRK2 mutant rodents correlates with dysregulation of Hedgehog signaling as monitored by in situ hybridization of Gli1 and Gdnf transcripts. Dopaminergic neurons of the substantia nigra secrete a Hedgehog signal that is sensed in the striatum to trigger neuroprotection; our data support a model in which LRRK2 and PPM1H mutant mice show altered responses to critical Hedgehog signals in the nigrostriatal pathway., Competing Interests: SK, YS, HD, FT, KB, DA, SP No competing interests declared, (© 2021, Khan et al.)

Rab8a is associated with the dynamic regulation of membrane protrusions in polarized cells. Rab8a is one of several Rab GTPases that are substrates of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase that is linked to Parkinson's disease. Rab8a is phosphorylated at T72 (pT72) in its switch 2 helix and recruits the phospho-specific effector RILPL2, which subsequently regulates ciliogenesis. Here, we report the crystal structure of phospho-Rab8a (pRab8a) in complex with the RH2 (RILP homology) domain of RILPL2. The complex is a heterotetramer with RILPL2 forming a central α-helical dimer that bridges two pRab8a molecules. The N termini of the α helices cross over, forming an X-shaped cap (X-cap) that orients Arg residues from RILPL2 toward pT72. X-cap residues critical for pRab8a binding are conserved in JIP3 and JIP4, which also interact with LRRK2-phosphorylated Rab10. We propose a general mode of recognition for phosphorylated Rab GTPases by this family of phospho-specific effectors. • Structure of LRRK2-phosphorylated Rab8a in complex with RH2 domain of effector RILPL2 • Phosphothreonine in switch 2 of Rab8a recognized by an arginine from RILPL2 • JIP3 and JIP4 have conserved arginine and bind to LRRK2-phosphorylated Rab10 • Conserved mode of phospho-Rab recognition by RH2 domain of effector proteins LRRK2 is a serine/threonine kinase associated with Parkinson disease. LRRK2 phosphorylates Rab8a (pRab8a), which subsequently binds effector RILPL2 and regulates ciliogenesis. Waschbüsch et al. describe the structure of the pRab8a:RILPL2 complex and how phosphothreonine is recognized by an arginine from RILPL2. The structure reveals how LRRK2 assembles a trafficking complex. [ABSTRACT FROM AUTHOR]

Mutations in the Leucine-rich repeat kinase 2 ( LRRK2 ) gene are the most prevalent cause of familial Parkinson's disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S +/- mice. However, a significant increase in ciliogenesis was observed in G2019S +/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

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. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)