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Andrew B. West - One of the best experts on this subject based on the ideXlab platform.
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Predictive Markers of LRRK2 Inhibition in Biofluids
bioRxiv, 2020Co-Authors: Shijie Wang, Kaela Kelly, Nathalie Schussler, Sylviane Boularand, Laurent Dubois, Frank Hsieh, Elizabeth Tengstrand, Jonathan M. Brotchie, James B. Koprich, Andrew B. WestAbstract:Hyper-activated LRRK2 is linked to Parkinsons disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, may be critical in the clinical development of successful LRRK2-targeting therapeutics. In this study, three structurally distinct, brain-penetrant, and selective LRRK2 small-molecule kinase inhibitors (PFE-360, MLi2, and RA283) were orally administered to groups of cynomolgus macaques at different doses. Biofluid markers with proposed predictive value for assessing LRRK2 inhibition were measured from samples of blood, urine, and cerebral-spinal fluid (CSF). LRRK2 kinase inhibition led to consistent reduced pS935-LRRK2 and pRab10 proteins in blood mononuclear cells, reduced exosome LRRK2 protein and di-docosahexaenoyl (22:6) bis (monoacylglycerol) phosphate in urine, and reduced exosome LRRK2 and autophosphorylated pS1292-LRRK2 protein in CSF. Incomplete LRRK2 kinase inhibition reduced LRRK2 protein secretion in exosomes whereas high drug exposures may reduce both exosome and tissue levels of LRRK2 protein. These orthogonal pairs of markers for LRRK2 inhibition in urine and CSF can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics in the brain and periphery.
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Caught in the act: LRRK2 in exosomes
Biochemical Society Transactions, 2019Co-Authors: Shijie Wang, Andrew B. WestAbstract:Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a frequent genetic cause of late-onset Parkinson9s disease (PD) and a target for therapeutic approaches. LRRK2 protein can influence vesicle trafficking events in the cytosol, with action both in endosomal and lysosomal pathways in different types of cells. A subset of late endosomes harbor intraluminal vesicles that can be secreted into the extracellular milieu. These extracellular vesicles, called exosomes, package LRRK2 protein for transport outside the cell into easily accessed biofluids. Both the cytoplasmic complement of LRRK2 as well as the exosome-associated fraction of protein appears regulated in part by interactions with 14-3-3 proteins. LRRK2 inside exosomes have disease-linked post-translational modifications and are relatively stable compared with unprotected proteins in the extracellular space or disrupted cytosolic compartments. Herein, we review the biology of exosome-associated LRRK2 and the potential for utility in diagnosis, prognosis, and theragnosis in PD and other LRRK2-linked diseases.
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LRRK2 phosphorylates membrane bound rabs and is activated by gtp bound rab7l1 to promote recruitment to the trans golgi network
Human Molecular Genetics, 2018Co-Authors: Nicole Bryant, Ravindran Kumaran, Alexandra Beilina, Asa Abeliovich, Mark R Cookson, Andrew B. WestAbstract:Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.
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Elevated LRRK2 autophosphorylation in brain-derived and peripheral exosomes in LRRK2 mutation carriers.
Acta neuropathologica communications, 2017Co-Authors: Shijie Wang, Tao Ye, Omar S. Mabrouk, Tyler Maltbie, Jan O. Aasly, Andrew B. WestAbstract:Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene can cause late-onset Parkinson disease (PD). LRRK2 mutations increase LRRK2 kinase activities that may increase levels of LRRK2 autophosphorylation at serine 1292 (pS1292) and neurotoxicity in model systems. pS1292-LRRK2 protein can be packaged into exosomes and measured in biobanked urine. Herein we provide evidence that pS1292-LRRK2 protein is robustly expressed in cerebral spinal fluid (CSF) exosomes. In a novel cohort of Norwegian subjects with and without the G2019S-LRRK2 mutation, with and without PD, we quantified levels of pS1292-LRRK2, total LRRK2, and other exosome proteins in urine from 132 subjects and in CSF from 82 subjects. CSF and urine were collected from the same morning clinic visit in 55 of the participants. We found that total LRRK2 protein concentration was similar in exosomes purified from either CSF or urine but the levels did not correlate. pS1292-LRRK2 levels were higher in urinary exosomes from male and female subjects with a LRRK2 mutation. Male LRRK2 mutation carriers without PD had intermediate pS1292-LRRK2 levels compared to male carriers with PD and controls. However, female LRRK2 mutation carriers without PD had the same pS1292-LRRK2 levels compared to female carriers with PD. pS1292-LRRK2 levels in CSF exosomes were near saturated in most subjects, ten-fold higher on average than pS1292-LRRK2 levels in urinary exosomes, irrespective of LRRK2 mutation status or PD diagnosis. These results provide insights into the effects of LRRK2 mutations in both the periphery and brain in a well-characterized clinical population and show that LRRK2 protein in brain exosomes may be much more active than in the periphery in most subjects.
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Urinary LRRK2 phosphorylation predicts parkinsonian phenotypes in G2019S LRRK2 carriers
Neurology, 2016Co-Authors: Kyle B. Fraser, Mark S. Moehle, Roy N. Alcalay, Andrew B. WestAbstract:Objective: To test whether phosphorylated Ser-1292 LRRK2 levels in urine exosomes predicts LRRK2 mutation carriers ( LRRK2 +) and noncarriers ( LRRK2 −) with Parkinson disease (PD+) and without Parkinson disease (PD−). Methods: LRRK2 protein was purified from urinary exosomes collected from participants in 2 independent cohorts. The first cohort included 14 men ( LRRK2 +/PD+, n = 7; LRRK2− /PD+, n = 4; LRRK2− /PD−, n = 3). The second cohort included 62 men ( LRRK2− /PD−, n = 16; LRRK2+ /PD−, n = 16; LRRK2 +/PD+, n = 14; LRRK2− /PD+, n = 16). The ratio of Ser(P)-1292 LRRK2 to total LRRK2 was compared between LRRK2 +/PD+ and LRRK2− in the first cohort and between LRRK2 G2019S carriers with and without PD in the second cohort. Results: LRRK2 +/PD+ had higher ratios of Ser(P)-1292 LRRK2 to total LRRK2 than LRRK2− /PD− (4.8-fold, p LRRK2− /PD+ (4.6-fold, p p Conclusion: Elevated ratio of phosphorylated Ser-1292 LRRK2 to total LRRK2 in urine exosomes predicted LRRK2 mutation status and PD risk among LRRK2 mutation carriers. Future studies may explore whether interventions that reduce this ratio may also reduce PD risk.
Dario R Alessi - One of the best experts on this subject based on the ideXlab platform.
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LRRK2 kinase in parkinson s disease
Science, 2018Co-Authors: Dario R Alessi, Esther M. SammlerAbstract:Despite intensive research, attempts to pause or even just slow the progression of Parkinson's disease (PD) have thus far failed. Although most cases of PD are idiopathic and with largely unknown aetiology, mutations in ∼20 genes, including LRRK2 (leucine-rich repeat kinase 2), cause rare genetic Parkinsonism. All pathogenic mutations in LRRK2 result in hyperactivation of the LRRK2 kinase, offering the prospect of elaborating disease-modifying treatments. Indeed, LRRK2 inhibitors have entered phase 1 clinical trials. Data are also emerging for LRRK2 involvement in idiopathic PD, suggesting that inhibitors may benefit patients beyond those carrying LRRK2 mutations. Recent advances point toward a role for LRRK2 in regulating autophagy, an intracellular process that delivers cytoplasmic constituents to the lysosome for degradation and recycling. LRRK2 phosphorylates a subgroup of RAB proteins and regulates their ability to bind cognate effector proteins. Additionally, LRRK2 is highly expressed in immune cells. Intriguing research indicates that, in early life, increased LRRK2 activity may protect against opportunistic pathogenic infection but then later increases the risk of developing PD, a concept called antagonistic pleiotropy.
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rab29 activation of the parkinson s disease associated LRRK2 kinase
The EMBO Journal, 2018Co-Authors: Elena Purlyte, Adil R. Sarhan, Terina N. Martinez, Herschel S Dhekne, Rachel C Gomez, Melanie Wightman, Francesca Tonelli, Suzanne R Pfeffer, Dario R AlessiAbstract:Abstract Parkinson9s disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector‐binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson9s patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans ‐Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild‐type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29‐mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2‐mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.
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Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils
Biochemical Journal, 2017Co-Authors: Ying Fan, Andrew J.m. Howden, Adil R. Sarhan, Pawel Lis, Genta Ito, Terina N. Martinez, Kathrin Brockmann, Thomas Gasser, Dario R Alessi, Esther M. SammlerAbstract:© 2018 The Author(s). There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson’s disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson’s as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson’s bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.
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Small-Molecule Inhibitors of LRRK2
Advances in neurobiology, 2017Co-Authors: John M. Hatcher, Dario R Alessi, Hwan Geun Choi, Nathanael S GrayAbstract:Mutations in the leucine-rich repeat kinase 2 (LRRK2) protein have been genetically and functionally linked to Parkinson’s disease (PD). The kinase activity of LRRK2 is increased by pathogenic mutations; therefore, modulation of LRRK2 kinase activity by a selective small-molecule inhibitor has been proposed as a potentially viable treatment for Parkinson’s disease. This chapter presents a historical overview of the development and bioactivity of several small-molecule LRRK2 inhibitors that have been used to inhibit LRRK2 kinase activity in vitro or in vivo. These compounds are important tools for understanding the cellular biology of LRRK2 and for evaluating the potential of LRRK2 inhibitors as disease-modifying PD therapies.
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gsk2578215a a potent and highly selective 2 arylmethyloxy 5 substitutent n arylbenzamide LRRK2 kinase inhibitor
Bioorganic & Medicinal Chemistry Letters, 2012Co-Authors: Alastair D Reith, Jinwei Zhang, Xianming Deng, Hwan Geun Choi, Pamela Dossang, Paul Bamborough, Karamjit S Jandu, Daniele Andreotti, Lucy Mensah, Dario R AlessiAbstract:Parkinson’s disease (PD) is a debilitating neurodegenerative disease that affects over one million Americans.1,2 Recent genetic studies have revealed an underlying genetic cause in at least 10% of all PD cases,3 which provides new opportunities for discovery of molecularly targeted therapeutics that may ameliorate neurodegeneration. Among the genes associated with PD, leucine-rich repeat kinase 2 (LRRK2) is unique because of a missense mutation, G2019S, that is frequently found not only in familial but also sporadic Parkinson’s disease cases.4,5 The G2019S mutation enhances kinase activity, suggesting that small molecule LRRK2 kinase inhibitors may be able to block aberrant LRRK2-dependent signaling in Parkinson’s disease.6,7 LRRK2 kinase inhibitors are being actively pursued and recently first-generation ‘tool’ inhibitors that exhibit good potency and reasonable selectivity for LRRK2 such as LRRK2-IN-18 and CZC-251469 have been reported. However, off-target activities of these tools may confound interpretation of data in biological systems and neither compound is able to achieve good exposure in mouse brains, which limits their utility in murine PD models and eventual translation into human clinical trials.8,9 Here we report GSK2578215A as an exemplar of a 2-(benzyloxy)-5-(2-fluoropyridin-4-yl)-N-(pyridin-3-yl)benzamide series.10 GSK2578215A is a potent and highly selective LRRK2 kinase inhibitor that possesses good blood-brain barrier (BBB) permeability with a high ratio of brain to plasma distribution in mice (Fig. 1). Figure 1 GSK2578215A inhibits LRRK2 in vitro. The discovery and optimization of the 2-(benzyloxy)-5-(2-fluoropyridin-4-yl)-N-(pyridin-3-yl)benzamide series of LRRK2 inhibitors will be described in detail elsewhere.11 Briefly, hits for this series were identified in a screen of GlaxoSmithKline’s KCS (a kinase-focused set of compounds for lead discovery) using a homogeneous time-resolved fluorescence (HTRF) assay that measured the inhibition of phosphorylation of the peptide substrate LRRKtide by baculoviral-derived recombinant 6His-Tev-LRRK2 (1326-2527). SAR and optimization of leads was performed using similar recombinant LRRK2 enzyme assays. GSK2578215A exhibited biochemical IC50s of 10.9 and 8.9 nM against wild-type LRRK2 and LRRK2[G2019S], respectively (Fig. 1). While the biochemical potency of GSK2578215A for inhibition of wild-type and G2019S LRRK2 is similar to LRRK2-IN-1, the potency of GSK2578215A for inhibition of A2016T mutant LRRK2 was reduced eightfold (Fig. 1). Such sensitivity to A2016T mutation is comparable to that reported previously for sunitinib, Y2763212 and TAE684.13 In contrast, the inhibitory activity of LRRK2-IN-1 was reported to be much more sensitive to the A12016T synthetic mutation, an observation that has been attributed to a steric clash of the anthranilic acid ring of LRRK2-IN-1 with the A2016T residue.8 A2016T-mediated changes in compound sensitivity were not attributable to changes in Km for ATP, since Km was found to be similar for normal and A2016T mutant LRRK2 enzymes (data not shown). An understanding of the more modest effect of A2016T mutation on the inhibitory activity of GSK2578215A was apparent from modeling studies. Docking of GSK2578215A into a previously described LRRK2 homology model8,12 predicts binding at the hinge of the ATP site and gives essentially the same results whether or not the A2016 residue is mutated to threonine, suggesting that GSK2578215A is able to avoid a steric clash (Fig. 2). Figure 2 LRRK2 homology model. The kinase selectivity of GSK2578215A was assessed using standard radioactivity-based enzymatic assays against a panel of 131 kinases (Dundee profiling)14 and kinase-binding assays against a non-redundant set of 329 additional kinases (KINOMEscan, Ambit Biosciences).15 Analysis of data from these 460 distinct non-LRRK2 kinases revealed that GSK2578215A demonstrated a selectivity profile superior to that of previously reported LRRK2 inhibitors. At a concentration of 10 μM GSK2578215A only one kinase (smMLCK) showed >50% inhibition in the 131 Dundee kinase panel, and only two kinases [ALK and FLT3(D835Y)] exhibited an ambit score of <10 in the KINOMEscan profile (see Supplementary data). We next examined the ability of GSK2578215A to inhibit LRRK2 in a cellular context in comparison to LRRK2-IN-1. As there are no validated direct phosphorylation substrates of LRRK2, we monitored phosphorylation of Ser910 and Ser935, two residues whose phosphorylation is known to be dependent upon LRRK2 kinase activity16 (Fig. 3). GSK2578215A induced a dose-dependent inhibition of Ser910 and Ser935 phosphorylation in both wild-type LRRK2 and LRRK2[G2019S] stably transfected into HEK293 cells (Fig. 3a). Significant dephosphorylation of Ser910 and Ser935 was observed at 0.3–1.0 μM of GSK2578215A for wild-type LRRK2 and at slightly higher doses for LRRK2[G2019S] (Fig. 3a), which is almost equivalent to that observed using LRRK2-IN-1 (compare Fig. 3a and 3b). Consistent with the biochemical results, GSK2578215A also induced dephosphorylation of Ser910 and 935 at a concentration of 1–3 μM in the inhibitor-resistant LRRK2[A2016T + G2019S] and LRRK2[A2016T] mutants (Fig. 3a), suggesting that GSK2578215A binds to LRRK2 differently relative to LRRK2-IN-1 (compare Fig. 3a and 3b). Figure 3 GSK2578215A inhibits LRRK2 in cells. We next examined the effect of GSK2578215A on endogenously expressed LRRK2 in human lymphoblastoid cells derived from a control and Parkinson’s disease patient homozygous for the LRRK2[G2019S] mutation (Fig. 4a). We found that increasing doses of GSK2578215A led to similar dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, as was observed in HEK293 cells stably expressing wild-type LRRK2 or LRRK2[G2019S] (compare Fig. 3a to Fig. 4a). Moreover, endogenous LRRK2 was equally sensitive to GSK2578215A and LRRK2-IN-1, which is consistent with the trend we observed in HEK293 cells. We also found that GSK2578215A induced similar dose-dependent Ser910 and Ser935 dephosphorylation of endogenous LRRK2 in mouse Swiss 3T3 cells (Fig. 4b). Figure 4 GSK2578215A inhibits endogenously expressed LRRK2. Evaluation of the pharmacokinetic profile of GSK2578215A in normal mice demonstrated that the compound achieves exposure in the brain with a brain to plasma ratio of 1.9. GSK2578215A exhibits low oral bioavailability (12.2%F), a half-life of 1.14 h and plasma exposure (635.3 h ng/mL, AUClast) (Table 1). Based on these pharmacokinetic properties, pharmacodynamic experiments examining inhibition of LRRK2 Ser910/Ser935 phosphorylation were conducted after intraperitoneal injection with 100 mg/kg of GSK2578215A to normal mice. We observed complete Ser910 and Ser935 dephosphorylation of LRRK2 in the kidney and spleen, which also demonstrated similar potency relative to LRRK2-IN-1 (Fig. 5).8 Again, despite the significant exposure of GSK2578215A in the brain, no inhibition of LRRK2 Ser910 or Ser935 phosphorylation was observed in the brain, a finding similar to that observed for TAE68413 (Fig. 5). We are currently investigating the reasons for this unexpected result. Figure 5 Pharmacodynamic analysis for GSK2578215A. Table 1 Pharmacokinetic parameters of GSK2578215A In summary, we have discovered that GSK2578215A is a potent biochemical and cellular inhibitor of LRRK2 kinase activity which represents a novel chemotype with respect to all previously reported inhibitors of LRRK2, or indeed of any protein kinase Importantly, GSK2578215A exhibits exquisite selectivity across the kinome that is superior to previously reported LRRK2 inhibitor tool compounds. As such, GSK2578215A provides a significant addition to the battery of available tools to be deployed for elucidation of the functions of LRRK2. Detailed characterization of GSK2578215A using LRRK2-IN-1 as a bench mark revealed that these two compounds had quite similar potency against wild-type LRRK2 and LRRK2[G2019S] mutant both in vitro and in vivo. The A2016T and G2019S + A2016T LRRK2 mutations induce significant resistance to LRRK2-IN-1 but not to GSK2578215A. The ability of GSK2578215A to reduce phosphorylation levels of Ser910 and Ser935 in peripheral tissues on dosing to mice supports the notion that these phosphoepitopes can serve as markers of LRRK2 inhibitor activity in animal studies. Interestingly, whilst GSK2578215A achieves good exposure to mouse brain following oral administration, it failed to induce significant inhibition of Ser910 or Ser935 phosphorylation of LRRK2 in brain. It remains to be determined whether this reflects some pharmacokinetic limitation of this tool or phosphorylation of these sites in brain by non-LRRK2 kinases. Further development of 2-arylmethyloxy-5-substitutent-N-arylbenzamides may result in the identification of pharmacological agents to investigate the impact of LRRK2 inhibition in brain in preclinical animal models and eventually in humans.
T. M. Dawson - One of the best experts on this subject based on the ideXlab platform.
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LRRK2 pathobiology in Parkinson's disease
Journal of Neurochemistry, 2014Co-Authors: Ian Martin, V. L. Dawson, T. M. DawsonAbstract:Mutations in the catalytic Roc-COR and kinase domains of leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson’s disease (PD). LRRK2 mutations cause PD with age-related penetrance and clinical features identical to late-onset sporadic PD. Biochemical studies support an increase in LRRK2 kinase activity and a decrease in GTPase activity for kinase domain and Roc-COR mutations, respectively. Strong evidence exists that LRRK2 toxicity is kinase-dependent leading to extensive efforts to identify selective and brain-permeable LRRK2 kinase inhibitors for clinical development. Cell and animal models of PD indicate that LRRK2 mutations affect vesicular trafficking, autophagy, protein synthesis and cytoskeletal function. Although some of these biological functions are affected consistently by most disease-linked mutations, others are not and it is currently unclear how mutations that produce variable effects on LRRK2 biochemistry and function all commonly result in the degeneration and death of dopamine neurons. LRRK2 is typically present in Lewy bodies and its toxicity in mammalian models appears to be dependent on the presence of α-synuclein, which is elevated in human iPS-derived dopamine neurons from patients harboring LRRK2 mutations. Here, we summarize biochemical and functional studies of LRRK2 and its mutations and focus on aberrant vesicular trafficking and protein synthesis as two leading mechanisms underlying LRRK2-linked disease.
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LRRK2 GTPase dysfunction in the pathogenesis of Parkinson's disease
Biochemical Society Transactions, 2012Co-Authors: Y. Xiong, V. L. Dawson, T. M. DawsonAbstract:Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene are the most frequent genetic cause of PD (Parkinson9s disease), and these mutations play important roles in sporadic PD. The LRRK2 protein contains GTPase and kinase domains and several protein–protein interaction domains. The kinase and GTPase activity of LRRK2 seem to be important in regulating LRRK2-dependent cellular signalling pathways. LRRK29s GTPase and kinase domains may reciprocally regulate each other to direct LRRK29s ultimate function. Although most LRRK2 investigations are centred on LRRK29s kinase activity, the present review focuses on the function of LRRK29s GTPase activity in LRRK2 physiology and pathophysiology.
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ArfGAP1 Is a GTPase Activating Protein for LRRK2: Reciprocal Regulation of ArfGAP1 by LRRK2
The Journal of Neuroscience, 2012Co-Authors: Y. Xiong, T. M. Dawson, C. Yuan, Rong Chen, V. L. DawsonAbstract:Both sporadic and autosomal dominant forms of Parkinson9s disease (PD) have been causally linked to mutations in leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains. The kinase domain plays an important role in LRRK2-mediated toxicity. Although a number of investigations have focused on LRRK2 kinase activity, less is known about the GTPase function of LRRK2. The activity of GTPases is regulated by GTPase activating proteins (GAPs) and GTP exchange factors. Here, we identify ArfGAP1 as the first GAP for LRRK2. ArfGAP1 binds LRRK2 predominantly via the WD40 and kinase domain of LRRK2, and it increases LRRK2 GTPase activity and regulates LRRK2 toxicity both in vitro and in vivo in Drosophila melanogaster . Unexpectedly, ArfGAP1 is an LRRK2 kinase substrate whose GAP activity is inhibited by LRRK2, whereas wild-type and G2019S LRRK2 autophosphorylation and kinase activity are significantly reduced in the presence of ArfGAP1. Overexpressed ArfGAP1 exhibits toxicity that is reduced by LRRK2 both in vitro and in vivo . Δ64–ArfGAP1, a dominant-negative ArfGAP1, and shRNA knockdown of ArfGAP1 reduce LRRK2 toxicity. Thus, LRRK2 and ArfGAP1 reciprocally regulate the activity of each other. Our results provide insight into the basic pathobiology of LRRK2 and indicate an important role for the GTPase domain and ArfGAP1 in LRRK2-mediated toxicity. These data suggest that agents targeted toward regulation of LRRK2 GTP hydrolysis might be therapeutic agents for the treatment of PD.
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ArfGAP1 Is a GTPase Activating Protein for LRRK2: Reciprocal Regulation of ArfGAP1 by LRRK2
Journal of Neuroscience, 2012Co-Authors: Y. Xiong, T. M. Dawson, C. Yuan, Rong Chen, V. L. DawsonAbstract:Both sporadic and autosomal dominant forms of Parkinson's disease (PD) have been causally linked to mutations in leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains. The kinase domain plays an important role in LRRK2-mediated toxicity. Although a number of investigations have focused on LRRK2 kinase activity, less is known about the GTPase function of LRRK2. The activity of GTPases is regulated by GTPase activating proteins (GAPs) and GTP exchange factors. Here, we identify ArfGAP1 as the first GAP for LRRK2. ArfGAP1 binds LRRK2 predominantly via the WD40 and kinase domain of LRRK2, and it increases LRRK2 GTPase activity and regulates LRRK2 toxicity both in vitro and in vivo in Drosophila melanogaster. Unexpectedly, ArfGAP1 is an LRRK2 kinase substrate whose GAP activity is inhibited by LRRK2, whereas wild-type and G2019S LRRK2 autophosphorylation and kinase activity are significantly reduced in the presence of ArfGAP1. Overexpressed ArfGAP1 exhibits toxicity that is reduced by LRRK2 both in vitro and in vivo. Delta64-ArfGAP1, a dominant-negative ArfGAP1, and shRNA knockdown of ArfGAP1 reduce LRRK2 toxicity. Thus, LRRK2 and ArfGAP1 reciprocally regulate the activity of each other. Our results provide insight into the basic pathobiology of LRRK2 and indicate an important role for the GTPase domain and ArfGAP1 in LRRK2-mediated toxicity. These data suggest that agents targeted toward regulation of LRRK2 GTP hydrolysis might be therapeutic agents for the treatment of PD
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gtpase activity plays a key role in the pathobiology of LRRK2
PLOS Genetics, 2010Co-Authors: Y. Xiong, T. M. Dawson, V. L. Dawson, Candice E Coombes, Austin S Kilaru, Xiaojie Li, Aaron D Gitler, William J Bowers, Darren J MooreAbstract:Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson's disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker's yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human α-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.
V. L. Dawson - One of the best experts on this subject based on the ideXlab platform.
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LRRK2 pathobiology in Parkinson's disease
Journal of Neurochemistry, 2014Co-Authors: Ian Martin, V. L. Dawson, T. M. DawsonAbstract:Mutations in the catalytic Roc-COR and kinase domains of leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson’s disease (PD). LRRK2 mutations cause PD with age-related penetrance and clinical features identical to late-onset sporadic PD. Biochemical studies support an increase in LRRK2 kinase activity and a decrease in GTPase activity for kinase domain and Roc-COR mutations, respectively. Strong evidence exists that LRRK2 toxicity is kinase-dependent leading to extensive efforts to identify selective and brain-permeable LRRK2 kinase inhibitors for clinical development. Cell and animal models of PD indicate that LRRK2 mutations affect vesicular trafficking, autophagy, protein synthesis and cytoskeletal function. Although some of these biological functions are affected consistently by most disease-linked mutations, others are not and it is currently unclear how mutations that produce variable effects on LRRK2 biochemistry and function all commonly result in the degeneration and death of dopamine neurons. LRRK2 is typically present in Lewy bodies and its toxicity in mammalian models appears to be dependent on the presence of α-synuclein, which is elevated in human iPS-derived dopamine neurons from patients harboring LRRK2 mutations. Here, we summarize biochemical and functional studies of LRRK2 and its mutations and focus on aberrant vesicular trafficking and protein synthesis as two leading mechanisms underlying LRRK2-linked disease.
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LRRK2 GTPase dysfunction in the pathogenesis of Parkinson's disease
Biochemical Society Transactions, 2012Co-Authors: Y. Xiong, V. L. Dawson, T. M. DawsonAbstract:Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene are the most frequent genetic cause of PD (Parkinson9s disease), and these mutations play important roles in sporadic PD. The LRRK2 protein contains GTPase and kinase domains and several protein–protein interaction domains. The kinase and GTPase activity of LRRK2 seem to be important in regulating LRRK2-dependent cellular signalling pathways. LRRK29s GTPase and kinase domains may reciprocally regulate each other to direct LRRK29s ultimate function. Although most LRRK2 investigations are centred on LRRK29s kinase activity, the present review focuses on the function of LRRK29s GTPase activity in LRRK2 physiology and pathophysiology.
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ArfGAP1 Is a GTPase Activating Protein for LRRK2: Reciprocal Regulation of ArfGAP1 by LRRK2
The Journal of Neuroscience, 2012Co-Authors: Y. Xiong, T. M. Dawson, C. Yuan, Rong Chen, V. L. DawsonAbstract:Both sporadic and autosomal dominant forms of Parkinson9s disease (PD) have been causally linked to mutations in leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains. The kinase domain plays an important role in LRRK2-mediated toxicity. Although a number of investigations have focused on LRRK2 kinase activity, less is known about the GTPase function of LRRK2. The activity of GTPases is regulated by GTPase activating proteins (GAPs) and GTP exchange factors. Here, we identify ArfGAP1 as the first GAP for LRRK2. ArfGAP1 binds LRRK2 predominantly via the WD40 and kinase domain of LRRK2, and it increases LRRK2 GTPase activity and regulates LRRK2 toxicity both in vitro and in vivo in Drosophila melanogaster . Unexpectedly, ArfGAP1 is an LRRK2 kinase substrate whose GAP activity is inhibited by LRRK2, whereas wild-type and G2019S LRRK2 autophosphorylation and kinase activity are significantly reduced in the presence of ArfGAP1. Overexpressed ArfGAP1 exhibits toxicity that is reduced by LRRK2 both in vitro and in vivo . Δ64–ArfGAP1, a dominant-negative ArfGAP1, and shRNA knockdown of ArfGAP1 reduce LRRK2 toxicity. Thus, LRRK2 and ArfGAP1 reciprocally regulate the activity of each other. Our results provide insight into the basic pathobiology of LRRK2 and indicate an important role for the GTPase domain and ArfGAP1 in LRRK2-mediated toxicity. These data suggest that agents targeted toward regulation of LRRK2 GTP hydrolysis might be therapeutic agents for the treatment of PD.
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ArfGAP1 Is a GTPase Activating Protein for LRRK2: Reciprocal Regulation of ArfGAP1 by LRRK2
Journal of Neuroscience, 2012Co-Authors: Y. Xiong, T. M. Dawson, C. Yuan, Rong Chen, V. L. DawsonAbstract:Both sporadic and autosomal dominant forms of Parkinson's disease (PD) have been causally linked to mutations in leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains. The kinase domain plays an important role in LRRK2-mediated toxicity. Although a number of investigations have focused on LRRK2 kinase activity, less is known about the GTPase function of LRRK2. The activity of GTPases is regulated by GTPase activating proteins (GAPs) and GTP exchange factors. Here, we identify ArfGAP1 as the first GAP for LRRK2. ArfGAP1 binds LRRK2 predominantly via the WD40 and kinase domain of LRRK2, and it increases LRRK2 GTPase activity and regulates LRRK2 toxicity both in vitro and in vivo in Drosophila melanogaster. Unexpectedly, ArfGAP1 is an LRRK2 kinase substrate whose GAP activity is inhibited by LRRK2, whereas wild-type and G2019S LRRK2 autophosphorylation and kinase activity are significantly reduced in the presence of ArfGAP1. Overexpressed ArfGAP1 exhibits toxicity that is reduced by LRRK2 both in vitro and in vivo. Delta64-ArfGAP1, a dominant-negative ArfGAP1, and shRNA knockdown of ArfGAP1 reduce LRRK2 toxicity. Thus, LRRK2 and ArfGAP1 reciprocally regulate the activity of each other. Our results provide insight into the basic pathobiology of LRRK2 and indicate an important role for the GTPase domain and ArfGAP1 in LRRK2-mediated toxicity. These data suggest that agents targeted toward regulation of LRRK2 GTP hydrolysis might be therapeutic agents for the treatment of PD
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gtpase activity plays a key role in the pathobiology of LRRK2
PLOS Genetics, 2010Co-Authors: Y. Xiong, T. M. Dawson, V. L. Dawson, Candice E Coombes, Austin S Kilaru, Xiaojie Li, Aaron D Gitler, William J Bowers, Darren J MooreAbstract:Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson's disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker's yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human α-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.
Xianting Li - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation dependent 14 3 3 binding to LRRK2 is impaired by common mutations of familial parkinson s disease
PLOS ONE, 2011Co-Authors: Xianting Li, Qing Jun Wang, Yingming Zhao, Brian T ChaitAbstract:Background Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown. Methodology/Principal Findings Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2. Conclusions/Significance LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.
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enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial parkinson s disease mutation g2019s
The Journal of Neuroscience, 2010Co-Authors: Xianting Li, Jyoti C Patel, Jing Wang, Marat V Avshalumov, Charles Nicholson, Joseph D Buxbaum, Gregory A Elder, Margaret E RiceAbstract:PARK8/LRRK2 ( leucine-rich repeat kinase 2 ) was recently identified as a causative gene for autosomal dominant Parkinson9s disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events.
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leucine rich repeat kinase 2 LRRK2 park8 possesses gtpase activity that is altered in familial parkinson s disease r1441c g mutants
Journal of Neurochemistry, 2007Co-Authors: Xianting Li, Shibu M Poulose, Warren C Olanow, Xinyun HuangAbstract:Mutations in Leucine-rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson’s disease (PD). The LRRK2 protein contains two well-known functional domains, MAPKKK-like kinase and Rab-like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD-associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope-tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full-length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain-specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2-associated neuronal functions or dysfunctions in the pathogenesis of PD.
