LRRK2 causes centrosomal deficits via phosphorylated Rab10 and RILPL1 at centriolar subdistal appendages

The Parkinson′s disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that various LRRK2 risk variants and all currently described regulators of the LRRK2 signaling pathway converge upon causing centrosomal cohesion deficits. The cohesion deficits do not require the presence of RILPL2 or of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendages of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab protein to cause the centrosomal defects. These data reveal a common molecular pathway by which alterations in the LRRK2 kinase activity impact upon centrosome-related events.

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Nanobodies as allosteric modulators of Parkinson′s disease-associated LRRK2

10.1101/2021.08.30.458082 ◽

2021 ◽

Author(s):

Ranjan K. Singh ◽

Ahmed Soliman ◽

Giambattista Guaitoli ◽

Eliza Störmer ◽

Felix von Zweydorf ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Common Disease ◽

Type I ◽

Rab Protein ◽

Lrrk2 Kinase ◽

Inhibitory Drug ◽

Lrrk2 Kinase Activity

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson′s disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

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RAB8, RAB10 and RILPL1 contribute to both LRRK2 kinase–mediated centrosomal cohesion and ciliogenesis deficits

Human Molecular Genetics ◽

10.1093/hmg/ddz201 ◽

2019 ◽

Vol 28 (21) ◽

pp. 3552-3568 ◽

Cited By ~ 13

Author(s):

Antonio Jesús Lara Ordónez ◽

Belén Fernández ◽

Elena Fdez ◽

María Romo-Lozano ◽

Jesús Madero-Pérez ◽

...

Keyword(s):

Rab Gtpases ◽

Common Cause ◽

Increase Risk ◽

Dividing Cells ◽

Mutant Lrrk2 ◽

Lrrk2 Kinase ◽

Relevant Context ◽

Primary Astrocytes ◽

Peripheral Cells ◽

Familial Parkinson’S Disease

Abstract Mutations in the LRRK2 kinase are the most common cause of familial Parkinson’s disease, and variants increase risk for the sporadic form of the disease. LRRK2 phosphorylates multiple RAB GTPases including RAB8A and RAB10. Phosphorylated RAB10 is recruited to centrosome-localized RILPL1, which may interfere with ciliogenesis in a disease-relevant context. Our previous studies indicate that the centrosomal accumulation of phosphorylated RAB8A causes centrosomal cohesion deficits in dividing cells, including in peripheral patient-derived cells. Here, we show that both RAB8 and RAB10 contribute to the centrosomal cohesion deficits. Pathogenic LRRK2 causes the centrosomal accumulation not only of phosho-RAB8 but also of phospho-RAB10, and the effects on centrosomal cohesion are dependent on RAB8, RAB10 and RILPL1. Conversely, the pathogenic LRRK2-mediated ciliogenesis defects correlate with the centrosomal accumulation of both phospho-RAB8 and phospho-RAB10. LRRK2-mediated centrosomal cohesion and ciliogenesis alterations are observed in patient-derived peripheral cells, as well as in primary astrocytes from mutant LRRK2 mice, and are reverted upon LRRK2 kinase inhibition. These data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are distinct cellular readouts of the same underlying phospho-RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD.

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Role of LRRK2 kinase activity in the pathogenesis of Parkinson's disease

Biochemical Society Transactions ◽

10.1042/bst20120054 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1058-1062 ◽

Cited By ~ 22

Author(s):

Elisa Greggio

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Activity ◽

Complex Molecule ◽

Disease Onset ◽

Missense Mutations ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Interest in studying the biology of LRRK2 (leucine-rich repeat kinase 2) started in 2004 when missense mutations in the LRRK2 gene were linked to an inherited form of Parkinson's disease with clinical and pathological presentation resembling the sporadic syndrome. LRRK2 is a complex molecule containing domains implicated in protein interactions, as well as kinase and GTPase activities. The observation that the common G2019S mutation increases kinase activity in vitro suggests that altered phosphorylation of LRRK2 targets may have pathological outcomes. Given that protein kinases are ideal targets for drug therapies, much effort has been directed at understanding the role of LRRK2 kinase activity on disease onset. However, no clear physiological substrates have been identified to date, indicating that much research is still needed to fully understand the signalling pathways orchestrated by LRRK2 and deregulated under pathological conditions.

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Pathogenic mutations in LRRK2 sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

10.1101/2020.07.27.219501 ◽

2020 ◽

Author(s):

Adamantios Mamais ◽

Natalie Landeck ◽

Rebekah G. Langston ◽

Luis Bonet-Ponce ◽

Nathan Smith ◽

...

Keyword(s):

Iron Uptake ◽

Kinase Activity ◽

Small Gtpase ◽

Kinase Substrate ◽

Lrrk2 Kinase ◽

Effect Of Pd ◽

Lrrk2 Kinase Activity

AbstractMutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s disease (PD) while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a are poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a into lysosomes in cells while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive accumulation of endocytosed transferrin into Rab8a-positive lysosomes leading to a dysregulation of iron transport. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

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

10.1101/2020.04.28.065664 ◽

2020 ◽

Author(s):

Izumi Yanatori ◽

Herschel S. Dhekne ◽

Edmundo G. Vides ◽

Yuriko Sobu ◽

Federico Diez ◽

...

Keyword(s):

Dependent Manner ◽

Rab Gtpases ◽

Tail Domain ◽

Binding Partner ◽

Activating Mutations ◽

High Affinity Binding Site ◽

Mother Centriole ◽

Myosin Va ◽

Lrrk2 Kinase ◽

Rab Effector

AbstractActivating mutations in LRRK2 kinase cause 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. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10, and certain tissue-specific Myosin Va isoforms strongly prefer to bind phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 relocalizes to the peri-centriolar region in a phosphoRab10- and Myosin Va-dependent manner. In the absence of phosphoRab10, expression of RILPL2 or depletion of Myosin Va increase centriolar RILPL2 levels, and either condition is sufficient to block ciliogenesis in RPE cells. These experiments show that LRRK2 generated phosphoRab10 dramatically redistributes Myosin Va-RILPL2 complexes to the mother centriole, which may sequester Myosin Va and RILPL2 in a manner that blocks their normal roles in ciliogenesis.

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Rab8 Regulates the Actin-based Movement of Melanosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e04-09-0770 ◽

2005 ◽

Vol 16 (4) ◽

pp. 1640-1650 ◽

Cited By ~ 49

Author(s):

Marion L. Chabrillat ◽

Claire Wilhelm ◽

Christina Wasmeier ◽

Elena V. Sviderskaya ◽

Daniel Louvard ◽

...

Keyword(s):

Rab Proteins ◽

Rab Gtpases ◽

Wild Type ◽

In Vitro Motility Assay ◽

Actin Bundles ◽

In Vitro Motility ◽

Rab Protein

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes.

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Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts

10.1101/2021.11.05.465894 ◽

2021 ◽

Author(s):

Lilian Peptropoulou-Vathi ◽

Athina M Simitsi ◽

Politymi-Eleni Valkimadi ◽

Maria Kedariti ◽

Lampros Dimitrakopoulos ◽

...

Keyword(s):

Outcome Measures ◽

Kinase Activity ◽

Disease Status ◽

Clinical Samples ◽

Rab Gtpase ◽

Rab Gtpases ◽

Comprehensive Picture ◽

Lrrk2 Kinase ◽

A53t Mutation ◽

Lrrk2 Kinase Activity

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in 2 separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005894118 ◽

2021 ◽

Vol 118 (10) ◽

pp. e2005894118

Author(s):

Yuriko Sobu ◽

Paulina S. Wawro ◽

Herschel S. Dhekne ◽

Wondwossen M. Yeshaw ◽

Suzanne R. Pfeffer

Keyword(s):

Kinase Activity ◽

Cell Types ◽

Ips Cells ◽

Serum Starvation ◽

Critical Detail ◽

Cilia Formation ◽

Mother Centriole ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity ◽

Protein Blocks

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.

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P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

Biochemical Journal ◽

10.1042/bcj20170699 ◽

2018 ◽

Vol 475 (7) ◽

pp. 1271-1293 ◽

Cited By ~ 26

Author(s):

Alexia F. Kalogeropulou ◽

Jing Zhao ◽

Marc F. Bolliger ◽

Anna Memou ◽

Shreya Narasimha ◽

...

Keyword(s):

Kinase Activity ◽

Rab Proteins ◽

Missense Mutations ◽

Leucine Rich Repeat ◽

Lrrk2 Gene ◽

Ubiquitin Binding Domain ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity ◽

In Cells

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

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

Life Science Alliance ◽

10.26508/lsa.202101050 ◽

2021 ◽

Vol 4 (5) ◽

pp. e202101050

Author(s):

Herschel S Dhekne ◽

Izumi Yanatori ◽

Edmundo G Vides ◽

Yuriko Sobu ◽

Federico Diez ◽

...

Keyword(s):

Dependent Manner ◽

Rab Gtpases ◽

Tail Domain ◽

Over Expression ◽

Activating Mutations ◽

High Affinity Binding Site ◽

Mother Centriole ◽

Myosin Va ◽

Lrrk2 Kinase ◽

Rab Effector

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. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va’s role in ciliogenesis.

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