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RESEARCH ARTICLE Open Access

Dysregulated phosphorylation of RabGTPases by LRRK2 inducesneurodegenerationGa Ram Jeong1, Eun-Hae Jang2,3,4, Jae Ryul Bae1, Soyoung Jun2,3,4, Ho Chul Kang5, Chi-Hu Park6, Joo-Ho Shin7,Yukio Yamamoto8, Keiko Tanaka-Yamamoto4,8, Valina L. Dawson9,10,11, Ted M. Dawson9,11,12, Eun-Mi Hur2,3,4*

and Byoung Dae Lee1,13*

Abstract

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadicParkinsons disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediateneurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membraneand vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potentialsubstrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.

Methods: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab familyproteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed itby assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation onneurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associatedviral vectors (AAV) expressing wild-type or phosphomutants of Rab35.

Results: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue inthe switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rabproteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authenticsubstrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-bindingproperty in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity,which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35-T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo.

Conclusions: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells.We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primaryneurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2toxicity in the progression of PD.

Keywords: LRRK2, Rab GTPases, Phosphorylation, Neurodegeneration, Parkinsons disease

* Correspondence: ehur@kist.re.kr; bdaelee@khu.ac.krEqual contributors2Center for Neuroscience, Brain Science Institute, Korea Institute of Scienceand Technology (KIST), 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792,South Korea1Department of Neuroscience, Graduate School, Kyung Hee University, Seoul,South KoreaFull list of author information is available at the end of the article

The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Jeong et al. Molecular Neurodegeneration (2018) 13:8 https://doi.org/10.1186/s13024-018-0240-1

http://crossmark.crossref.org/dialog/?doi=10.1186/s13024-018-0240-1&domain=pdfhttp://orcid.org/0000-0002-9248-9493mailto:ehur@kist.re.krmailto:bdaelee@khu.ac.krhttp://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/

BackgroundParkinsons disease (PD) is the second most prevalentneurodegenerative disease, affecting about 1-3% of theelderly population [1]. Mutations in Leucine-rich repeatkinase 2 (LRRK2) comprise the leading cause of familialPD, and genome-wide association studies have also identi-fied LRRK2 as a risk locus for sporadic PD [2, 3]. LRRK2is a large multi-domain protein that encompasses a kinasedomain, a GTPase domain composed of a Ras of complex(ROC) and a C-terminus of ROC region, and severalprotein-protein interaction domains. Multiple lines of evi-dence indicate that LRRK2 toxicity is kinase-dependent[47], and the most common LRRK2 mutation,LRRK2-G2019S shows increased kinase activity towardgeneric kinase substrates [4, 5, 811]. Therefore, to under-stand the physiological functions of LRRK2 as well as themechanism by which mutations in LRRK2 contribute toPD pathogenesis, one of the greatest challenges in the fieldhas been to identify authentic LRRK2 substrates that areassociated with neurodegeneration. A previous study hassuggested that ribosomal protein s15 is an endogenoussubstrate of LRRK2 which links LRRK2 toxicity to alteredprotein synthesis and neurodegeneration [12]. However,LRRK2 is thought to be a multivalent kinase and theprecise mechanisms by which dysregulation of the kinaseactivity of LRRK2 causes neuronal toxicity are not fullyunderstood.The physiological functions of LRRK2 in neurons

remain to be determined, but LRRK2 has been broadlyimplicated in membrane dynamics and vesicle trafficking.Previous studies have shown that LRRK2 localizes tomembranous or vesicular structures, such as endosomes,lysosomes, multivesicular bodies, and autophagic vesicles[1315], and increasing evidence suggests that LRRK2might have a role in endocytosis [16, 17], endolysosomalsorting [18], retromer-mediated trafficking [19], andautophagy [13, 14, 2023]. Rab GTPases, which comprisemore than 60 members in the human genome, serve asmultifaceted organizers in almost all membrane andvesicle trafficking processes [24, 25]. Since the identifica-tion of the first LRRK2-related Rab GTPase Rab5b, phys-ical, genetic and functional interactions between LRRK2and many Rab family members have been reported [18,19, 2628]. An unbiased search for interactors of LRRK2and a brain transcriptomics approach concurred thatLRRK2 interacts with Rab7L1 (also known as Rab29), anddysfunction of the LRRK2-Rab7L1 complex has beensuggested to cause neurotoxicity by disrupting the endoly-sosomal and autophagic pathways [13, 19, 23, 27]. Large-scale phosphoproteomic screens using fibroblasts fromknock-in mice that were genetically engineered to harboreither LRRK2-G2019S or inhibitor-resistantLRRK2-A2016T led to the identification of a singleLRRK2 substrate, Rab10 [29]. Further analysis with

selected Rab GTPases showed that a few more Rabs(Rab1b and Rab8a) were also directly phosphorylated byLRRK2 [29]. These findings are strongly suggestive of aphysiological role for Rab phosphorylation by LRRK2.Thus, it is worthwhile to undertake a systematic inquiryon the interplay of LRRK2 and Rab family GTPases, andperhaps an equally important task is to interrogate thefunctional consequence of Rab phosphorylation byLRRK2, particularly with respect to neurodegeneration.Here we performed an in vitro LRRK2 kinase assay

with forty-five human Rab family proteins to screen RabGTPases as potential substrates of LRRK2. Our screen-ing revealed that Rab1a, 1b, 3, 8a, 8b, and 35 are directlyphosphorylated by LRRK2, and we identified that Rabproteins are phosphorylated at a conserved threonineresidue in the switch II region. By using the kinase-inactive LRRK2-D1994A and the pathogenic mutantLRRK2-G2019S along with phosphomutants of Rabs, weconfirmed that a subset of Rab proteins, includingRab35, are authentic substrates of LRRK2 both in vitroand in cells. We also validated Rab phosphorylation atthe endogenous level by using kinase inhibitors ofLRRK2 and Lrrk2 knockout mice. Moreover, substitutionof the LRRK2 site in Rab1, 3, and 35 to either alanine oraspartate, but not the wild-types induced neurotoxicity,among which mutations in Rab35 caused the mostsevere phenotype. Furthermore, intracranial injection ofadeno-associated viral vectors (AAVs) expressingphosphomutants of Rab35 but not the wild-type into thesubstantia nigra induced degeneration of dopaminergicneurons in vivo. To the best of our knowledge, thisstudy is the first report on neurodegeneration of dopa-minergic neurons caused by a direct substrate of LRRK2in the mammalian brain.

MethodsGeneration of recombinant Rab proteinsEntry clone cDNA for each Rab GTPases was subclonedinto N-terminal GST tagged bacteria expression vector(pDEST15, Invitrogen, Carlsbad, CA, USA) using LRclonase (Invitrogen, Carlsbad, CA, USA) and the expres-sion vectors generated were transformed into BL21-AIOne Shot chemically competent E.coli (Invitrogen,Carlsbad, CA, USA). Protein expression was inducedusing 0.2% w/v L-arabinose (Sigma, St. Louis, MO, USA)for 1.5 h. Cells were pelleted (3000 g for 20 min) andthen lysed with sonication in the presence of chilled lysisbuffer (50 mM Tris-HCl (pH 7.5), 0.5% TX-100,150 mM NaCl, 0.5 mM EGTA, 2 mM DTT andComplete Protease Inhibitor Mixture). Cell lysates werecentrifuged at 15,000 g for 15 min, and the supernatantwas incubated with GSH-sepharose beads (GE health-care, Little Chalfont, UK) for overnight at 4C. Afterincubation, the beads were washed with PBS containing

Jeong et al. Molecular Neurodegeneration (2018) 13:8 Page 2 of 17

150 mM NaCl and 0.1% TX-100 three times. For thekinase assay, beads were stored in PBS containing 30%glycerol at 80C.

In vitro LRRK2 kinase assaysRecombinant LRRK2 (Invitrogen, Carlsbad, CA, USA)and Rab proteins were incubate

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