Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration

Ga Ram Jeong,Ho Chul Kang,Eun-Hae Jang,Keiko Tanaka-Yamamoto,Byoung Dae Lee,Jae Ryul Bae,Soyoung Jun,Valina L Dawson,Joo-Ho Shin,Yukio Yamamoto,Eun-Mi Hur,Chi-Hu Park,Ted M Dawson

doi:10.1186/s13024-018-0240-1

Abstract

BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.MethodsIn vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35.ResultsOur screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property 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.ConclusionsHere 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 primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

Highlights

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s disease (PD)
Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD
The extent of phosphorylation on each Rab was calculated by normalizing Rab phosphorylation against both the amount of Rab GTPases determined by Coomassie brilliant blue staining and the level of LRRK2 autophosphorylation determined by standard autoradiography as described in Methods

Summary

Introduction

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Mutations in Leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of familial PD, and genome-wide association studies have identified LRRK2 as a risk locus for sporadic PD [2, 3]. To understand the physiological functions of LRRK2 as well as the mechanism by which mutations in LRRK2 contribute to PD pathogenesis, one of the greatest challenges in the field has been to identify authentic LRRK2 substrates that are associated with neurodegeneration. LRRK2 is thought to be a multivalent kinase and the precise mechanisms by which dysregulation of the kinase activity of LRRK2 causes neuronal toxicity are not fully understood

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Results

Discussion

Conclusion