Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration

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.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0240-1) contains supplementary material, which is available to authorized users.

Neurodegeneration caused by LRRK2-G2019S requires Rab10 in select dopaminergic neurons

“…The existence of Rab10 in the tyrosine hydroxylase positive neurons controlling vision and proboscis movement argues that LRRK2 might indeed phosphorylate Rab10 directly. Thus our in vivo results both support the in vitro (biochemical and cell culture) data in which LRRK2 directly phosphorylates Rab10 (3)(4)(5)(6)(7)(8), but also suggest a separate pathway for LRRK2-G2019S action. Further, while Rab3 is also phosphorylated by LRRK2 in vitro (3), it did not synergise with LRRK2-G2019S in the visual assay, and was not detected in the MC neurons .…”

“…A number of studies highlighted a diverse range of >30 proteins that might be phosphorylated by LRRK2, suggesting it is a generalised kinase (2). However, several research teams have recently reported that LRRK2 is a more specific kinase, phosphorylating Rab GTPases (3)(4)(5)(6)(7)(8). However, it is not clear which of the more than 60 Rabs are actually phosphorylated in vivo.…”

“…Biochemically, about 8 seem to be directly phosphorylated by LRRK2 [ Rabs 3,5,8,10,12,29,35 and 43 (3)]. In mammals, analysis of the role of the Rabs is complex because individual Rabs may have similar, or even compensatory functions, which may differ by tissue (8,13).…”

Vesicular Dysfunction and the Pathogenesis of Parkinson’s Disease: Clues From Genetic Studies

“…Rab7L1 was found to be phosphorylated by LRRK2 and is involved in vesicular clearance through the trans-Golgi network (Liu et al, 2017). It was shown that LRRK2, phospho-Rab 8/10 together with Rab7L1 help to maintain homeostasis in stressed lysosomes Jeong et al, 2018) and phosphomutants of Rab proteins at conserved LRRK2 phosphorylation sites induces neurotoxicity and dopaminergic neuron degeneration in mice (Jeong et al, 2018). In human iPS neurons, in wild-type and LRRK2 mutated neurons, it was shown that LRRK2 may be playing a role by phosphorylating auxilin in its clathrin domain at Ser627 which is abolished upon kinase inhibition (Nguyen and Krainc, 2018), and transmission electron microscopy showed decreased synaptic vesicle density in presynaptic nerve terminals of R1441C dopaminergic neurons indicating defective synaptic vesicle endocytosis (Nguyen and Krainc, 2018).…”

Endogenous Rab29 does not impact basal or nigericin and monensin stimulated LRRK2 pathway activity

“…They influence biology by interacting with a series of effector proteins when complexed to GTP. LRRK2 phosphorylates Rab proteins at a highly conserved Ser/Thr residue located at the center of the effector binding region of these enzymes that is also known as the Switch-II motif [7, 26,28]. This phosphorylation event appears to act in two ways.…”