Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.DOI: http://dx.doi.org/10.7554/eLife.12813.001

Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).

SignificanceLRRK2, a protein kinase related to Parkinson’s disease, is implicated in the maintenance of lysosomes, and a subset of Rab GTPases has been identified as bona fide substrates of LRRK2. Here, we reveal a key stress-responsive pathway composed of Rab7L1, LRRK2, and phosphorylated Rab8/10 involved in lysosomal homeostasis. Lysosomal overload stress induces translocation of Rab7L1 and LRRK2 to lysosomes, where LRRK2 is activated, and stabilizes Rab8 and Rab10 through phosphorylation. The activation of this machinery protects against lysosomal enlargement and upregulates lysosomal secretion through Rab effectors, EHBP1 and EHBP1L1. These findings elucidate a novel regulatory mechanism of Rab GTPases by phosphorylation by LRRK2 in stressed lysosomes, which may also be involved in the pathomechanism of LRRK2-related disorders.

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.

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

Involvement of the peripheral autonomic nervous system is a core feature of Lewy body (LB) diseases, including Parkinson disease (PD), PD with dementia, and dementia with LBs. To investigate the potential use of skin biopsy for the diagnosis of LB diseases, we assessed anti-phosphorylated alpha-synuclein immunoreactivity in peripheral nerves in samples of skin from the abdominal wall and flexor surface of the upper arm in 279 prospectively studied consecutively autopsied patients whose data were registered at the Brain Bank for Aging Research between 2002 and 2005. Positive immunoreactivity was demonstrated in the unmyelinated fibers of the dermis in 20 of 85 patients with LB pathology in the CNS and the adrenal glands, the latter representing a substitute for peripheral autonomic nervous system sympathetic ganglia; no reactivity was seen in 194 patients without CNS LB pathology. In 142 retrospectively studied patients autopsied from 1995 onward who had subclinical or clinical LB disease, the sensitivity of the positive skin immunoreactivity was 70% in PD and PD with dementia and 40% in dementia with LBs. Skin immunoreactivity was absent in cases of multiple-system atrophy, progressive nuclear palsy, and corticobasal degeneration. We demonstrate for the first time that the skin is involved and may be a highly specific and useful biopsy site for the pathological diagnosis of LB diseases.

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Lewy body disease is defined as Lewy body-related neuronal degeneration involving the nigrostriatal system, limbic-neocortical system, and peripheral autonomic nervous system (PANS). We investigated whether the adrenal gland, which is evolutionarily related to sympathetic ganglia and is routinely examined in general autopsy, could be used to assess pathology of the PANS in Lewy body disease. Brains, spinal cords, and adrenal glands from 783 consecutive autopsy cases from a general geriatric hospital were examined immunohistochemically with antiphosphorylated alpha-synuclein antibodies and routine staining. Parkinson disease (PD) with dementia and dementia with Lewy bodies (DLB) were defined using 1996 Consensus Guidelines for DLB and the secondary Lewy body-related alpha-synucleinopathy or amygdala variants using previously established criteria. Lewy body-related alpha-synucleinopathy was found in 207 (26.4%) of 783 cases, with 1 case solely in the adrenal gland. In all 18 PD cases with or without dementia and in 33 of 38 DLB cases, the adrenal gland was involved, but it was spared in all cases of amygdala variants. Our results indicate that the adrenal gland can provide useful information for evaluation of the PANS in Lewy body disease.