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

Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Metabotropic glutamate receptors (mGluRs) have been implicated in regulating anxiety, stress responses, and the neurobehavioral effects of psychostimulants. The present study sought to determine whether group II mGluR activation by the potent mGlu2/3 receptor agonist, (Ϫ)-2-oxa-4-aminobicylco hexane-4,6-dicarboxylic acid (LY379268), antagonizes reinstatement of cocaine-seeking induced by cocaine-related stimuli and whether this effect extends to behavior induced by stimuli conditioned to a potent conventional reinforcer, sweetened condensed milk (SCM). Also, we tested whether the suppressant effects of LY379268 on conditioned reinstatement extend to the primary reinforcing effects of cocaine or SCM. Rats were trained to associate discriminative stimuli (S D ) with the availability of cocaine or SCM versus non-reward and then subjected to repeated extinction sessions during which the respective reinforcers and S D were withheld. Subsequent reexposure to the cocaine or SCM S D , but not the non-reward S D , produced recovery of responding at the previously active lever. LY379268 (0.3-3.0 mg/kg, s.c.) dose-dependently attenuated recovery of cocaine seeking but reduced conditioned reinstatement by the SCM S D only at the highest dose. LY379268 did not alter responding reinforced directly by SCM, and only the highest LY379268 dose reduced cocaine self-administration. The results suggest that the effects of LY379268 are selective for behavior maintained by cocaine as opposed to palatable conventional reinforcers. More importantly, the results show that LY379268 suppresses behavior motivated by stimuli conditioned to cocaine or SCM more effectively than consummatory behavior maintained by the unconditioned effects of these substances. As such, the results identify group II mGluRs as a pharmacotherapeutic target for craving and relapse prevention associated with cocaine cue exposure.

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.

The Pavlovian conditioning analysis of drug tolerance emphasizes that cues present at the time of drug administration become associated with drug-induced disturbances. These disturbances elicit unconditional responses that compensate for the pharmacological perturbation. The drug-compensatory responses eventually come to be elicited by drug-paired cues. These conditional compensatory responses (CCRs) mediate tolerance by counteracting the drug effect when the drug is administered in the presence of cues previously paired with the drug. If the usual predrug cues are presented in the absence the drug, the unopposed CCRs are evident as withdrawal symptoms. Recent findings elucidate intercellular and intracellular events mediating CCRs and indicate the importance of internal stimuli (pharmacological cues and interoceptive cues inherent in self-administration) to the acquisition of drug tolerance and the expression of withdrawal symptoms. Early chroniclers of drug effects noted that responsivity to drugs often decreased as a function of experience with the drug. For example, in 1612, Jean Mousin, physician to the King of France, wondered why individuals sometimes became progressively more sober while they were continuing to drink alcoholic beverages. Although the term tolerance was not used until some years later, it appears that Mousin observed the phenomenon now termed acute tolerance-decreased responsiveness to a drug within the course of a single administration (Kalant, 1998). Acute Tolerance and Withdrawal Acute tolerance has been investigated extensively with respect to ethanol (e.g., see LeBlanc, Kalant, & Gibbins, 1975) as well as other drugs, such as opiates. For example, over the course of a single, long administration of morphine, accomplished by gradual infusion via an implanted morphine pellet, the analgesic effect of the drug decreases (e.g., see Tilson, Rech, & Stolman, 1973; Wei & Way, 1975). The existence of acute tolerance is evidence that pharmacological stimulation initiates adaptive responses that compensate for the primary drug effect (

Major precipitating factors for relapse to drug use are stress and exposure to drug-related environmental stimuli. Group II (mGlu 2/3 ) metabotropic glutamate receptors (mGluRs) are densely expressed within circuitries mediating the motivating effects of stress and drug cues and, therefore, may participate in regulating drug-seeking linked to both of these risk factors. Thus, we tested the hypothesis that pharmacological activation of group II mGluRs modifies both stress-and cue-induced ethanol-seeking, using reinstatement models of relapse. In parallel, brain c-fos expression was examined to identify neural substrates for the behavioral effects of group II mGluR activation. The selective mGlu 2/3 agonist LY379268 (1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate) (0.3, 1.0, and 3.0 mg/kg, s.c.) dose dependently blocked the recovery of extinguished ethanol-seeking induced by either footshock stress or ethanolassociated discriminative stimuli. These effects were accompanied by modulation of c-fos expression in the hippocampus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and medial parvocellular paraventricular nucleus of the hypothalamus. The results implicate group II mGluRs as a shared neuropharmacological substrate for ethanol-seeking elicited by both drug cues and stress and identify group II mGluRs as promising treatment targets for relapse prevention.

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson’s disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype.  In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats [1] and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson’s disease.