Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies

The Parkinson's disease VPS35[D620N] mutation enhances LRRK2 mediated Rab protein phosphorylation in mouse and human

Missense mutations in the LRRK2 and VPS35 genes result in autosomal dominant Parkinson’s disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation, strikingly elevates LRRK2 mediated phosphorylation of Rab8A, Rab10 and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (lung, kidney, spleen and brain). Furthermore, LRRK2 mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson’s patients with a heterozygous VPS35[D620N] mutation compared to healthy donors and idiopathic Parkinson’s patients. LRRK2 mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild type, LRRK2[R1441C] or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson’s patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing Parkinson’s disease through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson’s might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans

Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase

Mutations that activate the LRRK2 protein kinase, predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector binding Switch-II motif. In this study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against 9 different LRRK2 phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2 phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2 controlled phosphorylation of a range of endogenous Rab proteins including Rab8A, Rab10 and Rab35. The antibodies described in this study will help with assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess impact of LRRK2 inhibitors in future clinical trials

Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons

BACKGROUND: Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson’s disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity. RESULTS: Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons. CONCLUSIONS: We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD

Analysis of inflammation-related nigral degeneration and locomotor function in DJ-1−/− mice

Abstract Background Complex interactions involving genetic susceptibility and environmental factors are thought to underlie the pathogenesis of Parkinson’s disease (PD). Although the role of inflammatory processes in modulating risk for development of PD has yet to be fully understood, prospective studies suggest that chronic use of NSAIDs reduce the incidence of PD. Loss-of-function mutations in the DJ-1 gene cause a rare form of familial PD with an autosomal recessive pattern of inheritance; however, DJ-1−/− mice do not display nigrostriatal pathway degeneration, suggesting that additional factors such as inflammation may be needed to induce neurodegeneration on the background of DJ-1 gene mutations. Neuroinflammation causes oxidative stress and, based on evidence that DJ-1 plays a protective role against oxidative stress, we investigated whether DJ-1−/− mice display increased vulnerability to inflammation-induced nigral degeneration. Methods We exposed adult wild-type and DJ-1−/− mice to repeated intranasal administration of soluble TNF (inTNF) or repeated intraperitoneal injections of low-dose lipopolysaccharide (LPS) or saline vehicle. We measured locomotor performance using a variety of behavior tasks, striatal dopamine (DA) content by HPLC, DA neuron (TH+ cells) and total neuron (NeuN+ cells) number in the substantia nigra pars compacta and ventral tegmental area by unbiased stereology, number of Iba1-positive microglia, and mRNA levels of inflammatory and oxidative stress genes by quantitative PCR in the midbrain, cortex and isolated peritoneal macrophages of DJ-1−/− and wild-type mice. Results We found that chronic LPS injections induced similar neuroinflammatory responses in the midbrains of DJ-1−/− mice and wild-type mice and neither group developed locomotor deficits or nigral degeneration. inTNF administration did not appear to induce neuroinflammatory responses in LPS-treated wild-type or DJ-1−/− mice. The lack of vulnerability to inflammation-induced nigral degeneration was not due to enhanced anti-oxidant gene responses in the midbrains of DJ-1−/− mice which, in fact, displayed a blunted response relative to that of wild-type mice. Peripheral macrophages from wild-type and DJ-1−/− mice displayed similar basal and LPS-induced inflammatory and oxidative stress markers in vitro. Conclusions Our studies indicate that DJ-1−/− mice do not display increased vulnerability to inflammation-related nigral degeneration in contrast to what has been reported for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. We conclude that either DJ-1 does not have a critical role in protecting DA neurons against inflammation-induced oxidative stress and/or there is compensatory gene expression in the midbrain of DJ-1−/− mice that renders them resistant to the cytotoxic effects triggered by chronic peripheral inflammation

DJ-1 −/ − mice

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