Impact of Type II LRRK2 inhibitors on signaling and mitophagy

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study, we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild-type LRRK2 compared with pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast with Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aid with the development of more selective Type II LRRK2 inhibitors

Pharmacological rescue of impaired mitophagy in Parkinson's disease-related LRRK2 G2019S knock-in mice

Parkinson’s disease (PD) is a major and progressive neurodegenerative disorder, yet the biological mechanisms involved in its aetiology are poorly understood. Evidence links this disorder with mitochondrial dysfunction and/or impaired lysosomal degradation – key features of the autophagy of mitochondria, known as mitophagy. Here, we investigated the role of LRRK2, a protein kinase frequently mutated in PD, in this process in vivo. Using mitophagy and autophagy reporter mice, bearing either knockout of LRRK2 or expressing the pathogenic kinase-activating G2019S LRRK2 mutation, we found that basal mitophagy was specifically altered in clinically relevant cells and tissues. Our data show that basal mitophagy inversely correlates with LRRK2 kinase activity in vivo. In support of this, use of distinct LRRK2 kinase inhibitors in cells increased basal mitophagy, and a CNS penetrant LRRK2 kinase inhibitor, GSK3357679A, rescued the mitophagy defects observed in LRRK2 G2019S mice. This study provides the first in vivo evidence that pathogenic LRRK2 directly impairs basal mitophagy, a process with strong links to idiopathic Parkinson’s disease, and demonstrates that pharmacological inhibition of LRRK2 is a rational mitophagy-rescue approach and potential PD therapy

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associ- ated with Parkinson’s disease, chronic inflammation and mycobac- terial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobac- terial control independently of autophagy. In vivo, LRRK2 defi- ciency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2- dependent cellular pathway that controls M. tuberculosis replica- tion by regulating phagosome maturation

Phos-tag analysis of Rab10 phosphorylation by LRRK2:a powerful assay for assessing kinase function and inhibitors

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 Thr/Ser 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] knock-in 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) knock-in 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] knock-in 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 aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

AbstractBackground: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3.Results: SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3α in vitro, with Kis of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3β with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a β-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase.Conclusions: SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Inhibition of VEGF-Dependent Multistage Carcinogenesis by Soluble EphA Receptors

Elevated expression of Eph receptors has long been correlated with the growth of solid tumors. However, the functional role of this family of receptor tyrosine kinases in carcinogenesis and tumor angiogenesis has not been well characterized. Here we report that soluble EphA receptors inhibit tumor angiogenesis and tumor progression in vivo in the RIP-Tag transgenic model of vascular endothelial growth factor (VEGF)-dependent multistage pancreatic islet cell carcinoma. Soluble EphA receptors delivered either by a transgene or an osmotic minipump inhibited the formation of angiogenic islet, a premalignant lesion, and reduced tumor volume of solid islet cell carcinoma. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor volume but increased tumor and endothelial cell apoptosis in vivo. In addition, soluble EphA receptors inhibited VEGF and βTC tumor cell-conditioned medium-induced endothelial cell migration in vitro and VEGF-induced cornea angiogenesis in vivo. A dominant negative EphA2 mutant inhibited—whereas a gain-of-function EphA2 mutant enhanced—tumor cell-induced endothelial cell migration, suggesting that EphA2 receptor activation is required for tumor cell-endothelial cell interaction. These data provide functional evidence for EphA class receptor regulation of VEGF-dependent tumor angiogenesis, suggesting that the EphA signaling pathway may represent an attractive novel target for antiangiogenic therapy in cancer