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Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

ABSTRACT: Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene belong to the most common genetic causes of inherited Parkinson's disease (PD) and variations in its locus increase the risk to develop sporadic PD. Extensive research efforts aimed at understanding how changes in the LRRK2 function result in molecular alterations that ultimately lead to PD. Cellular LRRK2-based models revealed several potential pathophysiological mechanisms including apoptotic cell death, LRRK2 protein accumulation and deficits in neurite outgrowth. However, highly variable outcomes between different cellular models have been reported. Here, we have investigated the effect of different experimental conditions, such as the use of different tags and gene transfer methods, in various cellular LRRK2 models. Readouts included cell death, sensitivity to oxidative stress, LRRK2 relocalization, ?-synuclein aggregation and neurite outgrowth in cell culture, as well as neurite maintenance in vivo. We show that overexpression levels and/or the tag fused to LRRK2 affect the relocalization of LRRK2 to filamentous and skein-like structures. We found that overexpression of LRRK2 per se is not sufficient to induce cellular toxicity or to affect ?-synuclein-induced toxicity and aggregate formation. Finally, neurite outgrowth/retraction experiments in cell lines and in vivo revealed that secondary, yet unknown, factors are required for the pathogenic LRRK2 effects on neurite length. Our findings stress the importance of technical and biological factors in LRRK2-induced cellular phenotypes and hence imply that conclusions based on these types of LRRK2-based assays should be interpreted with caution.

SUBMITTER: Lobbestael E

PROVIDER: S-EPMC7560525 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

Lobbestael Evy E Van den Haute Chris C Macchi Francesca F Taymans Jean-Marc JM Baekelandt Veerle V

Bioscience reports 20201001 10

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene belong to the most common genetic causes of inherited Parkinson's disease (PD) and variations in its locus increase the risk to develop sporadic PD. Extensive research efforts aimed at understanding how changes in the LRRK2 function result in molecular alterations that ultimately lead to PD. Cellular LRRK2-based models revealed several potential pathophysiological mechanisms including apoptotic cell death, LRRK2 protein accumu ...[more]

PMID: 32975566

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Project description:BackgroundStem cell characteristics are an important feature of human cancer cells and play a major role in the therapy resistance of tumours. Strategies to target cancer stem cells are thus of major importance for cancer therapy. Differentiation therapy by nucleoside drugs represents an attractive approach for the elimination of cancer stem cells. However, even if it is generally assumed that the activity of these drugs is mediated by their ability to modulate epigenetic pathways, their precise mode of action remains to be established. We therefore analysed the potential of three nucleoside analogues to induce differentiation of the embryonic cancer stem cell line NTERA 2 D1 and compared their effect to the natural ligand retinoic acid.Methodology/principal findingsAll nucleoside analogues analyzed, but not retinoic acid, triggered proteolytic degradation of the Polycomb group protein EZH2. Two of them, 3-Deazaneplanocin A (DZNep) and 2'-deoxy-5-azacytidine (decitabine), also induced a decrease in global DNA methylation. Nevertheless, only decitabine and 1beta-arabinofuranosylcytosine (cytarabine) effectively triggered neuronal differentiation of NT2 cells. We show that drug-induced differentiation, in contrast to retinoic acid induction, is caused by caspase activation, which mediates depletion of the stem cell factors NANOG and OCT4. Consistent with this observation, protein degradation and differentiation could be counteracted by co-treatment with caspase inhibitors or by depletion of CASPASE-3 and CASPASE-7 through dsRNA interference. In agreement with this, OCT4 was found to be a direct in-vitro-target of CASPASE-7.Conclusions/significanceWe show that drug-induced differentiation is not a consequence of pharmacologic epigenetic modulation, but is induced by the degradation of stem-cell-specific proteins by caspases. Our results thus uncover a novel pathway that induces differentiation of embryonic cancer stem cells and is triggered by the established anticancer drugs cytarabine and decitabine. These findings suggest new approaches for directly targeting the stem cell fraction of human tumours.

Project description:Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.

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Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

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Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

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