Blauwendraat C, Reed X, Kia DA, Gan-Or Z, Lesage S, Pihlstrøm L, Guerreiro R, Gibbs JR, Sabir M, Ahmed S, Ding J, Alcalay RN, Hassin-Baer S, Pittman AM, Brooks J, Edsall C, Hernandez DG, Chung SJ, Goldwurm S, Toft M, Schulte C, Bras J, Wood NW, Brice A, Morris HR, Scholz SW, Nalls MA, Singleton AB, Cookson MR, COURAGE-PD (Comprehensive Unbiased Risk Factor Assessment for Genetics and Environment in Parkinson’s Disease) Consortium, the French Parkinson’s Disease Consortium, and the International Parkinson’s Disease Genomics Consortium (IPDGC). Frequency of Loss of Function Variants in LRRK2 in Parkinson Disease. JAMA Neurol. 2018 Nov 1;75(11):1416-1422. PubMed.
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IPBLN, CSIC (Spanish National Research Council)
Two recent studies report on the role of LRRK2 in altering endolysosomal structure/function and lysosomal secretion, albeit coming to different conclusions. Eguchi et al. used various tissue culture cell lines treated with chloroquine, a lysosomotropic agent that enlarges lysosomal structures and causes lysosomal overload stress, as measured by the secretion of cathepsin D. Their data indicate that knockdown of endogenous LRRK2 or inhibition of the LRRK2 kinase activity by pharmacological means causes a further increase in the size of lysosomal structures in the presence of chloroquine, associated with a decrease in the secretion of cathepsin D. They further report that overexpression of pathogenic LRRK2 mutants rescues the chloroquine-induced lysosomal structural alterations, even though the effect of these mutants on cathepsin D secretion was not addressed. The authors further link these effects to a Rab7L1-mediated recruitment of LRRK2 to the chloroquine-enlarged lysosomes, as well as to the involvement of Rab8/Rab10, two protein substrates for the kinase activity of LRRK2. The latter link requires further validation, since knockdown of Rab8 was found to cause lysosomal enlargement without altered cathepsin D secretion, whilst knockdown of Rab10 caused no lysosomal enlargement but had an effect on cathepsin D secretion, suggesting that these two readouts may not go hand in hand. In addition, the nature of the enlarged structures needs further clarification using additional lysosomal markers apart from LAMP1. This is especially important since the chloroquine-mediated release of intermediates of cathepsin D processing suggests that the released species may reside in compartments other than lysosomes. Whilst it will be important to determine whether the same findings can be observed with other, more physiological stimuli to induce lysosomal overload, these data suggest that inhibiting LRRK2 kinase impairs lysosomal secretion under lysosomal overload stress conditions.
Similarly, Bae et al., using different model systems and technical approaches, showed that knockout of LRRK2 in either C. elegans or rat models impairs α-synuclein propagation, which involves exocytosis of α-synuclein from donor cells and subsequent endocytosis by recipient cells. Importantly, in cultured cells, their data further indicate that pathogenic LRRK2 increases uptake in recipient cells as well as release from donor cells. These studies were performed using a bimolecular fluorescence complementation assay, which, due to the irreversible nature of the interactions, may overestimate the observed effects. In addition, the α-synuclein species which is transmitted from one cell to another in those assays remains unclear. Nevertheless, an LRRK2 kinase inhibitor was found to reverse the effects of pathogenic LRRK2 on α-synuclein propagation in vitro, and importantly also decreased the α-synuclein aggregates in the brains of α-synuclein transgenic mice. The authors further show that the effects on α-synuclein propagation in worms and in cell culture models are modulated when altering the levels and/or activity of Rab35, another reported protein substrate for the LRRK2 kinase activity. Their data altogether suggest that pathogenic LRRK2 causes increased α-synuclein propagation, presumably via increased lysosomal exocytosis, even though this is not formally demonstrated.
The two studies differ in the set of LRRK2 Rab protein substrates that they nominate as regulators for the trafficking events analyzed. It remains to be seen whether this is due to studying distinct intracellular compartments (e.g., lysosomes versus phagolysosomes versus late endosomes) and/or distinct cell types. In addition, caution should be exercised when expressing phosphomimetic or phosphodeficient Rab variants, as such mutations can cause loss-of-function protein variants in all cases. Therefore, formal proof that the observed intracellular trafficking deficits are mediated by LRRK2-mediated phosphorylation of these distinct Rab proteins is largely lacking. Whilst both reports clearly nominate LRRK2 as a regulator of proper lysosomal functioning, additional cellular studies performed under endogenous levels of pathogenic LRRK2 expression and measuring endogenous α-synuclein accumulation and secretion, as recently reported (Schapansky et al., 2018), combined with a careful analysis of Rab protein substrate phosphorylation and localization using appropriate tools such as phospho-state-specific antibodies, may shed conclusive insights into the mechanism(s) by which pathogenic LRRK2 regulates endolysosomal homeostasis and α-synuclein propagation.
References:
Schapansky J, Khasnavis S, DeAndrade MP, Nardozzi JD, Falkson SR, Boyd JD, Sanderson JB, Bartels T, Melrose HL, LaVoie MJ. Familial knockin mutation of LRRK2 causes lysosomal dysfunction and accumulation of endogenous insoluble α-synuclein in neurons. Neurobiol Dis. 2018 Mar;111:26-35. Epub 2017 Dec 12 PubMed.
View all comments by Sabine HilfikerIndiana University School of Medicine
Both Bae et al. and Eguchi et al. show LRRK2 functioning in the endocytic recycling pathway, which is consistent with an earlier study by Mark Cookson’s group showing that LRRK2 interacts with Rab7L1—a protein involved in endosomal recycling (Beilina et al., 2014). A number of other subsequent studies reported similar findings, albeit some studies located LRRK2 functioning at different steps in the endocytic recycling pathway.
These subtle differences are also apparent in these two papers, where Bae et al. show LRRK2 functioning at the endosome in concert with Rab35, while Eguchi et al. show LRRK2 interacting with stressed lysosome via Rab7L1. However, both papers show that LRRK2 kinase activity enhances exocytosis—although of different organelles. It is not immediately clear what caused the difference in LRRK2 localization between these studies; however, since they used different cells lines and studied under different conditions—one used chloroquine to induce lysosomal stress and the other used exogenous α-synuclein aggregates to track propagation—they might have been observing different LRRK2 conformations (potentially a GTP-bound versus a GDP-bound conformation).
References:
Beilina A, Rudenko IN, Kaganovich A, Civiero L, Chau H, Kalia SK, Kalia LV, Lobbestael E, Chia R, Ndukwe K, Ding J, Nalls MA, International Parkinson’s Disease Genomics Consortium, North American Brain Expression Consortium, Olszewski M, Hauser DN, Kumaran R, Lozano AM, Baekelandt V, Greene LE, Taymans JM, Greggio E, Cookson MR. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2626-31. Epub 2014 Feb 7 PubMed.
View all comments by Quyen HoangEvotec
We think that the papers are very nice! Eguchi et al. confirm all our recently published findings and they observe an increase in Rab10 phosphorylation upon lysosomal activation, which is very intriguing. This is the very first time that we have seen a stimulus of endogenous LRRK2 activity (recent work from Dario Alessi's group shows that Rab29 overexpression stimulates LRRK2).
They also find that the EHBPL1 protein is important for their lysosomal phenotype and, interestingly, we identified this protein as phospho-specific interactor (Steger et al., 2017). Bae et al. show that increased LRRK2 activity correlates with increased α-syn deposition and that this is related to defects in the lysosomal machinery (Rab35-mediated). It would be nice to test whether Rabs other than Rab35, especially Rab3a (strongly expressed in the brain), as well as Rab10 and Rab29, are important regulators of this phenotype. Also, it would be important to show that Rab35 is directly phosphorylated by LRRK2 in this system by using a global/targeted phosphoproteomics approach. There are also phospho-specific Rab antibodies available that could be used to partially answer this question (Lis et al., 2018).
Overall, we don't think that the conclusions of the two papers are very different. Yes, they use different models, but they both show that LRRK2 is involved in the lysosomal pathway and that pathogenic LRRK2 promotes the secretion of lysosomal contents (hence kinase activity-dependent). They are very nice follow ups of our published work (Steger et al., 2017; Steger et al., 2016).
We think that one should analyze the Rab-LRRK2 pathway in more detail in more tissues and cell types. For example, why are dopaminergic neurons of the substantia nigra preferentially affected by Parkinson's disease? There might be specific Rab subtypes (and effectors) that are responsible for this. Moreover, we know that LRRK2 is expressed at high levels in tissues other than brain. We think that LRRK2 function in these cell types (e.g., immune cells, intestine, and lungs) might be important for PD pathogenesis and that proteomic and cellular approaches are needed to answer these open questions. Finally, because of these recent studies, we think that LRRK2 might be linked to PARKIN-PINK1 mediated mitophagy.
— Mathias Mann is the co-author of this comment.
References:
Steger M, Diez F, Dhekne HS, Lis P, Nirujogi RS, Karayel O, Tonelli F, Martinez TN, Lorentzen E, Pfeffer SR, Alessi DR, Mann M. Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis. Elife. 2017 Nov 10;6 PubMed.
Lis P, Burel S, Steger M, Mann M, Brown F, Diez F, Tonelli F, Holton JL, Ho PW, Ho SL, Chou MY, Polinski NK, Martinez TN, Davies P, Alessi DR. Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase. Biochem J. 2018 Jan 2;475(1):1-22. PubMed.
Steger M, Tonelli F, Ito G, Davies P, Trost M, Vetter M, Wachter S, Lorentzen E, Duddy G, Wilson S, Baptista MA, Fiske BK, Fell MJ, Morrow JA, Reith AD, Alessi DR, Mann M. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases. Elife. 2016 Jan 29;5 PubMed.
View all comments by Martin StegerMake a Comment
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