Skip to main content
SpringerLink
Log in

Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson’s disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Kumaran R, Cookson MR (2015) Pathways to Parkinsonism Redux: convergent pathobiological mechanisms in genetics of Parkinson’s disease. Hum Mol Genet 24:R32–R44. https://doi.org/10.1093/hmg/ddv236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zimprich A, Biskup S, Leitner P et al (2004) Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44:601–607. https://doi.org/10.1016/j.neuron.2004.11.005

    Article  CAS  PubMed  Google Scholar 

  3. Paisán-Ruíz C, Jain S, Evans EW et al (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 44:595–600. https://doi.org/10.1016/j.neuron.2004.10.023

    Article  PubMed  Google Scholar 

  4. Funayama M, Hasegawa K, Ohta E et al (2005) An LRRK2 mutation as a cause for the parkinsonism in the original PARK8 family. Ann Neurol 57:918–921. https://doi.org/10.1002/ana.20484

    Article  CAS  PubMed  Google Scholar 

  5. Hernandez DG, Reed X, Singleton AB (2016) Genetics in Parkinson disease: Mendelian versus non-Mendelian inheritance. J Neurochem 139(Suppl 1):59–74. https://doi.org/10.1111/jnc.13593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cookson MR (2010) The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson’s disease. Nat Rev Neurosci 11:791–797. https://doi.org/10.1038/nrn2935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Greggio E, Jain S, Kingsbury A et al (2006) Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol Dis 23:329–341. https://doi.org/10.1016/j.nbd.2006.04.001

    Article  CAS  PubMed  Google Scholar 

  8. Skibinski G, Nakamura K, Cookson MR, Finkbeiner S (2014) Mutant LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein but not kinase activity or inclusion bodies. J Neurosci 34:418–433. https://doi.org/10.1523/JNEUROSCI.2712-13.2014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lee BD, Shin J-H, VanKampen J et al (2010) Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson’s disease. Nat Med 16:998–1000. https://doi.org/10.1038/nm.2199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yao C, Johnson WM, Gao Y et al (2013) Kinase inhibitors arrest neurodegeneration in cell and C. elegans models of LRRK2 toxicity. Hum Mol Genet 22:328–344. https://doi.org/10.1093/hmg/dds431

    Article  CAS  PubMed  Google Scholar 

  11. Liu Z, Hamamichi S, Lee BD et al (2011) Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson’s disease models. Hum Mol Genet 20:3933–3942. https://doi.org/10.1093/hmg/ddr312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Li T, He X, Thomas JM et al (2015) A novel GTP-binding inhibitor, FX2149, attenuates LRRK2 toxicity in Parkinson’s disease models. PloS One 10:e0122461. https://doi.org/10.1371/journal.pone.0122461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. West AB (2015) Ten years and counting: moving leucine-rich repeat kinase 2 inhibitors to the clinic. Mov Disord 30:180–189. https://doi.org/10.1002/mds.26075

    Article  CAS  PubMed  Google Scholar 

  14. Rudenko IN, Kaganovich A, Hauser DN et al (2012) The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson’s disease is a partial loss-of-function mutation. Biochem J 446:99–111. https://doi.org/10.1042/BJ20120637

    Article  CAS  PubMed  Google Scholar 

  15. Rudenko IN, Chia R, Cookson MR (2012) Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson’s disease? BMC Med 10:20. https://doi.org/10.1186/1741-7015-10-20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Langston RG, Rudenko IN, Cookson MR (2016) The function of orthologues of the human Parkinson’s disease gene LRRK2 across species: implications for disease modelling in preclinical research. Biochem J 473:221–232. https://doi.org/10.1042/BJ20150985

    Article  CAS  PubMed  Google Scholar 

  17. Yue M, Hinkle KM, Davies P et al (2015) Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice. Neurobiol Dis 78:172–195. https://doi.org/10.1016/j.nbd.2015.02.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tong Y, Pisani A, Martella G et al (2009) R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice. Proc Natl Acad Sci USA 106:14622–14627. https://doi.org/10.1073/pnas.0906334106

    Article  PubMed  PubMed Central  Google Scholar 

  19. Greggio E, Cookson MR (2009) Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: three questions. ASN Neuro. https://doi.org/10.1042/AN20090007

    Article  PubMed  PubMed Central  Google Scholar 

  20. West AB, Moore DJ, Biskup S et al (2005) Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci USA 102:16842–16847. https://doi.org/10.1073/pnas.0507360102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chia R, Haddock S, Beilina A et al (2014) Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7. Nat Commun 5:5827. https://doi.org/10.1038/ncomms6827

    Article  CAS  PubMed  Google Scholar 

  22. Beilina A, Rudenko IN, Kaganovich A et al (2014) Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. Proc Natl Acad Sci USA 111:2626–2631. https://doi.org/10.1073/pnas.1318306111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nichols RJ, Dzamko N, Hutti JE et al (2009) Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson’s disease. Biochem J 424:47–60. https://doi.org/10.1042/BJ20091035

    Article  CAS  PubMed  Google Scholar 

  25. Rudenko IN, Kaganovich A, Langston RG et al (2017) The G2385R risk factor for Parkinson’s disease enhances CHIP-dependent intracellular degradation of LRRK2. Biochem J 474:1547–1558. https://doi.org/10.1042/BCJ20160909

    Article  CAS  PubMed  Google Scholar 

  26. Steger M, Tonelli F, Ito G et al (2016) Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. eLife. https://doi.org/10.7554/eLife.12813

    Article  PubMed  PubMed Central  Google Scholar 

  27. Ding X, Goldberg MS (2009) Regulation of LRRK2 stability by the E3 ubiquitin ligase CHIP. PLoS ONE 4:e5949. https://doi.org/10.1371/journal.pone.0005949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nichols RJ, Dzamko N, Morrice NA et al (2010) 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson’s disease-associated mutations and regulates cytoplasmic localization. Biochem J 430:393–404. https://doi.org/10.1042/BJ20100483

    Article  CAS  PubMed  Google Scholar 

  29. Wang L, Xie C, Greggio E et al (2008) The chaperone activity of heat shock protein 90 is critical for maintaining the stability of leucine-rich repeat kinase 2. J Neurosci 28:3384–3391. https://doi.org/10.1523/JNEUROSCI.0185-08.2008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jorgensen ND, Peng Y, Ho CC-Y et al (2009) The WD40 domain is required for LRRK2 neurotoxicity. PLoS ONE 4:e8463. https://doi.org/10.1371/journal.pone.0008463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Greggio E, Zambrano I, Kaganovich A et al (2008) The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation. J Biol Chem 283:16906–16914. https://doi.org/10.1074/jbc.M708718200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Klein CL, Rovelli G, Springer W et al (2009) Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment. J Neurochem 111:703–715. https://doi.org/10.1111/j.1471-4159.2009.06358.x

    Article  CAS  PubMed  Google Scholar 

  33. Zhao J, Molitor TP, Langston JW, Nichols RJ (2015) LRRK2 dephosphorylation increases its ubiquitination. Biochem J 469:107–120. https://doi.org/10.1042/BJ20141305

    Article  CAS  PubMed  Google Scholar 

  34. Ito G, Okai T, Fujino G et al (2007) GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson’s disease. Biochemistry 46:1380–1388. https://doi.org/10.1021/bi061960m

    Article  CAS  PubMed  Google Scholar 

  35. West AB, Moore DJ, Choi C et al (2007) Parkinson’s disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum Mol Genet 16:223–232. https://doi.org/10.1093/hmg/ddl471

    Article  CAS  PubMed  Google Scholar 

  36. West AB, Cowell RM, Daher JPL et al (2014) Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents. J Comp Neurol 522:2465–2480. https://doi.org/10.1002/cne.23583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Matthew S. Goldberg (University of Texas Southwestern Medical Center, USA) for sharing a mouse Lrrk2 cDNA construct. This work was supported by the Intramural Research Program of the National Institute on Aging, NIH, by The Michael J. Fox Foundation for Parkinson’s Research, and by a Parkinson’s Foundation- American Parkinson Disease Association Summer Student Fellowship, PF-APDA-SFW-1742.

Author information

Author notes

  1. Iakov N. Rudenko

    Present address: Department of Neurology, SUNY at Stony Brook, Health Science Center, T12-020, Stony Brook, NY, 11794-8121, USA

  2. Kelechi Ndukwe

    Present address: Medical College of Wisconsin, Medical School, 8701 W Watertown Plank Rd, Milwaukee, WI, 53226, USA

  3. Allissa A. Dillman

    Present address: Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

  4. David N. Hauser

    Present address: Sanford Burnham Prebys Medicial Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA

Authors and Affiliations

  1. Laboratory of Neurogenetics, Cell Biology and Gene Expression Section, NIA, NIH, Bethesda, MD, 20892, USA

    Rebekah G. Langston, Iakov N. Rudenko, Ravindran Kumaran, David N. Hauser, Alice Kaganovich, Luis Bonet Ponce, Adamantios Mamais, Kelechi Ndukwe, Allissa A. Dillman, Amr M. Al-Saif, Aleksandra Beilina & Mark R. Cookson

  2. Laboratory of Neurogenetics, Cell Biology and Gene Expression Section, National Institute on Aging, NIH, 35 Convent Drive, Room 1A-116, Bethesda, MD, 20892-3707, USA

    Mark R. Cookson

Authors
  1. Rebekah G. Langston
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Iakov N. Rudenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ravindran Kumaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David N. Hauser
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alice Kaganovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Luis Bonet Ponce
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Adamantios Mamais
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kelechi Ndukwe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Allissa A. Dillman
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Amr M. Al-Saif
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Aleksandra Beilina
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Mark R. Cookson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark R. Cookson.

Additional information

Special Issue: In honor of Prof. Anthony J. Turner.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11064_2018_2650_MOESM1_ESM.pdf

Supplementary figure S1. Higher mouse LRRK2 protein expression is not explained by increased protein stability. A. HEK293FT cells were transfected with Flag-tagged human or mouse LRRK2 and subjected to a 35S-cysteine/35S-methionine “pulse” followed by a “chase” with media enriched with “cold” cysteine and methionine. Cell lysates were subjected to immunoprecipitation (IP) for Flag and exposed to a storage phosphor screen (upper panel), then blotted for Flag (lower panel). The representative blots shown are taken from an experiment in which cells were collected at 32 hours. In a separate experiment cells were collected at 2 hours, and not at 32 hours. B. Quantification of 35S-LRRK2 relative to Flag-LRRK2 for human and mouse LRRK2 from n=3 independent experiments, with technical n=3 for each construct at each time point. Error bars indicate SEM. The best-fit lines shown are semilog lines, where X is linear, and Y is log. A one phase decay non-linear regression equation was used to calculate half-life of each protein (Human LRRK2: half-life = 3.83h, 95%CI=2.71-5.68, R2=0.84. Mouse Lrrk2: half-life = 3.36h, 95%CI=2.60-4.38, R2=0.904). (PDF 252 KB)

11064_2018_2650_MOESM2_ESM.pdf

Supplementary figure S2. Mouse LRRK2 is expressed at higher levels than human protein in mouse cells. A-C. Primary mouse glial cells (A), N2a cells (B) or NIH 3t3 cells (C) were transfected with Flag-tagged human or mouse LRRK2, or mock transfected and protein levels measured by western blot using a flag antibody. Cyclophilin B is used as a loading control for each lane. (PDF 550 KB)

11064_2018_2650_MOESM3_ESM.pdf

Supplementary figure S3. Mouse and human LRRK2 self-interact at similar levels. A. HEK293FT cells were co-transfected with GFP-tagged LRRK2 and indicated Flag-tagged human or mouse LRRK2 constructs, with GUS as a negative control. Cell lysates were subjected to immunoprecipitation (IP) for GFP and blotted for GFP (upper panel) and Flag (lower panels). Inputs for the IP are shown on the left. B. Quantification of IP for Flag-tagged proteins relative to inputs for indicated human and mouse constructs from n=3 independent experiments. Error bars indicate SEM, **, p < 0.01; ns, non-significant by Tukey’s post-hoc test from one way ANOVA compared to WT human LRRK2. (PDF 104 KB)

11064_2018_2650_MOESM4_ESM.pdf

Supplementary figure S4. Enzyme activity of mouse and human LRRK2 estimated using model peptide, Nictide. Quantification of n=3 independent experiments using indicated constructs of the phosphorylation of the Nictide peptide. *, p < 0.05; ***, p < 0.001; ns, non-significant by Tukey’s post-hoc test compared to WT human LRRK2 from one way ANOVA (F10,55=20.87, p < 0.001, n=6 samples per construct). (PDF 58 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Langston, R.G., Rudenko, I.N., Kumaran, R. et al. Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2. Neurochem Res 44, 1446–1459 (2019). https://doi.org/10.1007/s11064-018-2650-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-018-2650-4

Keywords

Search

Navigation