Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Abstract

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing1,2,3,4. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson’s disease as well as impairment of adult neurogenesis in mice5. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson’s disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson’s disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson’s disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s disease pathology and may help to open new avenues for Parkinson’s disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: LRRK2 (G2019S) mutation results in progressive deterioration of nuclear architecture in ipsNSCs.
Figure 2: LRRK2 (G2019S) mutant ipsNSCs show deficiency in clonal expansion, spontaneous neuronal differentiation at the late passages and exhibit enhanced susceptibility to proteasomal stress-induced apoptosis.
Figure 3: Phenotypic analyses of isogenic iPSC and ESC lines in the presence or absence of the LRRK2 (G2019S) mutation.
Figure 4: Rescue of LRRK2 (G2019S)-associated phenotypic defects in NSCs by inhibition of LRRK2 kinase activity and morphological analysis of nuclear envelope in Parkinson’s disease brain slices.

References

  1. 1

    Liu, G. H. et al. Recapitulation of premature ageing with iPSCs from Hutchinson–Gilford progeria syndrome. Nature 472, 221–225 (2011)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Dechat, T. et al. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 22, 832–853 (2008)

    CAS  Article  Google Scholar 

  3. 3

    Kudlow, B. A., Kennedy, B. K. & Monnat, R. J., Jr Werner and Hutchinson–Gilford progeria syndromes: mechanistic basis of human progeroid diseases. Nature Rev. Mol. Cell Biol. 8, 394–404 (2007)

    CAS  Article  Google Scholar 

  4. 4

    Worman, H. J., Ostlund, C. & Wang, Y. Diseases of the nuclear envelope. Cold Spring Harb. Perspect. Biol. 2, a000760 (2010)

    Article  Google Scholar 

  5. 5

    Winner, B. et al. Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice. Neurobiol. Dis. 41, 706–716 (2011)

    CAS  Article  Google Scholar 

  6. 6

    Chang, K. H. et al. Nuclear envelope dispersion triggered by deregulated Cdk5 precedes neuronal death. Mol. Biol. Cell 22, 1452–1462 (2011)

    Article  Google Scholar 

  7. 7

    Tran, D., Chalhoub, A., Schooley, A., Zhang, W. & Ngsee, J. K. A mutation in VAPB that causes amyotrophic lateral sclerosis also causes a nuclear envelope defect. J. Cell Sci. 125, 2831–2836 (2012)

    CAS  Article  Google Scholar 

  8. 8

    Padiath, Q. S. et al. Lamin B1 duplications cause autosomal dominant leukodystrophy. Nature Genet. 38, 1114–1123 (2006)

    CAS  Article  Google Scholar 

  9. 9

    Woulfe, J. M. Abnormalities of the nucleus and nuclear inclusions in neurodegenerative disease: a work in progress. Neuropathol. Appl. Neurobiol. 33, 2–42 (2007)

    CAS  PubMed  Google Scholar 

  10. 10

    Cookson, M. R. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease. Nature Rev. Neurosci. 11, 791–797 (2010)

    CAS  Article  Google Scholar 

  11. 11

    Cookson, M. R. & Bandmann, O. Parkinson's disease: insights from pathways. Hum. Mol. Genet. 19, R21–R27 (2010)

    CAS  Article  Google Scholar 

  12. 12

    Deng, X. et al. Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nature Chem. Biol. 7, 203–205 (2011)

    CAS  Article  Google Scholar 

  13. 13

    Lee, B. D. et al. Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease. Nature Med. 16, 998–1000 (2010)

    CAS  Article  Google Scholar 

  14. 14

    Li, W. et al. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc. Natl Acad. Sci. USA 108, 8299–8304 (2011)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Krishnan, V. et al. Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice. Proc. Natl Acad. Sci. USA 108, 12325–12330 (2011)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Cheung, I. et al. Developmental regulation and individual differences of neuronal H3K4me3 epigenomes in the prefrontal cortex. Proc. Natl Acad. Sci. USA 107, 8824–8829 (2010)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Xie, W. et al. Proteasome inhibition modeling nigral neuron degeneration in Parkinson's disease. J. Neurochem. 115, 188–199 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Liu, G. H. et al. Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell 8, 688–694 (2011)

    CAS  Article  Google Scholar 

  19. 19

    Suzuki, K. et al. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc. Natl Acad. Sci. USA 105, 13781–13786 (2008)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Li, M. et al. Efficient correction of hemoglobinopathy-causing mutations by homologous recombination in integration-free patient iPSCs. Cell Res. 21, 1740–1744 (2011)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Aizawa, E. et al. Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors. Mol. Ther. 20, 424–431 (2012)

    CAS  Article  Google Scholar 

  22. 22

    Rudenko, I. N., Chia, R. & Cookson, M. R. Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson's disease? BMC Med. 10, 20 (2012)

    CAS  Article  Google Scholar 

  23. 23

    Kanao, T. et al. Activation of FoxO by LRRK2 induces expression of proapoptotic proteins and alters survival of postmitotic dopaminergic neuron in Drosophila . Hum. Mol. Genet. 19, 3747–3758 (2010)

    CAS  Article  Google Scholar 

  24. 24

    Gehrke, S., Imai, Y., Sokol, N. & Lu, B. Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression. Nature 466, 637–641 (2010)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Nichols, R. J. et al. 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization. Biochem. J. 430, 393–404 (2010)

    CAS  Article  Google Scholar 

  26. 26

    Dzamko, N. et al. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14–3-3 binding and altered cytoplasmic localization. Biochem. J. 430, 405–413 (2010)

    CAS  Article  Google Scholar 

  27. 27

    Scaffidi, P. & Misteli, T. Lamin A-dependent nuclear defects in human aging. Science 312, 1059–1063 (2006)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Poulopoulos, M. et al. Clinical and Pathological Characteristics of LRRK2 G2019S Patients with PD. J. Mol. Neurosci. 47, 139–143 (2012)

    CAS  Article  Google Scholar 

  29. 29

    Thaler, A., Mirelman, A., Gurevich, T., Simon, E., Orr-Urtreger, A., Marder, K., Bressman, S. & Giladi, N. Lower cognitive performance in healthy G2019S LRRK2 mutation carriers. Neurology 79, 1027–1032 (2012)

    CAS  Article  Google Scholar 

  30. 30

    Tiscornia, G., Vivas, E. L. & Belmonte, J. C. Diseases in a dish: modeling human genetic disorders using induced pluripotent cells. Nature Med. 17, 1570–1576 (2011)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We would like to thank K. Mitani, P. Ng, A. Lieber, Y. Imai, M. A. Miyawaki, Filocamo, S. Goldwurm, Telethon Genetic Biobank Network for providing constructs and cells (the fibroblast samples were obtained from the “Cell Line and DNA Biobank from patients affected by Genetic Diseases” (G. Gaslini Institute)-Telethon Genetic Biobank Network (project no. GTB07001)); Neurological Tissue Bank of the Biobank-Hospital Clínic-IDIBAPS for providing human brain tissue; F. Gage, M. Hetzer, J. Yao, Y. Mu, D. Yu, E. Gelpí, X. M. Wang, X. Wang, G. Bai and Z. J. Liu for helpful discussions; M. Joens and J. Fitzpatrick of the Waitt Advanced Biophotonics Core Facility for performing TEM analysis; M. Marti for imaging, teratoma and karyotyping analysis; F. Osakada for statistics analysis; and M. Schwarz, P. Schwarz and L. Laricchia-Robbio for administrative help. G.-H.L. is supported by the Thousand Young Talents program of China, the National Laboratory of Biomacromolecules, the Strategic Priority Research Program of the Chinese Academy of Sciences, the National Natural Science Foundation of China (NSFC) (81271266 and 31222039), and the Beijing Municipal Natural Science Foundation. J.Q. was partly supported by an AFAR/Ellison Medical Foundation postdoctoral fellowship. K.S. was partly supported by a Uehara Memorial Foundation research fellowship. E.N. was partly supported by an F.M. Kirby Foundation postdoctoral fellowship. X.X. is supported by NSFC (31201111). B.R. was supported by a US National Institute of Health (NIH) grant (ES017166) and the Ludwig Institute for Cancer Research. J.Y. was supported by an NIH grant (P41 RR011823). J.C.I.B. was supported by grants from the Glenn Foundation, G. Harold and Leila Y. Mathers Charitable Foundation, Sanofi, the California Institute of Regenerative Medicine, the Ellison Medical Foundation, the Helmsley Charitable Trust, ERA-Net Neuron, MINECO and Fundacion Cellex.

Author information

Affiliations

Authors

Contributions

G.-H.L., J.Q., K.S. prepared the figures, designed and performed all in vitro experiments. E.N. and N.M. designed and performed in vivo experiments. A.G., J.K., R.D.S., X.X., W.Z., Y.L., S.R. and C.R.E. provided technical assistance. I.D. performed teratoma studies. F.Y. generated microarray data. M.L. performed FISH and DNA methylation assays. B.R., U.W. and A.K. performed and analysed epigenetic studies. J.T. and J.Y.III performed proteomic studies. G.-H.L., J.Q., K.S., E.N., I.S.-M. and J.C.I.B. wrote the manuscript.

Corresponding authors

Correspondence to Guang-Hui Liu or Juan Carlos Izpisua Belmonte.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1-18 and Supplementary references. (PDF 3162 kb)

Supplementary Data

This file contains Supplementary Tables 1-5. (XLS 135 kb)

In-1 mediated restoration of cellular morphology

The In-1 mediated restoration of cellular morphology in late passage LRRK2 G2019S NSCs. 5 mM In-1 was added to passage 18 ipsNSCs-LK2(GS/GS), and then the cells were cultured for 5 days. Cells were imaged every 10 min for the 5 day duration. (MOV 18568 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, GH., Qu, J., Suzuki, K. et al. Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature 491, 603–607 (2012). https://doi.org/10.1038/nature11557

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing