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. 2012 Nov 22;491(7425):603-7.
doi: 10.1038/nature11557. Epub 2012 Oct 17.

Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Guang-Hui Liu  1 , Jing Qu, Keiichiro Suzuki, Emmanuel Nivet, Mo Li, Nuria Montserrat, Fei Yi, Xiuling Xu, Sergio Ruiz, Weiqi Zhang, Ulrich Wagner, Audrey Kim, Bing Ren, Ying Li, April Goebl, Jessica Kim, Rupa Devi Soligalla, Ilir Dubova, James Thompson, John Yates 3rd, Concepcion Rodriguez Esteban, Ignacio Sancho-Martinez, Juan Carlos Izpisua Belmonte
Affiliations

Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Guang-Hui Liu et al. Nature. .

Abstract

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. 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 mice. 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.

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Figures

Fig.1
Fig.1. LRRK2(G2019S) mutation results in progressive deterioration of nuclear architecture in ipsNSCs
a-b, Immunofluorescence analysis of the indicated neural progenitor markers and proliferation markers Ki67 at passage 14 (p14). Nuclei were stained with Hoechst. Scale bars, 20 μm (a), and 10 μm (b). c, Immunofluorescence of Lamin B1 and LAP2 at passage 14. Nuclei were stained with Hoechst. Projections of y-z axes from representative nuclei are shown on the right of each panel. Scale bars, 10 μm. d-e, Quantifications of nuclear area (d) and nuclear envelope (NE) circularity (e) in differentiated NSCs at passage 6, 14, and 19. a.u., arbitrary units. At least 100 randomly chosen cells were counted. #p<10−15. f, Subcellular distributions of Lamin B1, Lamin B2, LAP2β*** and H3K9me3 were determined at passage 15. Arrows indicate nuclear envelope microdomains deficient for Lamin B types. Scale bars, 5 μm. g, Quantifications of local loss of Lamin B1 in ipsNSCs. Data are shown as mean±s.d. n=3. **p<0.01. h, Extracts from wild-type esNSCs transduced with HDAdV(venus) vector (HDAdV) or HDAdV(venus)-Flag-LRRK2(G2019S)(Fl-LK2(GS)) were immunoprecipitated (IP) with an anti-Flag antibody, followed by immunoblotting analysis for the indicated proteins. Asterisks indicate non-specific bands, and arrowheads indicate Lamin B2.
Fig. 2
Fig. 2. LRRK2(G2019S) mutant ipsNSCs show deficiency in clonal expansion, spontaneous neuronal differentiation, and exhibit enhanced susceptibility to proteasomal stress-induced apoptosis
a, Pair-wise comparisons of quantile normalized log2-read counts within +/− 2.5 kb of the Transcription Start Site (TSS) or RefSeq genes. b, passage 16 ipsNSCs-LK2(GS/GS) demonstrated significantly lower colony forming capacity. #1 and #2 represent two independent NSC lines. Data are shown as mean±s.d. n=3. **p<0.01. c, Immunofluorescence analysis of the neuronal marker MAP2 in spontaneous differentiation experiments. Arrows indicate deformed nuclei. Percentages of neuronal differentiation efficiency, indicated by the ratio of MAP2 positive cells to total cell nuclei, are shown in corners. See Supplementary Fig. 11a (left panels) for wide field images. Scale bar, 20 μm. d, 14 days after ipsNSC seeding, ipsNSCs-LK2(GS/GS) at passage 16 demonstrated significantly lower neuronal differentiation capability as compared to wild-type counterparts. Data are shown as mean±s.d. n=3. **p<0.01. e. Cell death assays upon MG132 (10 μM, 20 h)-treatment in ipsNSCs-LK2(+/+) and ipsNSCs-LK2(GS/GS) at passage 15. Representative results from three independent experiments are shown.
Fig. 3
Fig. 3. Phenotypic analyses of isogenic iPSC and ESC lines in the presence or absence of the LRRK2(G2019S) mutation
a, Schematic representation of HDAdV-based correction of the G2019S mutation in the LRRK2 gene locus. Half arrows indicate the primer sites for PCR (P1, P2, P3 and P4). The probes for Southern analysis are shown as black bars (a, 5′ probe; b, neo probe; c, 3′ probe). Red X indicates the mutation site in exon 41. Blue triangles indicate the FLPo recognition target (FRT) site. P indicates the PflFI sites. b, Sequencing results of the G2019S mutation site in exon 41 of the LRRK2 gene in LRRK2(G2019S) heterozygous mutant iPSCs before (left; LK2(GS/+)) and after (right; C-LK2(GS/+) ) gene correction. c, Southern blot analysis of LRRK2(G2019S) heterozygous mutant iPSCs (LK2(GS/+)) and their gene-corrected counterparts bearing neo cassette (C-LK2(neo/+)). d, Subcellular distributions of Lamin B1 and Lamin B2 in representative ipsNSCs-LK2(GS/+) and ipsNSCs-C-LK2(GS/+) at passage 19. e, cell death assays upon MG132 treatment in corrected and uncorrected ipsNSCs. f-g, Colony formation (f) and neuronal differentiation (g) assay of ipsNSCs at passage 19. h, Immunofluorescence analysis for Lamin B1 and H3K9me3 in esNSCs-H9 and esNSCs-H9-LK2(GS/+) at passage 14. i, cell death assays upon MG132 treatment in knock-in differentiated ESCs. j, Colony formation assay on esNSCs-H9 and esNSCs-H9-LK2(GS/+) at passage 14. k, Spontaneous neuronal differentiation assay of esNSCs-H9 and esNSCs-H9-LK2(GS/+) at passage 14. For e-g and i-k, data are shown as mean±s.d. n=3.** p<0.01. For d and h, scale bars, 10 μm. Nuclei were stained with Hoechst. Arrowheads denote nuclear microdomains deficient for Lamin B1.
Fig. 4
Fig. 4. Inhibition of LRRK2 kinase activity rescues LRRK2(G2019S)-associated phenotypic defects in NSCs and morphological analysis of nuclear envelope in PD brain slices
a, Immunoblotting analysis of passage 15 ipsNSCs-LK2(+/+) and ipsNSCs-LK2(GS/GS) Data are shown as mean±s.d. n=3, **p<0.01. b, Immunoblotting analysis of indicated phosphorylated protein substrates in ipsNSCs. Data are shown as mean±s.d. n=3, **p<0.01. c, Nuclear morphology analysis in ipsNSCs-LK2(GS/GS) at passage 15 treated with In-1 for 4 days, compared to untreated controls. Nuclei were stained with Hoechst. Arrows indicate nuclear microdomains deficient for Lamin B1. Scale bars, 5 μm. d-e, Quantification of nuclear area (d) and nuclear envelope (NE) circularity (e) in ipsNSCs at passage 15 treated and untreated with In-1 for 4 days. a.u., arbitrary units. #p<10−15. f, ipsNSCs-LK2(GS/GS) at passage 17 were treated with or without In-1 for 8 days, followed by treatment with MG132 for 20 h. Data are shown as mean±s.d. n=3. **p<0.01. g, Hierarchical clustering of gene expression profiles in esNSCs-H9, esNSCs-H9-LK2(GS/+) and esNSCs-H9-LK2(GS/+) treated with 3 μM In-1 for 5 days at passage 14. h, Immunofluorescence analysis in PD patients bearing the LRRK2(G2019S) mutation (Right panel) and age-matched healthy individuals (Left panel). Data are shown as mean±s.e.m. n=5. Scale bars, 25 μm.
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References

    1. Liu GH, et al. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature. 2011;472:221–225. - PMC - PubMed
    1. Dechat T, et al. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 2008;22:832–853. - PMC - PubMed
    1. Kudlow BA, Kennedy BK, Monnat RJ., Jr. Werner and Hutchinson-Gilford progeria syndromes: mechanistic basis of human progeroid diseases. Nat Rev Mol Cell Biol. 2007;8:394–404. - PubMed
    1. Worman HJ, Ostlund C, Wang Y. Diseases of the nuclear envelope. Cold Spring Harb Perspect Biol. 2010;2:a000760. - PMC - PubMed
    1. Winner B, et al. Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice. Neurobiol Dis. 2011;41:706–716. - PMC - PubMed

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