Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 12 (3), 518-531

Modeling G2019S-LRRK2 Sporadic Parkinson's Disease in 3D Midbrain Organoids

Affiliations

Modeling G2019S-LRRK2 Sporadic Parkinson's Disease in 3D Midbrain Organoids

Hongwon Kim et al. Stem Cell Reports.

Abstract

Recent advances in generating three-dimensional (3D) organoid systems from stem cells offer new possibilities for disease modeling and drug screening because organoids can recapitulate aspects of in vivo architecture and physiology. In this study, we generate isogenic 3D midbrain organoids with or without a Parkinson's disease-associated LRRK2 G2019S mutation to study the pathogenic mechanisms associated with LRRK2 mutation. We demonstrate that these organoids can recapitulate the 3D pathological hallmarks observed in patients with LRRK2-associated sporadic Parkinson's disease. Importantly, analysis of the protein-protein interaction network in mutant organoids revealed that TXNIP, a thiol-oxidoreductase, is functionally important in the development of LRRK2-associated Parkinson's disease in a 3D environment. These results provide proof of principle for the utility of 3D organoid-based modeling of sporadic Parkinson's disease in advancing therapeutic discovery.

Keywords: Parkinson's disease; disease modeling; iPSC; midbrain; organoids.

Figures

Figure 1
Figure 1
Generation of Midbrain 3D Organoids from hiPSCs (A) Bright-field microscopy images of the stages of 3D organoid generation for 2 months. Scale bars, 200 μm. (B) qRT-PCR analysis of a pluripotency marker (OCT4), a neural progenitor marker (SOX1), and dopaminergic neuronal markers (TH and VMAT2) at different time points. Data represent the mean ± SEM. p < 0.05, ∗∗p < 0.01 by ANOVA. (C) Immunofluorescence for MAP2, MASH1, TUJ1, VMAT2, TH, DAT, and GIRK2 to confirm the presence of midbrain dopaminergic neurons on day 60. Scale bars, 50 μm. (D) Schematic image of midbrain development. The marginal zone (MZ) contains midbrain dopaminergic neurons that differentiate from radial glia cells in the ventricular zone (VZ). (E) Percentage of MAP2/MASH1-positive cells in the MZ- and VZ-like zones at day 60. (F) Gene expression profiling using qRT-PCR from 1 to 45 days. Red and green represent higher and lower gene expression levels, respectively; n = 3 per sample. (G) Fluorescence-activated cell sorting analysis of synapsin-RFP-positive cells from midbrain 3D organoids. (H and I) KCL-induced dopamine levels in midbrain 3D organoids using liquid chromatography-mass spectrometry analysis. Data represent the mean ± SEM. ∗∗p < 0.01 by ANOVA. (J) Gene set enrichment analysis of the microarray data from midbrain 3D organoids compared with that of 2D cultures.
Figure 2
Figure 2
Generation of Midbrain 3D Organoids from LRRK2-G2019S hiPSCs (A) Immunofluorescence staining of midbrain 3D organoids from LRRK2-G2019S hiPSCs. Scale bars, 100 μm. (B) qRT-PCR analysis of midbrain 3D organoids and LRRK2-G2019S 3D organoids regarding dopaminergic neuronal markers TH, AADC, and DAT at 60 days. Data represent the mean ± SEM. p < 0.05, ∗∗p < 0.01 by ANOVA (n = 3 per sample). (C) Immunostaining of TH-positive and cleaved caspase-3-positive cells in midbrain 3D organoids and LRRK2-G2019S 3D organoids. Scale bars, 20 μm. (D) Percentage of cleaved caspase-3/TH-positive cells in midbrain 3D organoids and LRRK2-G2019S 3D organoids treated with 0.5 mM MPTP. Data represent the mean ± SEM. p < 0.05, ∗∗p < 0.01 by ANOVA (n = 3 per sample). (E and F) Western blot analysis (E) and quantification (F) show an increase in cleaved caspase-3 levels after treatment with MPTP. Data represent the mean ± SEM. p < 0.05, ∗∗p < 0.01 by ANOVA.
Figure 3
Figure 3
Pathological Analyses of LRRK2-G2019S Knockin 3D Organoids (A) Representative images showing colocalization analysis of EEA1-positive endosomes with pS129-α-synuclein-positive puncta in wild-type and LRRK2-G2019S 3D organoids. Scale bar, 20 μm. (B) Percentage of pS129-α-synuclein-positive and EEA1-positive puncta among all EEA1-positive puncta (left). Number of pS129-α-synuclein/EEA1-positive endosomes per cell in LRRK2-G2019S 3D organoids (right). Data represent the mean ± SEM. p < 0.05, ∗∗p < 0.01 by ANOVA (n = 10 per sample). (C) Thioflavin T (50 μM) staining of wild-type and LRRK2-G2019S 3D organoids. The arrow indicates α-synuclein deposits. Scale bars, 20 μm. (D) Percentage of thioflavin T-positive cells among all TH-positive cells in the wild-type and LRRK2-G2019S 3D organoids. Data represent the mean ± SEM. ∗∗p < 0.01 by ANOVA (n = 3 per sample). (E) Quantification of the mean area of thioflavin T-positive deposits in midbrain 3D organoids at 60 days. Data represent the mean ± SEM. ∗∗p < 0.01 by ANOVA (n = 5 per sample). (F and G) (F) Treatment with the LRRK2 kinase inhibitor (GSK2578215A, 1 μM) significantly reduces phosphorylated α-synuclein oligomer levels in LRRK2-G2019S 3D organoids. (G) Intensity of pS129-α-synuclein in LRRK2-G2019S 3D organoids treated with the LRRK2 kinase inhibitor. Data represent the mean ± SEM. p < 0.05 by ANOVA (n = 3 per sample). (H) Real-time qPCR analysis of dopaminergic neuron markers (TH, AADC, and DAT) in wild-type and LRRK2-G2019S 3D organoids treated with the LRRK2 kinase inhibitor GSK2578215A. Data represent the mean ± SEM. p < 0.05 by ANOVA (n = 3 per sample).
Figure 4
Figure 4
Global Gene Expression Analyses of LRRK2-G2019S Knockin 3D Organoids and Midbrain 3D Organoids (A) Venn diagram showing the overlap of differentially expressed genes between LRRK2-G2019S knockin 2D cultures compared with wild-type 2D cultures and LRRK2-G2019S knockin 3D organoids compared with wild-type 3D organoids. Number of genes up- or downregulated by 1.5-fold are presented in the Venn diagram. (B) Differentially expressed genes in control and LRRK2-G2019S under 2D or 3D culture conditions. Heatmap and density color code of the 3,965 genes showing differential expression in the microarray analysis under wild-type and LRRK2-G2019S. One sample from each condition was prepared. (C) Scatterplots of the microarray data for LRRK2-G2019S knockin 3D organoids compared with wild-type 3D organoids (top) and LRRK2-G2019S knockin 2D cultures compared with wild-type 2D cultures (bottom). (D) Gene set enrichment analysis of the microarray expression data from LRRK2-G2019S knockin 3D organoid compared with wild-type 3D organoids. (E) Graph of the TXNIP protein interaction network related to PD risk factors. Red color indicates high expression and blue color represents low expression. Rectangular-shaped nodes represent proteins that have PD genetic risk factors.
Figure 5
Figure 5
Knockdown of TXNIP Rescues α-Synuclein Oligomers in LRRK2-G2019S Knockin 3D Organoids (A) Validation of microarray and real-time qPCR gene expression data. (B) Real-time qPCR analysis of TXNIP expression in LRRK2-G2019S knockin 3D organoids and midbrain 3D organoids. Data represent the mean ± SEM. ∗∗p < 0.01 by ANOVA (n = 3 per sample). (C) Real-time qPCR analysis of TXNIP expression in control and LRRK2-G2019S 3D organoids compared with control and LRRK2-G2019S 2D cultures. Data represent the mean ± SEM. ∗∗p < 0.01 by ANOVA (n = 3 per sample). (D) Validation of TXNIP gene expression in LRRK2-G2019S knockin 3D organoids treated with TXNIP-short hairpin RNA (TXNIP-shRNA) (target sequence: agt gga ggt gtg tga agt tac tcg tgt ca, i026466a) via real-time qPCR. Data represent the mean ± SEM. p < 0.05, ∗∗p < 0.01 by ANOVA (n = 3 per sample). (E and F) Western blot analysis (E) and quantification (F) of pS129-α-synuclein reveals a reduction in pS129-α-synuclein oligomers in LRRK2-G2019S knockin 3D organoids treated with TXNIP-shRNA. Data represent the mean ± SEM. p < 0.05 by ANOVA (n = 3 per sample). (G) Representative immunofluorescence images of LAMP1 and pS129-α-synuclein puncta in LRRK2-G2019S knockin 3D organoids. Scale bar, 10 μm. (H and I) Quantification (H) of LAMP1+ and pS129-α-synuclein+ puncta showing a reduction (I) of pS129-α-synuclein inclusions upon treatment with TXNIP-shRNA. Data represent the mean ± SEM. p < 0.05 by ANOVA (n = 5 per sample).

Similar articles

See all similar articles

Cited by 4 PubMed Central articles

References

    1. Arenas E. Wnt signaling in midbrain dopaminergic neuron development and regenerative medicine for Parkinson's disease. J. Mol. Cell Biol. 2014;6:42–53. - PubMed
    1. Barral S., Beltramo R., Salio C., Aimar P., Lossi L., Merighi A. Phosphorylation of histone H2AX in the mouse brain from development to senescence. Int. J. Mol. Sci. 2014;15:1554–1573. - PMC - PubMed
    1. Beal M.F. Experimental models of Parkinson's disease. Nat. Rev. Neurosci. 2001;2:325–334. - PubMed
    1. Bershteyn M., Nowakowski T.J., Pollen A.A., Di Lullo E., Nene A., Wynshaw-Boris A., Kriegstein A.R. Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell. 2017;20:435–449.e4. - PMC - PubMed
    1. Byers B., Lee H.L., Reijo Pera R. Modeling Parkinson's disease using induced pluripotent stem cells. Curr. Neurol. Neurosci. Rep. 2012;12:237–242. - PMC - PubMed

Publication types

LinkOut - more resources

Feedback