Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons

Abstract

Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Keywords: ATP13A2; CRISPR; DJ1; Parkin; Parkinson's disease; disease modeling; genome editing; human pluripotent stem cells; proteomics; transcriptomics.

Figure 1

Spin Culture Differentiation of TH Reporter hPSCs into Midbrain DNs (A) Experimental scheme depicting the CRISPR-mediated TH reporter knockin strategy. (B) Donor plasmid containing the targeting vector TH with a 5′TH homology arm followed by a 2A self-cleaving peptide sequence, a WPRE sequence, floxed selection cassette, and 3′ TH homology arm. Genomic locus indicating the targeting area in exon 14 of the TH gene, red star representing the position of the stop codon. Schematic representation of successful targeting post-CRE excision. (C) Experimental scheme showing the culture conditions in spin culture. (D) Bright-field images of WA01-WT spheres at time points d0, d15, d22, and d38. Scale bars, 500 μm (4×). (E) Immunohistochemistry of sectioned organoids/spheres. Each panel organized as (top left, 4× d0; bottom left, 4× d15. Scale bar, 500 μm (4×); top middle, 20× d0; bottom middle, 20× d15; top right, 20× d22; and bottom right, 20× d38. Scale bar, 50 μm (20×) using validated antibodies against: top left panel (TRA-1-60), top right panel (OCT4), bottom left panel (FOXA2 and LMX1A), and bottom right panel (dsRed and TH). Nuclei were counterstained using HOECHST. (F) Fluorescence images of WA01-WT spheres at d38 showing TH:TdTomato reporter expression. Scale bars, 500 μm, 4× (n = 3 differentiation experiments). (G) Maximum projection image of d22 sphere from differentiated BJ-SIPS WT TH cells showing TH:TdTomato expression. z stack images acquired using spinning disc confocal CX7 at 10×. Scale bar, 100 μm. (H) Fluorescence microscopy panel, showing DNs at d38 from WA01-TH-WT cell lines, dissociated on d25 and plated in low density on glial cells. Scale bars, 50 μm, 20× (top left, Hoechst; top right, a-dsRed; bottom left, a-TH [MAB318]; bottom right, merge).

Figure 2

Generation, and Molecular and Functional Characterization of Knockout Cell Lines in the PD Model (A) Experimental Scheme depicting the CRISPR knockout mutagenesis strategy. (B) Graphical display of chromosome positions, targeted exons, and CRISPR sequences. (C) Table summarizing knockouts indicating targeted exons, and coloring scheme used in this study in isogenic HUES1, WA01 and BJ-SIPS lines: WT, PARKIN^−/−, DJ-1^−/−, and ATP13A2^−/−. (D) Fluorescence microscopy panel, showing HUES1-derived DNs at d35 from all isogenic reporter cell lines, dissociated on d25 and plated in low density on mouse glial cells. Scale bars, 100 μm, 40× (left, ICC stained with TH antibody AB152; middle, TH:TdTomato reporter fluorophore expression, right merge). (E) Fluorescence microscopy images of flow sorted TH⁺ neurons from all isogenic lines 4 weeks post plating on glial cells before electrophysiological analysis. Scale bars, 50 μm. (F) Whole-cell patch clamp recordings in TH⁺-labeled DNs derived from all four iPSC lines (n = 6–9 for each line) showing voltage-gated sodium and potassium currents (top row), evoked action potentials (middle), and spontaneous action potentials (bottom). (G) Quantification of PARKIN abundance in DNs from WT, PARKIN^−/−, and DJ-1^−/− lines using AQUA peptides. (H) Western blot of WT, PARKIN^−/−, and ATP13A2^−/− lines using PARKIN antibody and GAPDH as a control, at three time points, indicated as pluripotent at d0, NPC at d12, midbrain DN at d75, and mouse whole-brain lysate as control. (I) Scree plot showing the percentage of variances explained by each principal component. (J) PCA plot of components 1 and 2 at d35. (K) PCA plot of components 2 and 3 at d35. (L) Bar chart showing normalized relative abundance of DJ1 protein, WT samples set to 1. (M) Bar chart showing normalized relative abundance of ATP13A2 protein, WT samples set to 1. Statistical significance was analyzed by one-way ANOVA followed by post hoc test (1% FDR)-Bonferroni-Holm for multiple comparisons.

Figure 3

OS, Mitochondrial Dysfunction and Lysosomal Dysregulation Are Shared Phenotypes in all Early-Onset PD DNs (A) The canonical oxidative phosphorylation pathway created using IPA software. log₂ fold changes were plotted. Purple outlines indicate significantly dysregulated proteins/pathways (based on −0.7/0.7 log₂ fold change). Red color indicates a positive change or protein enrichment in the PARKIN^−/− line (none noted), while blue represents a depletion. (B, D, G) Heatmap.2 function across all samples using specified gene lists, default clustering and row scaling. Columns represent samples, rows represent genes, and color intensity represents column Z score, where red indicates enriched and blue depleted proteins. (B) Heatmap analysis of 179 Mito Carta 2.0 proteins with mitochondrial function or localization that shows differential protein abundance between any cell line in d35 DNs. (C) Subset of Mito Carta 2.0 analysis showing all quantified proteins that are part of the NDUF-complex I in d35 DNs. (D) Heatmap of all proteins that were quantified with at least two unique peptides that are part of the NDUF-complex I in hPSCs. (E) Dissociated spheres at d35 were stained with CellROX green to detect mROS production in the basal state. mROS-G⁺ was quantified in TH⁻ as well as TH⁺ cells. Histograms of mROS-G positivity overlaid with no staining control, stained WT (blue) and PARKIN^−/− (red) lines, respectively. (F) Averaged mROS percentage, quantified as mROS-G⁺ using synchronized bisector gates (FloJo, LLC) to divide the x axis into GFP⁻ and GFP⁺ non-overlapping populations based on the background expression in our unstained control. Statistical significance was analyzed by one-way ANOVA followed by Bonferroni-Holm multiple comparison test (n = 4 independent staining and flow experiments significance indicated in tables [bottom]). (G) Heatmap analysis of proteins with an annotated lysosomal function that were quantified with at least two unique peptides.

Figure 4

Quantification of Cell Death Events in TH⁺ DNs (A) Dissociated spheres were analyzed via flow cytometry, TH quantification in isogenic HUES1 lines at d35 showing decreased numbers of TH⁺ cells in the PARKIN^−/− line. Statistical significance p value derived from one-way ANOVA followed by Bonferroni-Holm multiple comparison test (n = 6, 6, 3, and 3 independent differentiation experiments). (B) TH quantification in WT and PARKIN^−/− isogenic lines in a time course experiment in HUES1 line (n = 1 except d35 n = 6). (C) TH quantification in WT and PARKIN^−/− isogenic lines in a time course experiment in WA01 line (d0–d21, WT, n = 3 differentiation replicates; PARKIN^−/−, n = 3 independent clones; d26–d38, WT, n = 4 differentiation replicates; PARKIN^−/−, n = 5 or 3 independent clones, 2 differentiation replicates; d38, ^∗p < 0.05, unpaired two-sided t test). (D–F) Nikon Biostation CT live cell fluorescence imaging of TH:TdTomato expression during differentiation of WT and PARKIN^−/− lines after dissociation at d15. Image acquisition starts 24 h post plating. Images were acquired every 6 h, time points indicated. Squares depict areas of interest that are shown in higher magnification. (D) WT cell line. (E) PARKIN^−/− cell line. (F) Representative images of cell death events in the PARKIN^−/− cell line. Zoomed images with red arrows indicating cells of interest before and after cell death. (G) Cell death quantification of burst events in WT and PARKIN^−/− cell lines at the indicated time points, shown as log₂ fold change between PARKIN^−/− and WT cell lines (n = 3 wells per cell line). ^∗p < 0.05, unpaired two-sided t test. (H) Bar chart showing normalized relative abundance of SNCA protein across all samples, WT samples set to 1. (I) Bar chart showing normalized relative abundance of CALB1 protein across all samples. WT samples set to 1. Statistical significance was analyzed by one-way ANOVA followed by post hoc test (1% FDR) Bonferroni-Holm for multiple comparisons.

Figure 5

Transcriptomics Analysis of Sorted DNs Highlights Common and Distinct Dysregulation of PD-Relevant Genes and Pathways: HUES1 PD Lines (A) Multidimensional scaling plot of all isogenic cell lines in which distance corresponds to leading log fold changes between each pair of RNA samples. Dimension 1 separates PARKIN^−/− line from all other lines and dimension 3 separates the ATP13A2^−/− line from WT and DJ-1^−/− lines. (B) Table showing the number of significant DEGs between WT and isogenic lines (FDR = 0.01), the number of up- and downregulated genes, and the resulting ratio. Overlap analysis as a three-way Venn diagram intersecting DEGs between WT and all isogenic lines. (C) Heatmap across all samples using the 24 intersecting genes between WT versus PARKIN^−/− and WT versus DJ-1^−/− analyses. (D) Heatmap across all samples using the 64 intersecting genes between WT versus PARKIN^−/− and WT versus ATP13A2^−/− analyses. (E) Bar chart of d35 global transcriptomics comparisons between WT and all PD cell lines showing select altered disease or biological function pathways determined using IPA curated sets. The y axis shows the significance (–log(p value)) and the orange line shows the significance threshold cutoff of −log(p value = 0.05), sorted by significance in PARKIN^−/− versus WT comparison.