Figures and data
BPAN and control iPSC line differentiation into mDA neurons
(A) Immunofluorescence analysis of MAP2/TH in patient and control lines at day 65 of differentiation. Mature mDA neurons exhibit typical morphology and express pan-neuronal (MAP2) and mDA-specific (TH) markers. Nuclei were counterstained with DAPI. Scale bar, 200 μm.
(B) Quantification of MAP2/TH abundance in control and patient-derived neurons (n=3 biological replicates per line, 3 individual images analysed per replicate). Percentages were calculated after manual counting of cells on ImageJ/Fiji (approximately 500 nuclei counted per image, followed by counting of cells also staining positive for TH and/or MAP2).
(C) Chromatograms from WDR45 cDNA sequencing in female BPAN iPSC lines. In Patient 01, the cDNA has 28 additional bp, while for Patient 03, the c.700C>T leading to an early stop codon is retained.
(D) Relative Fluorescence Units of Androgen Receptor CAG repeat PCR products from female BPAN lines, in the presence or absence of methylation-sensitive restriction enzyme Hpall. Patient 01 fibroblasts (BUCL01) exhibit a random XCI pattern, with two PCR bands detectable in the presence and absence of Hpall. For patient 03 (535-201), there is practically only one detectable band in fibroblasts (535-201), signifying a skewing of XCI towards the expression of only one allele. For both patients, iPSC lines and derived neurons have very skewed patterns of XCI, with PCR bands practically undetectable in the presence of Hpall.
(E) Cropped immunoblot of total WDR45 and beta actin protein expression at Day 65, and quantification of WDR45 relative to the loading control. n=3 biological replicates for each line.
Error bars represent the Standard Error of Mean. Statistics were calculated using ANOVA.
RNASeq at Day 65 of differentiation.
(A) Volcano plots comparing gene expression profiles patient lines versus controls. Overexpressed genes are shown on the right of the X axis while underexpressed genes of the left.
(B) Heat maps depicting gene underexpression and overexpression in gradients of green and red, respectively.
(C) GO Term and KEGG pathway enrichment analysis depicting intracellular pathways affected in BPAN. Pathways with the most significant Fold Enrichment (Y axis) are shown; differentially expressed gene count is depicted on the X axis.
n=3 biological replicates for all lines, analysis based on median TPM values. P-values of <0.05 and fold change of <0.5 or >2 [-1<log2(FC)>1] (Student’s t-test) were set as statistically significant cut-offs. The top 40 genes (as per lowest p-values) are labelled in volcano plots and heat maps. ATG= autophagy-related gene, TPM= transcript per million.
High content imaging LC3 assay for drug screening.
(A) High Content Imaging immunofluorescence analysis of LC3/ FOXA2 in patient and control ventral midbrain progenitors at day 11 of differentiation. Representative images. Cell density at 15,000 cells/well. n=8 independent differentiations/ biological replicates for each line.
(B) Quantification of LC3 puncta/ nuclei in control and patient-derived neurons DMSO and Torin 1 treatments were of 3-hour durations. For statistical analysis, the Student’s unpaired two tailed t-test was used. Error bars represent the Standard Error of Mean.
Drug Screening
(A) Screening protocol. Patient 02 cells used for the screen. The process was optimised and, wherever possible, automated to allow large-scale compound testing.
(B) Source plate layout (left) and a representative heatmap (right) are shown. N and P represent negative and positive controls, respectively. Increasing puncta numbers per nuclei (in hits and positive controls) are depicted in increasingly darker shade of red.
(C) Representative images. Immunofluorescence analysis of LC3/ DAPI after treatments with DMSO, compound hit digoxin and Bafilomycin A1. Digoxin increases LC3 puncta production to levels higher than in Bafilomycin-treated cells. Scale bar, 200 µm
(D) Distribution of hits with the highest z-scores. There is an even distribution across all 16 destination plates.
(E) Quantile-Quantile plot for screened compounds. Several compound hits have z-scores and sample quantiles that are much higher than their theoretical values, as well as values from positive controls (in red).
P62 hit validation assay.
(A) Immunofluorescence analysis of p62/DAPI in Patient 02 Day 11 ventral midbrain progenitors after treatments with Prestwick screen hits with the 26 highest z-scores. Representative images from negative controls (DMSO), positive controls (Torin 1), autophagy inducers and blockers, as well as ‘false hits’ (resulting in reduced cell viability).
(B) Quantification of cell viability (fold change of nuclei number per well, compared to DMSO) and p62 degradation (fold change of p62 puncta per nuclei, compared to DMSO; green bars) for each hit, as well as Torin 1 and DMSO.
(C) 5 cardiac glycosides significantly degrade p62 when compared to DMSO and are associated with acceptable cell viability. There is no statistical difference in p62 puncta per cell, or nuclei per well, when comparing Torin 1 and the 5 cardiac glycosides. There is also no statistical significance between DMSO-treated and glycoside-treated nuclei per well (statistics not shown).
n=3 technical replicates for each compound, n=9 for DMSO and Torin 1. Error bars represent Standard Deviation. Statistics were calculated using ANOVA. Abbreviations: FC = fold change, Nr= number
Correction of intracellular pathways after compound hit 48-hour treatment.
(A) Volcano plots comparing Patient 02, Patient 02 compound-treated and Patient 02 CRISPR-corrected (CRISPR 01) mDA neuronal gene expression.
(B) Intracellular pathways jointly corrected by both WDR45 CRISPR-mediated mutation correction and compound treatments.
(C) ATG differential expression and correction after CRISPR-genome editing and compound treatments. ATGs with consistent differential (over- or under) expression after CRISPR correction and compound treatments, when compared to untreated Patient 02 neurons, are marked with *. White= not mapped during RNAseq.
(D), (E) ClueGO analysis of GO term enrichment of differentially expressed genes, showing network graphs of differentially expressed genes between Patient 02, CRISPR01, and compound-treated Patient 02 mDA neurons. Cellular components (CC) (C), and molecular functions (MF) (D) are shown. Network graph nodes represent GO terms (the most significant are named) and edges indicate shared genes between GO terms. Functional groups of GO terms are indicated by the same colour. GO functional groups exhibiting statistically significant differences (p< 0.05) are shown.
n=3 biological for all lines, analysis based on median TPM values. P-values of <0.05 and fold change of <0.5 or >2 [-1<log2(FC)>1] (Student’s t-test) were set as statistically significant cut-offs. The top 40 genes (as per lowest p-values) are labelled in volcano plots.
iPSC characterisation of pluripotency.
(A) Immunofluorescence staining at Day 16 of the Spontaneous in vitro differentiation protocol. SOX17 (SRY-BOX 17; endoderm-related), TUJ1 (Neuronal Class III β-Tubulin; ectoderm-related), and SMA (Alpha Smooth Muscle Actin; mesoderm-related) markers are shown. Nuclei were counterstained with DAPI. Scale bar, 500 µm.
(B) Immunofluorescence for pluripotency markers NANOG, OCT4, TRA-1-60, TRA-1-81 in iPSC lines. Nuclei were counterstained with DAPI. Scale bar, 200 µm.
(C) Epi-Pluri-Score testing for iPSC lines. DNA methylation profiles (β-values) in genes ANKRD46, C14orf115, and POU5F1 for all iPSC lines match profiles of pluripotent samples (red cloud).
(D) RT PCR in iPSC lines for expression of pluripotency markers SOX2, KLF4, NANOG, OCT4. H9 human embryonic stem cell (H9 hESC) line and human dermal fibroblasts were used as positive and negative controls, respectively. GAPDH was used as a housekeeping gene.
(E) RT PCR in iPSC lines for detection of Sendai virus genome and pluripotency transgenes. A positive (+ve) control (SeV DNA) and a negative control (cDNA from the H9 human embryonic stem cell line, H9 hESC) were also analysed. GAPDH was used as a housekeeping gene.
(F) Chromatograms from genomic DNA sequencing in BPAN iPSC lines. iPSC lines maintain disease-causing mutations. WDR45 disease-causing mutations are highlighted in the red rectangles.
(G) SNP array analysis of all iPSC lines used for downstream experiments, including the two isogenic controls. Representative images. All deletions/ gains in iPSCs used for downstream experiments were small (<5Mb) and deemed as non-pathogenic by BlueFuse Multi.
(H) Alignment of wild type, patient 02 (c.19C>T), patient 03 (c.700C>T) and CRISPR corrected WDR45 genomic DNA (above) and amino acid (below) sequences. Premature protein truncation results from both c.19C>T and c.700C>T mutations. For each CRISPR-corrected line, three nucleotide substitutions have occurred after HDR (red rectangles). For both corrections, the first two are silent/ synonymous changes and, overall, the sequence leads to translation of a full-length WDR45 protein.
Generation and basic characterisation of mDA model.
(A) Protocol for A9-type mDA differentiation.
(B) Immunofluorescence for ventral midbrain progenitor-specific markers FOXA2 and LMX1A at Day 11 of mDA differentiation. Nuclei were counterstained with DAPI. Scale bar, 500 µm.
(C) Quantification of FOXA2 and LMX1A abundance in Day 11 progenitors. n=3 biological replicates for all lines, 3 individual images from random areas of a well for each biological replicate. Percentages were calculated after manual counting of cells on ImageJ/Fiji (approximately 500 nuclei counted per image, followed by counting of cells also staining positive for FOXA2 and/or LMX1A).
(D) qRT-PCR at d11 for pluripotency markers OCT4 and NANOG, and midbrain related markers FOXA2, LMX1A, LMX1B, EN1, EN2, relative to housekeeping gene (GAPDH) and normalised to their respective iPSCs (n = 1 for each line, 3 technical replicates). Error bars indicate the Standard Error of Mean.
(E) qRT-PCR for TH, SNCA, NURR1, DAPT and DAT at day 65. mRNA values are relative to the housekeeping gene and normalised to the corresponding iPSCs (n = 3-5 per line).
(F) Cropped immunoblot of total WDR45 and beta actin protein expression at Day 11, and relevant quantification. n=3-4 biological replicates for each line.
Error bars represent the Standard Error of Mean. Statistics were calculated using ANOVA. Abbreviations: EBs= embryoid bodies. FC= fold change
RNASeq at Day 65 of differentiation
(A) List of differentially expressed genes when comparing Patient 01, Patient 02, Patient 03 versus Control 01, Control 02, CRISPR 01 and CRISPR 02 mDA neurons.
(B) ClueGO analysis of GO terms enrichment of differentially expressed genes, showing pie charts for cellular component (CC), and molecular function (MF).
(C) Volcano plots of differentially expressed genes when comparing Patient 02 and corresponding CRISPR line (CRISPR 01), as well as Patient 03 versus corresponding CRISPR line (CRISPR 02). The top 40 genes (as per lowest p-values) are labelled. Right: GO Term and KEGG pathway enrichment analysis depicting intracellular pathways jointly corrected in both Patients 02 and 03, when compared to CRISPR 01 and 02.
(D) List of intracellular pathways and genes corrected in both Patients 02 and 03, when compared to CRISPR 01 and 02.
(E) List of differentially expressed genes and involved pathways when comparing Patient 02, versus CRISPR 01 (Patient 02 Corrected) and Patient 02 Torin 1- and Digoxin-treated mDA neurons.
(F) ClueGO analysis of GO terms enrichment of differentially expressed genes, showing pie charts for cellular component (CC), and molecular function (MF).
n=3 for all lines, median TPM values analysed. Network graph nodes represent GO terms (the most significant are named) and edges indicate shared genes between GO terms. Functional groups of GO terms are indicated by the same colour. Pie charts show the percentages of each functional group representation. GO functional groups exhibiting statistically significant differences (p< 0.05) are shown.
Defective autophagy flux in BPAN cells.
(A) Patient and control fibroblasts imaged after 3-hour treatments with DMSO, autophagy flux inducers (Torin 1) and/ or inhibitors (Bafilomycin A1). Representative images. Cells were plated in 96-well plates at a density of 15,000 cells/well. n=5 biological replicates for each line. For each biological replicate, all lines were seeded on the same 96-well plate.
(B) Quantification of LC3 puncta/ nuclei in control and patient-derived fibroblasts. For statistical analysis, the Student’s unpaired two tailed t-test was used. Error bars represent the Standard Error of Mean.
(C) Quantification of LC3 puncta/ nuclei in control and patient-derived neuronal progenitors, at basal (DMSO-treated) conditions. Experiment identical to the one depicted in Fig. 3A-B, but with more independent biological replicates (n=11). Additional replicates enhance the statistical significance of previous findings. For statistical analysis, the Student’s unpaired two tailed t-test was used. Error bars represent the Standard Error of Mean.
(D) Day 11 ventral progenitors imaged after 3-hour autophagy flux induction or inhibition. Representative images. Cells were plated in 96-well plates at a density of 15,000 cells/well. n=7 independent differentiations/ biological replicates for each line. For each biological replicate, all 5 lines had the same start date of differentiation and were seeded on the same 96-well plate.
(E) Quantification of LC3 puncta/ nuclei in control and patient-derived neurons. For statistical analysis, the Student’s unpaired two tailed t-test was used. Error bars represent the Standard Error of Mean.
Fibroblast and corresponding iPSC lines used.
sgRNA and HDR donor templates used for CRISPR/Cas9 genome editing in patient lines.
PCR primers for WDR45 gene sequencing.
Primers used for WDR45 cDNA sequencing.
Primer pairs for detection of pluripotency marker expression via RT PCR.
Primers used for Sendai Virus Clearance-related RT PCR experiments.
qRT PCR primers used for Day 11 and Day 65 characterisation.
Primary and corresponding secondary antibodies used for immunofluorescence and western blotting experiments.
Hits from Prestwick screen with the 200 highest z-scores.
