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. 2018 Jun 13;8(1):9033.
doi: 10.1038/s41598-018-27058-0.

A Single Cell High Content Assay Detects Mitochondrial Dysfunction in iPSC-derived Neurons With Mutations in SNCA

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Free PMC article

A Single Cell High Content Assay Detects Mitochondrial Dysfunction in iPSC-derived Neurons With Mutations in SNCA

Daniel Little et al. Sci Rep. .
Free PMC article

Abstract

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of midbrain dopaminergic neuronal differentiation. iPSC-derived neurons stained for neuron specific tubulin beta 3 (TuJ1) and tyrosine hydroxylase (TH, A) or for neuron specific microtubule-associated protein 2 (MAP2) and α-synuclein, (B) in control and patient lines. Scale bar represents 50 μm. The percentage of cells expressing each neuronal marker for each line relative to the total number of nuclei was calculated (C). Immunoblot of control 1 and α-synuclein triplication patient line showing α-synuclein over expression with GAPDH as a loading control (D). Figure shows cropped blot, uncropped version is shown in Supplementary Figure 1. Bars represent mean + SD, no significant difference between lines, 2 way ANOVA, n = 2 independent plates stained and analysed.
Figure 2
Figure 2
Mitochondrial function assay workflow and image analysis. Outline of assay workflow (A). iPSC-derived neurons stained with Hoechst (shown in blue), Calcein (shown in green) and TMRM (shown in red) (B), scale bar represents 50 μm. Nuclei identified from Hoechst staining were segmented using CellProfiler software. Each object is shown in a different arbitrary colour (C,i). Cell soma identified by Calcein staining were then segmented by propagation from each nuclei; each object is shown in a different arbitrary colour (C,ii). Hoechst-positive cells with no Calcein staining, assumed to be dead cells (arrows, Ci,ii) were removed from analysis (C,iii), leaving the final cells to be analysed, each shown in a different arbitrary colour (C,iii). For mitochondrial identification, the TMRM image (D,i) was enhanced with a white top-hat filter (D,ii), mitochondria were then segmented from the enhanced image, each shown in a different arbitrary colour (E,iii). Mitochondria were then associated with their related soma. For some measurements all mitochondria within a cell were classified as one object, shown as one colour for each associated cell (D,iv). Fluorescence intensity was measured from the original TMRM image, analysing either all pixels within the segmented cell soma (E,i, green outlines) or all pixels within the segmented mitochondria (E,ii, red outlines).
Figure 3
Figure 3
Comparison of TMRM intensity between control and patient lines. TMRM fluorescence intensity measured in the whole cell (A) or in mitochondria (E) in two control lines and two patient lines, normalized to Control 1 p < 0.05–p < 0.0001 Kruskal-Wallis test and relative frequency distribution of same data (B and F). Intensity data from both control lines and both patient lines combined measured in the whole cell (C) or in mitochondria (G), p < 0.0001 Kolmogrov-Smirnov test. Change in intensity in CCCP treated cells, normalized to basal for each line at a cellular level (D) and a mitochondrial level (H), p < 0.0001 compared to basal for each line, Kruskal-Wallis test. Dots represent mean data for each image, line and bars represent median ± interquartile range, n = 3 independent experiments.
Figure 4
Figure 4
Effect of oligomycin and rotenone on TMRM intensity. TMRM fluorescence intensity measured in cells following exposure to oligomycin (A) p < 0.05 Kruskal-Wallis test, or rotenone (B), p < 0.05–p < 0.001 Kruskal-Wallis test, normalised to basal for each line. TMRM fluorescence intensity measured in mitochondria following exposure to oligomycin (C), p < 0.01 Kruskal-Wallis test, or rotenone (D) p < 0.05–p < 0.0001 Kruskal-Wallis test normalised to basal for each line. Dots represent mean data for each image, all data normalised to basal for each cell line, basal mean represented by dotted line, error line and bars represent median ± interquartile range, n = 3 independent experiments.
Figure 5
Figure 5
Effect of oligomycin and rotenone on difference in TMRM intensity between control and patient lines. TMRM intensity in cells exposed to oligomycin at a whole cell level, normalised to Control 1 basal (A), p < 0.0001, Kruskal-Wallis test compared to Control 1 or Control 2 exposed to oligomycin, at a mitochondrial level (E), p < 0.05–p < 0.0001 Kruskal-Wallis test compared to Control 1 or Control 2 exposed to oligomycin. Cumulative frequency of TMRM intensity following oligomycin exposure normalised to Control 1 basal at a cellular (C) and mitochondrial (G) level. Change in TMRM intensity in cells exposed to rotenone at a whole cell level, normalised to Control 1 basal (B), p < 0.05 Kruskal-Wallis test compared to Control 2 exposed to rotenone, no significant difference between Control 1 and Patient 1 or 2. Change in TMRM intensity at a mitochondrial level in cells exposed to rotenone (F), no significant difference Kruskal-Wallis test. Cumulative frequency of TMRM intensity following rotenone exposure normalised to Control 1 basal at a cellular level (D) and mitochondrial level (H). Dots represent mean data for each image, line and bars represent median ± interquartile range, n = 3 independent experiments.
Figure 6
Figure 6
Comparison of TMRM intensity measurements and variation in fibroblasts and iPSC-derived neurons. Fibroblasts stained with Hoechst, Calcein, and TMRM (A). Comparison of variation in well to well data (B) and cell to cell data (C) between fibroblasts and Control 1 neurons by coefficient of variation (CoV) not significant, unpaired t test, dots represent individual experiments, bars represent mean ± SEM. Whole cell TMRM intensity at a single cell level for Control 1 neurons and fibroblasts, with or without exposure to CCCP, normalised to basal for each cell type. p < 0.0001 Kuskal-Wallis test comparing basal with CCCP for each cell type, or comparing neuron CCCP with fibroblast CCCP (D), dots represent individual cells, bars represent median ± interquartile range. n = 3, scale bar represents 50 μm.
Figure 7
Figure 7
Mitochondrial morphology assessment in control and patient neurons. The mean area of individual mitochondrion objects, normalised to Control 1, under basal conditions (A), p < 0.0001 Kruskal-Wallis test, compared to Control 1 or Control 2. Mean area of mitochondria following exposure to oligomycin (B) p < 0.05, Kuskal-Wallis test, compared to Control 1 or Control 2, and following exposure to rotenone (C) p < 0.01, Kruskal-Wallis test, Patient 1 compared to Control 2 only. The total area of mitochondria within a cell, divided by the area of the cell, normalised to control 1 under basal conditions (D), p < 0.05–p < 0.0001 Kruskal-Wallis test compared to Control 1 and Control 2. Total mitochondrial area following exposure to oligomycin (E), p < 0.0001 Kruskal-Wallis test compared to Control 1 and Control 2 and following exposure to rotenone (F) p < 0.05 for Patient 1 and 2 compared to Control 2 only. The aspect ratio of individual mitochondrion objects, normalised to Control 1, under basal conditions (G) no significant difference, Kruskal-Wallis test. Aspect ratio of mitochondria following exposure to oligomycin (H), p < 0.05, Kruskal-Wallis test, Control 1 compared to Control 2 only and following exposure to rotenone (I) no significant difference, Kruskal-Wallis test. The length of the major axis of individual mitochondrion objects, normalised to Control 1, under basal conditions (J), p < 0.05, Kruskal-Wallis test for Patient 1 and Patient 2 compared to Control 1 only. Major axis length following oligomycin exposure (K), p < 0.0001, Kruskal-Wallis compared to Control 1 or 2 or following rotenone exposure (L), p < 0.05 Kruskal-Wallis test for Patient 2 only compared to Control 1 or 2. Dots represent mean data for each image, line and bars represent median ± interquartile range, n = 3 independent experiments.
Figure 8
Figure 8
Effect of oligomycin and rotenone on mitochondrial morphology. Mean mitochondrial area measured in cells exposed to oligomycin (A), no significant difference compared to basal conditions for any line, Kruskal-Wallis test, or rotenone (B), p < 0.01–p < 0.0001 compared to basal conditions for Control 1 and 2 only, Kruskal-Wallis test. Total mitochondrial area, divided by cell area, following exposure to oligomycin (C), p < 0.0001 compared to basal conditions for Patient 1 and 2 only, Kruskal-Wallis test, or rotenone (D) p < 0.001–p < 0.0001 compared to basal conditions, Kruskal-Wallis test. Mitochondrial aspect ratio measured in cells exposed to oligomcyin (E) or rotenone (F) p < 0.0001 compared to basal conditions, Kruskal-Wallis test. Major aspect length of mitochondria exposed to oligomcyin (G), p < 0.05 for Patient 2 only compared to basal conditions, Kruskal-Wallis test, not significantly different to cells exposed to rotenone (H). Dots represent mean data for each image, all data normalised to basal for each cell line, basal mean represented by dotted line, error line and bars represent median ± interquartile range, n = 3 independent experiments.
Figure 9
Figure 9
Assessment of cell viability in control and patient lines within mitochondrial function assay. Cell viability was analysed by 2 measures: (1) the number of live cells analysed, (2) the percentage of dead cells identified. Change in number of live cells (A) or percentage of dead cells (B) assessed following exposure to CCCP, oligomycin or rotenone, normalized to basal for each line for live cell change. No significant difference from basal for each condition, n = 3 individual experiments. Effect of H2O2 on the number of live cells (C) and percentage of dead cells (D), one-way ANOVA p < 0.005–0.001 for dead cells exposed to H2O2 compared to basal for each line, n = 2 independent experiments. Dots represent data for individual experiments, lines represent mean ± SEM.

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