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. 2022 Dec 2;15:966209. doi: 10.3389/fnmol.2022.966209

Figure 4.

Figure 4

Impairment of mitochondrial calcium links to mitochondrial respiration and dopamine handling. (A) Complex V protein levels in hDaN lysates. Representative blot showing Complexes V, IV and III and quantification of intensity of Complex V bands relative to a-tubulin. nDiff = 8, data displayed as mean ± SD; paired t test (two-tailed). (B) Respiratory analysis of hDaNs treated 2 h with 20 μM Mitoxantrone. Injection of medium, Oligomycin, CCCP and Rotenone with Antimycin A as indicated. Oxygen consumption rate was normalized to number of cells seeded and to mean of t = 0 in isogenic control. nDiff = 3, data displayed as mean ± SD. (C) Basal respiration and spare respiratory capacity in response to injection of either medium or 5 μM ionomycin in hDaNs treated 2 h with 20 μM Mitoxantrone calculated from respiratory analysis. nDiff = 3, data displayed as mean ± SD; basal respiration: Two-way ANOVA with Šídák’s multiple comparisons; spare respiratory capacity: Wilcoxon matched-pairs signed rank test with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. (D) Neurotransmitter transporter uptake assay. hDaNs were treated either for 24 h with 50 μM L-DOPA, for 30 min with 5 μM Ionomycin or for 2 h with 20 μM Mitoxantrone. Slopes was calculated from uptake normalized to t = 0. nDiff = 3, data displayed as mean ± SD; Wilcoxon matched-pairs signed rank test with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. (E) Representative image of untreated hDaNs stained with Dopamine, DAT and MAP2. nDiff = 3. Scale bar: 50 μM. (F) Quantification of corrected total Dopamine and DAT fluorescence normalized to MAP2. hDaNs were treated either for 30 min with 5 μM Ionomycin or for 24 h with 5 μM L-DOPA. nDiff = 3 (nimages = 30), data displayed as mean ± SD; Kruskal-Wallis with Dunn’s multiple comparisons. (G) MAO-B protein levels in hDaN lysates. Representative blot and quantification of intensity of MAO-B bands relative to a-vinculin. nDiff = 5, data displayed as mean ± SD; paired t test (two-tailed).