Figure 3.
LMX1A and LMX1B control basal autophagy and mitochondrial function to protect mDANs. (A) Representative immunofluorescence images (left) and quantitation (right) of basal WIPI2 puncta in TH+ve/GFP+ve iPSC-derived mDAN cell bodies (day 30–45) following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction. Mean ± SD (>30 neurons/condition; three independent experiments); one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test: ****P < 0.0001 vs. shRNA control. Bars = 20 µm, full field; 10 µm, inset zoom. (B) MitoSOX intensity in TH-positive iPSC-derived mDANs transduced with hsyn-GFP-U6-shControl/shLMX1A/shLMX1B (day 30–50). Cells were treated with carbonyl cyanide chlorophenylhydrazone (10 µM, 4 h) as a positive control. Mean ± SD of individual cell bodies from four independent experiments; one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test: ****P < 0.0001. (C and D) Seahorse mitochondrial respiration profiles in iPSC-derived mDAN cultures (day 30–50) transduced with (C) hsyn-GFP-U6-shRNA LMX1A or (D) hsyn-GFP-U6-shRNA LMX1B lentiviruses. Pairwise comparisons were performed against non-targeting shRNA control. Oxygen consumption rate (OCR) was normalized to protein levels after sequential exposure to oligomycin (Oligo; 1 µM), FCCP (1 µM), and rotenone/antimycin (R+A; 0.5 µM) as indicated (n = 3). Mean ± SD; Student’s t test: *P < 0.05, **P < 0.01. (E) Basal mDAN cell death analysis following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction (day 25–50 mDANs). Fluorometric caspase assay normalized to total protein (n = 2–3). Mean ± SD; one-way ANOVA followed by Fisher’s LSD test for planned comparisons. (F) Rotenone-induced cell death in iPSC-derived mDANs (day 25–50) transduced with TRE-empty (control), TRE-LMX1A, and/or TRE-LMX1B; expression was induced by DOX (500 ng/ml, 3 d). mDANs were treated with rotenone (15 µM) for 24 h. Caspase activity was normalized to total protein and measured relative to empty vector control (n = 2–4). Mean ± SD; one-way ANOVA followed by Fisher’s LSD test for planned comparisons: **P < 0.01, ***P < 0.001.

LMX1A and LMX1B control basal autophagy and mitochondrial function to protect mDANs. (A) Representative immunofluorescence images (left) and quantitation (right) of basal WIPI2 puncta in TH+ve/GFP+ve iPSC-derived mDAN cell bodies (day 30–45) following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction. Mean ± SD (>30 neurons/condition; three independent experiments); one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test: ****P < 0.0001 vs. shRNA control. Bars = 20 µm, full field; 10 µm, inset zoom. (B) MitoSOX intensity in TH-positive iPSC-derived mDANs transduced with hsyn-GFP-U6-shControl/shLMX1A/shLMX1B (day 30–50). Cells were treated with carbonyl cyanide chlorophenylhydrazone (10 µM, 4 h) as a positive control. Mean ± SD of individual cell bodies from four independent experiments; one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test: ****P < 0.0001. (C and D) Seahorse mitochondrial respiration profiles in iPSC-derived mDAN cultures (day 30–50) transduced with (C) hsyn-GFP-U6-shRNA LMX1A or (D) hsyn-GFP-U6-shRNA LMX1B lentiviruses. Pairwise comparisons were performed against non-targeting shRNA control. Oxygen consumption rate (OCR) was normalized to protein levels after sequential exposure to oligomycin (Oligo; 1 µM), FCCP (1 µM), and rotenone/antimycin (R+A; 0.5 µM) as indicated (n = 3). Mean ± SD; Student’s t test: *P < 0.05, **P < 0.01. (E) Basal mDAN cell death analysis following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction (day 25–50 mDANs). Fluorometric caspase assay normalized to total protein (n = 2–3). Mean ± SD; one-way ANOVA followed by Fisher’s LSD test for planned comparisons. (F) Rotenone-induced cell death in iPSC-derived mDANs (day 25–50) transduced with TRE-empty (control), TRE-LMX1A, and/or TRE-LMX1B; expression was induced by DOX (500 ng/ml, 3 d). mDANs were treated with rotenone (15 µM) for 24 h. Caspase activity was normalized to total protein and measured relative to empty vector control (n = 2–4). Mean ± SD; one-way ANOVA followed by Fisher’s LSD test for planned comparisons: **P < 0.01, ***P < 0.001.

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