Fig 1: MAO-A is decreased in Arhgef10 −/− mice. The main degradation enzymes 5-HT and NE were analyzed by Western blotting. a MAO-A expression was decreased in the frontal cortex and amygdala of Arhgef10 −/− mice. Another metabolizing enzyme, MOA-B, did not show a significant difference between WT and KO mice b. The main enzymes for the synthesis of the amine neurotransmitters were also analyzed by Western blotting. The protein levels of both DBH c and TPH d were similar between WT and KO mice in the examined brain areas. *p < 0.05 compared with WT. MAO-A monoamine oxidase A, MAO-B monoamine oxidase B, DBH dopamine β-hydroxylase, TPH tryptophan hydroxylase
Fig 2: Effect of MMPP on MPTP-induced protein expressions. The effects of MMPP on dopaminergic neuronal cell marker and inflammatory marker proteins were measured by western blotting. Expressions of TH+, MAO-B, and iNOS in the substantia nigra were measured (A and B). Expressions of p-p38 and p-STAT3 in the substantia nigra were measured (C and D). Values are presented as mean ± SD from 10 mice. * p < 0.05, significant difference from saline-injected mice; # p < 0.05, significant difference between MPTP-injected groups.
Fig 3: Effect of MMPP against dopaminergic neurodegeneration in vitro. The protective effects of MMPP against neurodegeneration, and expressions of TH, MAO-B, and inflammatory marker proteins were measured by western blotting analysis (A, D, E, and F). STAT3 luciferase activity was determined as described in the Methods (B). To evaluate the protective effects of MMPP, cell viability was measured using the LDH assay kit (C). Activation of STAT3, p38, and JNK were detected by western blotting (D). To evaluate the involvement of JNK (E) and p-38 (F) pathways in the inhibitory effect of MMPP on 1-methyl-4-phenylpyridinium (MPP+)-induced neuroinflammation, cultured primary cells were incubated with specific JNK and p-38 inhibitors (10 μM) for 24 h and co-treated with MMPP (5 μg/mL) at 37 °C. These effects were investigated by western blot analysis. Expression levels of MAO-B, STAT3, and inflammatory markers (GFAP and iNOS) were measured (E and F). *p < 0.05, significant difference from control astrocytes; #p < 0.05, significant difference from the MPP+-treated astrocytes.
Fig 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).
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