Fig 1: Plasmatic concentrations of (A) monoamine oxidase (MAO), (B) diamine oxidase (DAO) and (C) total antioxidant capacity (TAC) of dogs after 30 days of feeding with poultry by-product meal (PBPM, red columns) or hydrolyzed chicken liver (HCLP, black columns) diet at three crude protein concentrations (24, 32 or 40% CP). Results were expressed as mean ± standard deviation. Results were analyzed by two-way ANOVA. p1: comparison between protein sources (PBPM or HCLP); p2: comparison between different protein concentrations; p3: interaction between types of protein source and protein concentration. Different lowercase letters indicate significant differences according to Tukey post hoc test (p < 0.05 or 0.01).
Fig 2: Cell viability, MAO-B and ROS tests. (A) GDNF and its relationship with GFRα1 and RET (scheme based on (Duarte Azevedo et al., 2020): GDNF forms a complex with the GFRα1 receptor, which in turn binds to RET, thereby activating signaling pathways associated with cell survival, differentiation, and growth. Under conditions of disease, when neurons die, the GDNF-GFRα1 receptor complex forms in astrocytes, and the RET receptor is found in microglia. (B) Cell viability with different treatments (MPTP 500 µM model). (C) Cell viability with different treatments (1.25 mM MPTP model). (D) MAO-B activity under different treatments. (E) Representative confocal images showing the ROS levels with different treatments. Scale bar 40 μm. (F)Quantification of the fluorescence associated with ROS levels (RFU: Relative Fluorescence Units). ANOVA and Tukey’s test were conducted for statistical analysis. ** indicates p-values <0.01, *** p-values <0.001, and **** p-values <0.0001. The dashed line indicates a cell viability of 70%.
Fig 3: A model for the function of the redox molecule Parkin around mitochondria: When depolarization is triggered in the mitochondrial membrane, all ions, proteins, and ROS in the mitochondria leak or are exposed to the outer membrane. Parkin from the cytoplasm reacts with leaked H2O2 to aggregate and autoubiquitinate, and non-specifically precipitate into both the inner and outer membrane. PINK1 is exposed to the outer membrane and phosphorylates Parkin and ubiquitin; even when phosphorylated, Parkin does not ubiquitinate the substrate, but rather causes autoubiquitination. Parkin is also presumed to react with H2O2 generated by MAO-A/B on the outer membrane and to eliminate H2O2 in dopaminergic neurons. In addition, H2O2 leaking into the cytoplasm directly stimulates mitophagy (Suppl-Figures S1 and S2). Parkin aggregates also positively modulate mitophagy (Suppl-Figures S1 and S2).
Fig 4: Characterization of the basic properties of the redox molecule Parkin and its substrate FAF1. (A1): MAO Assay: Parkin reacts with and reduces H2O2, a by-product of the chemical reaction between MAO-A and tyramine. Error bars represent the mean ± SD values of three experiments; statistical analysis was calculated using one-way ANOVA, followed by the Bonferroni Multiple Comparison test. *** p < 0.0001; * p < 0.05. (A2): Chemical reaction equations of MAO with tyramine and of Parkin protein with H2O2. (B): WB for FAF1 Polyubiquitination Assay using the Parkin E3 ligase; E3 activity does not allow autoubiquitination. (B1): WB analysis using anti-Parkin antibodies. (B2): WB analysis using anti-FLAG antibodies for FAF1. (B3): WB analysis using anti-Ubi antibodies. The same WB membrane was used for all of (B1–B3). (C): Overexpression of Parkin/FAF1 in HEK293 (HEK) cells with/without H2O2: WB for soluble fractions. 20 mM H2O2 or 100 mM DTT were exposed for 30 min at room temperature. DTT was added 5 min before collecting the cells.
Supplier Page from Abcam for Monoamine Oxidase (MAO) Assay Kit