H₂O₂-induced mitochondrial size decrease is prevented by CIG pre-treatment in Hippocampal neurons.

<p>A, B, C and D, are representative microphotographs of mitochondrial population under different experimental conditions. E, F, G: image enlargement to show the variations in the mitochondrial morphology observed under different experimental conditions. H, I, J: Normalized mitochondrial size quantification (black: control; gray: H₂O₂; green: CIG+H₂O₂; blue: CIG+H₂O₂+ GW). H) * and *’ indicate statistically significant differences between H₂O₂ and Control (*: <i>F 6.542, d.f. 3, C.I. 0.07389 to 0.6266, t 3.856, p<0.001; *’: C.I. 0.08504 to 0.6519, t 3.955, p<0.001</i>); I) * and *’ indicate statistically significant differences between H₂O₂ and Control (<i>*: F 17.8, d.f. 3, C.I. −0.3665 to −0.1151, t 5.829, p<0.001; *’: C.I. −0.3805 to −0.1227, t 5.937, p<0.001</i>). J) * and *’ indicate statistically significant differences between Control and all other experimental conditions (<i>*: F 18.99, d.f. 3. H₂O₂ C.I. −0.1696 to −0.04917, t 5.527, p<0.001; H₂O₂+ CIG C.I. −0.1708 to −0.04728, t 5.371, p<0.001; *’: H₂O₂ C.I. −0.1787 to −0.05512, t 5.757, p<0.001; H₂O₂+ CIG C.I. −0.1595 to −0.03905, t 5.015, p<0.001</i>).</p

WY pre-treatment induces mitochondrial morphology changes in hippocampal neurons after H₂O₂ challenge.

<p>A) representative microphotographs of hippocampal neurons showing color merge under specific experimental conditions. B) mitochondrial morphology registered after H₂O₂ challenge and with WY pre-treatment.</p

PPAR-mediated mitochondrial dynamics modulation.

<p>We have previously proposed a direct link between antioxidant PPARs activity and Wnt signaling pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064019#pone.0064019-Itoh1" target="_blank">[19]</a>. Based on our present results we hypothesize that PPARs are able to modulate mitochondrial dynamics trough different cellular mechanisms. Complementary to the Wnt signaling pathway (<i>FZD</i>, frizzled; <i>LRP 5/6</i>, low density lipoprotein receptor-related protein; <i>DVL</i>, disheveled; <i>βcat</i>, β-catenin), PPARs might act inducing directly the expression of several mitochondrial-related proteins (<i>UCP</i>, uncoupled protein; <i>DRP1</i>, dynamin related protein 1; <i>Mfn</i>, mitofusin; <i>mtTFA</i>, mitochondrial transcription factor). Additionally, the PPARs-mediated calcium balance might also offer another control point for mitochondrial dynamics modulation (<i>VDCC</i>, voltage dependent calcium channels; <i>NMDAR</i>, NMDA receptor; <i>VGCC</i>, voltage-gated calcium channels; <i>PKC</i>, protein kinase C).</p

H2O2-induced PGC-1α signal intensity loss is prevented by WY pre-treatment in hippocampal neurons.

<p>A–E representative microphotographs of PGC-1α immunodetection in hippocampal neurons. F (cytoplasmic) and G (nuclear), normalized PGC-1α signal intensity quantification by means of the corrected total cell fluorescence (CTCF) (black: control; white: H₂O₂; green: 50 µM WY+H₂O₂; blue: 100 µM WY+H₂O₂; red: 150 µM WY+H₂O₂). F) * and *’ indicates statistically significant differences between Control and 50 µM and 100 µM WY (<i>F 5.131, d.f. 4. 50 µM WY C.I. −0.08866 to −0.007103, t 2.545, p<0.05, and C.I. −0.07239 to −0.000643, t 3.041, p<0.05; 100 µM WY C.I. −0.6401 to −0.001898, t 2.816, p<0.05, and C.I. −0.05095 to 0.02356, t 2.580, p<0.05</i>). *” indicates statistically significant differences between H₂O₂ and Control (<i>F 5.131, d.f. 4, C.I. −0.07803 to −0.02051, t 5.118, p<0.001)</i>. G) At 30 and 60 min (* and *’) only 150 µM WY did not show statistically significant differences respect Control situation. Interestingly, at 120 min (*”) also 150 µM WY exhibits significant differences regarding Control, showing an increment of the PGC-1α signal intensity (<i>F 5.131, d.f. 4, C.I. 0.002515 to 0.09452, t 3.448, p<0.01</i>).</p

H₂O₂-induced mitochondrial size decrease is prevented by WY pre-treatment in hippocampal neurons.

<p>A, D, G, J microphotographs of mitochondrial population under different experimental conditions. B, E, H, K: image enlargement to show variations in mitochondrial morphology observed under different experimental conditions. C, F, I: Normalized mitochondrial size quantification (black: control; gray: H₂O₂; green: 50 µM WY+H₂O₂; blue: 100 µM WY+H₂O₂; red: 150 µM WY+H₂O₂). C) * indicates statistically significant differences between H₂O₂ and Control (<i>F 9.275, d.f. 4, C.I. 0.06947 to 0.631, t 4.078, p<0.001</i>); *’ indicates statistically significant differences between H₂O₂ and Control (<i>F 9.275, d.f. 4, C.I. 0.08051 to 0.6564, t 4.183, p<0.001.</i>), interestingly at 60 min only 100 µM WY shows significant differences respect Control (<i>F 9.275, d.f. 4, C.I. −0.0009977 to 0.6653, t 3.259, p<0.01)</i>; *” indicates significant differences between H₂O₂ and all other treatments (<i>F 9.275, d.f. 4. Control C.I. 0.1069 to 0.6828, t 4.482, p<0.001; 50 µM WY C.I. −0.617 to 0.01, t 3.165, p<0.01; 100 µM WY C.I. −0.7046 to −0.06217, t 3.902, p<0.001; 150µM WY C.I. −0.6306 to 0–0303, t 2.969, p<0.05</i>). F) *, *’ and *” indicate significant differences between H₂O₂ and Control (<i>F 21.3, d.f. 4. Control: * C.I. −0.3851 to −0.09656, t 5.457, p<0.001; *’ C.I. −0.3995 to −0.1036, t 5.558, p<0.001; *” C.I. −0.4178 to −0.1218, t 5.961, p<0.001</i>). No significant differences were observed between Control and WY concentrations, except for 100 µM WY at 60 min (<i>F 21.3, d.f. 4, C.I. −0.386 to −0.04365, t 4.102, p<0.001</i>). I) At each single time point significant differences were registered between Control and all other experimental conditions; however, at 120 min was possible to observe a different tendency for WY pre-treated curves and that of H₂O₂.</p