RSV does not prevent stress-induced inhibition of the respiratory chain.

<p>Brain mitochondria of young (A) and old (B) Ctl (open bars) and RSV treated (hatched bars) mice were incubated 30 minutes with 10 nM H₂O₂ (light grey) or 10 mM succinate (dark grey) without rotenone. Respiration rates were then measured using different substrates of the respiratory chain complex I to IV and saturating ADP concentration (0.5 mM). Data are represented as mean ± <i>sem</i> of five animals for each group. The (#) indicated a significant effect of H₂O₂ or succinate treatment, the (*) indicated a significant effect of the RSV diet (p<0.05).</p

RSV increases the expression of anti-oxidant enzymes in brain mitochondria.

<p>Expression of MnSOD was analyzed by Western blotting (normalised to the quantity of VDAC protein) on mitochondrial fractions from young (Y) and old (O) control (Ctl) and RSV-treated mice. (A) Representative blot of two duplicate experiments on n = 4 animals. (B) Quantification of MnSOD expression. Data are represented as means ± <i>sem</i> of four animals in duplicate. The (+) indicates significant differences (p<0.05) between young and old animals and the (*) shows a significant effect of the RSV-diet (p<0.05).</p

RSV binds to complex I at the nucleotide binding site.

<p><b>(A) NADH binding site of complex I,</b> according to Sazanov et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144290#pone.0144290.ref037" target="_blank">37</a>]. The bound NADH molecule is colored in pink. The FMN and amino acids that interact with the bound nucleotide are colored in yellow. The hydrophobic stacking interactions between the three phenylalanines and the adenosine moiety are represented by dashed gray lines. <b>(B) Boxshade alignement of the amino acid sequence of NDUFV1 (human nomenclature) from Homo sapiens, Bos taurus, Mus musculus and Thermus thermophilus</b> showing the conservation of the NADH and FMN binding sites. The conserved amino acids are highlighted in dark, the NADH binding site is boxed in purple, the FMN binding site in red, and the amino acids interacting with RSV are boxed in yellow. <b>(C) Occlusion of the flavin site through the binding of NAD(H) or RSV</b>. RSV bound to active FMN site in complex I from <i>Thermus thermophilus</i> (Protein Data Bank ID: 3IAM). The FMN and bound nucleotide are shown as sticks. RSV is colored in green. Amino acids that interact with the bound nucleotide are colored in yellow, with hydrogen bonding interactions shown by dashed red lines and hydrophobic stacking interactions by dashed dark lines.</p

RSV increases the NFR and NUR activities but decreases the NCCR activity.

<p>Control young mice brain mitochondria were incubated with either DMSO (1/2000, vehicle) or 5 μM RSV (30 minutes) and NADH Ubiquinone Reductase (NUR), NADH:FeCN reductase (NFR), and NADH cytochrome C reductase (NCCR) activities were measured on incubated mitochondria. Data are represented as mean <i>± sem</i> of n = 3 animals in duplicate. The (*) indicates significant differences (p<0.05) between vehicle and RSV treated mitochondria.</p

RSV specifically targets Complex I activity in brain mitochondria.

<p><b>(A) Maximal activity of complexes in mitochondria incubated with 5</b> μ<b>M RSV. (B) Maximal activity of complexes in mitochondria incubated with 50</b> μ<b>M RSV.</b> Mouse brain mitochondria disrupted by frozen/thawed cycles were incubated with either vehicle (DMSO 1/2000) or RSV (50 μM) just prior analyzing complex I to IV activities. Data are expressed as the percentage of complex activities in vehicle treated mitochondria and are represented as mean <i>± sem</i> of n = 5 animals in duplicate. The (*) indicates significant differences (p<0.05) between vehicle and RSV treated mitochondria.</p

RSV increases oxidative stress markers.

<p><b>(A) Protein thiols content</b> measured on brain homogenates of n = 4 animals for each group <b>(B) HEL-modified protein content on brain homogenates of young (Y) and old (O) control (Ctl) and RSV-treated mice</b>. Left, representative blot of two duplicate experiments on n = 2 animals; right, quantitation of HEL content by Western blotting on n = 6 animals. Data are represented as means ± <i>sem</i>. The (*) showed a significant effect of the RSV-diet (p<0.05).</p

Functional consequences of RSV treatment on mitochondrial energy metabolism in brain mitochondria.

<p><b>(A) Maximal enzymatic activities of respiratory chain complexes</b> were measured on isolated brain mitochondria from young (Y, white bars) and old (O, grey bars) control (Ctl, open bars) and RSV-treated (hatched bars) mice. Results are normalised to citrate synthase activity and represented as mean <i>± sem</i> of n = 5 animals in duplicate. The (*) indicates significant differences (p<0.05) between Ctl and RSV fed mice. <b>(B) Respiration rates measured on brain mitochondria</b> of young (Y) and old (O) Ctl (open bars) and RSV (hatched bars) treated-mice using different substrates of the respiratory chain and saturating ADP concentration (0.5 mM. Data are represented as mean <i>± sem</i> of n = 6 (young) or n = 5 (old) animals for each group. The (+) indicated a significant effect of aging (p<0.05).</p