Mitochondrial impairment and melatonin protection in parkinsonian mice do not depend of inducible or neuronal nitric oxide synthases

<div><p>MPTP-mouse model constitutes a well-known model of neuroinflammation and mitochondrial failure occurring in Parkinson’s disease (PD). Although it has been extensively reported that nitric oxide (NO^●) plays a key role in the pathogenesis of PD, the relative roles of nitric oxide synthase isoforms iNOS and nNOS in the nigrostriatal pathway remains, however, unclear. Here, the participation of iNOS/nNOS isoforms in the mitochondrial dysfunction was analyzed in iNOS and nNOS deficient mice. Our results showed that MPTP increased iNOS activity in substantia nigra and striatum, whereas it sharply reduced complex I activity and mitochondrial bioenergetics in all strains. In the presence of MPTP, mice lacking iNOS showed similar restricted mitochondrial function than wild type or mice lacking nNOS. These results suggest that iNOS-dependent elevated nitric oxide, a major pathological hallmark of neuroinflammation in PD, does not contribute to mitochondrial impairment. Therefore, neuroinflammation and mitochondrial dysregulation seem to act in parallel in the MPTP model of PD. Melatonin administration, with well-reported neuroprotective properties, counteracted these effects, preventing from the drastic changes in mitochondrial oxygen consumption, increased NOS activity and prevented reduced locomotor activity induced by MPTP. The protective effects of melatonin on mitochondria are also independent of its anti-inflammatory properties, but both effects are required for an effective anti-parkinsonian activity of the indoleamine as reported in this study.</p></div

Lack of DMQ₉ and increase of CoQ₉ in <i>Coq9</i>^<i>R239X</i> mHPCs after transduction with CCoq9WP vector.

<p>Levels of CoQ₉ (<i>A</i>) and DMQ₉/CoQ₉ ratio (<i>B</i>). Representative chromatographs of the three different groups (<i>C-E</i>). U: 1 hit in HSCs, 400 μl, 23.5x-concentrated; V: 2 hits in HPCs 400 μl, 23.5x-concentrated; 1: 7–9 days after transduction in HPCs; 2: 12–16 days after transduction in HSCs. Data are expressed as mean ± SD. *<i>P</i> < 0.05, <i>Coq9</i>^<i>R239X</i> and <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>+/+</i> cells; **<i>P</i> < 0.01, <i>Coq9</i>^<i>R239X</i> and <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>+/+</i> cells; ***<i>P</i> < 0.001, <i>Coq9</i>^<i>R239X</i> and <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>+/+</i> cells; +++<i>P</i> < 0.001, <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>R239X</i> cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group).</p

Overexpression of COQ9 in <i>Coq9</i>^<i>R239X</i> MEFs and mHPCs increases the levels of COQ7.

<p>Levels of COQ7 in MEFs (<i>A</i>) and mHSCs (<i>B</i>). R: 1 hit in MEFs, 100 μl, 10x-concentrated; U: 1 hit in HPCs, 400 μl, 23.5x-concentrated; V: 2 hits in HPCs 400 μl, 23.5x-concentrated; 1: 7–9 days after transduction in HSCs; 2: 12–16 days after transduction in HSCs. Data are expressed as mean ± SD. **<i>P</i> < 0.01, <i>Coq9</i>^<i>R239X</i> and <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>+/+</i> cells; ***<i>P</i> < 0.001, <i>Coq9</i>^<i>R239X</i> and <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>+/+</i> cells; +<i>P</i> < 0.05, <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>R239X</i> cells; +++<i>P</i> < 0.001, <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>R239X</i> cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group).</p

Transduction with CCoq9WP vector increases the levels of Coq9 mRNA and COQ9 protein in MEFs and mHPCs from <i>Coq9</i>^<i>R239X</i> mice.

<p>Coq9 mRNA levels in MEFs (<i>A</i>) and mHPCs (<i>B</i>). COQ9 protein levels in MEFs (<i>C</i>) and mHPCs (<i>D</i>). Q: 1 hit in MEFs, 200 μl, non-concentrated; R: 1 hit in MEFs, 100 μl, 10x-concentrated; S: 1 hit in MEFs, 25 μl, 10x-concentrated; T: 1 hit in MEFs, 5 μl, 10x-concentrated; U: 1 hit in HSCs, 400 μl, 23.5x-concentrated; V: 2 hits in HSCs 400 μl, 23.5x-concentrated; 1: 7–9 days after transduction in HSCs; 2: 12–16 days after transduction in HSCs. Data are expressed as mean ± SD. ***<i>P</i> < 0.001, <i>Coq9</i>^<i>R239X</i> and <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>+/+</i> cells; +++<i>P</i> < 0.001, <i>Coq9</i>^{<i>R239X-CCoq9WP</i>} cells versus <i>Coq9</i>^<i>R239X</i> cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group). #: a non-specific band is detected in <i>Coq9</i>^<i>R239X</i> cells with the anti-COQ9 antibody.</p

Mitochondrial oxygen consumption decreased sharply after MPTP administration in SN and ST of all strains, an effect prevented by melatonin treatment.

<p>More significant changes were found in respiration through complexes I+II. The graphs show the changes in oxygen consumption at state 3 and 4 in SN (A) and ST (B) of iNOS^+/+, iNOS ^-/-, nNOS^+/+ and nNOS ^-/- mice respectively, through complexes I and II (dark bars) and through complex II (white bars). Results are shown as the mean ± SD of 4 animals per group, triplicated. <i>*P < 0</i>.<i>05</i>, <i>**P < 0</i>.<i>01</i>, <i>***P<0</i>.<i>001 and ****P < 0</i>.<i>0001 vs</i>. <i>saline-injected control;</i> ^<i>##</i><i>P < 0</i>.<i>01</i>, <i>and</i> ^<i>####</i><i>P < 0</i>.<i>0001 MPTP vs</i>. <i>MPTP+aMT;</i> ^Φ<i>P</i> < 0.05, ^ΦΦ<i>P</i> < 0.01, ^ΦΦΦ<i>P</i> < 0.001 and ^ΦΦΦΦ<i>P</i> < 0.0001 CII <i>vs</i>. CI+II.</p

The treatment with MPTP induced a drastic decrease in the activity of the complex I in SN and ST independently of the absence/presence of iNOS/nNOS.

<p>The administration of melatonin counteracted this effect. The graphs show the changes in the activity of complex I in SN and ST of iNOS^+/+ (above left) and iNOS^-/- (above right) and SN and ST of nNOS^+/+ (down left) and nNOS^-/- (down right). Mean ± SD of 6 animals per group, triplicated; *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ****<i>P</i> < 0.0001 <i>vs</i>. control; <i>P</i> < 0.05 ^## and ^####<i>P</i> < 0.0001 <i>vs</i>. MPTP; ^ΦΦ<i>P</i> < 0.05 and ^ΦΦΦΦ<i>P</i> < 0.0001 <i>vs</i>. basal complex I in SN.</p

Values of the ADP:O ratio in ST and SN mitochondria.

<p>Values of the ADP:O ratio in ST and SN mitochondria.</p

MPTP induced dramatic changes in mice locomotor activity, which were prevented by melatonin.

<p>Graphs show the changes in trajectories (A) and travelled distance (B) of iNOS^+/+, iNOS ^-/-, nNOS^+/+ and nNOS ^-/- mice respectively, during 4 hr at night, in control, MPTP and aMT treated groups. Mean ± SD of 8 animals per group. ***<i>P</i> <0.001 and ****<i>P</i> <0.0001 <i>vs</i>. saline-injected control; ^####<i>P</i> < 0.0001 <i>vs</i>. MPTP treatment.</p

The MPTP treatment caused an increase in the activity of iNOS but not nNOS in SN and ST whereas melatonin treatment restored the activity to control levels.

<p>The increase in NOS activity after MPTP administration is abolished in mice lacking iNOS. The graphs show the changes in total NOS and iNOS activities in ST and SN of control mice (left) vs. deficient mice (right) (Mean ± SD of 6 animals per group, triplicated). ****<i>P</i> < 0.0001 <i>vs</i>. control; ^####<i>P</i> < 0.0001 <i>vs</i>. MPTP.</p

Control respiratory index (RCR) in ST and SN mitochondria.

<p>Control respiratory index (RCR) in ST and SN mitochondria.</p