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

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

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

Scheme summarizing the main findings reported in this study.

<p>The glial metabolite of MPTP, MPP+, enters into dopaminergic neurons in the SN and accumulates in the mitochondria. where it specifically binds to and inhibits complex I of the ETC. This leads to mitochondrial respiration failure, reducing ETC-OXPHOS coupling as well as the efficiency of the oxidative phosphorylation. MPP+ also promotes an increase in the production of NO^● by activation of the inducible isoform iNOS, whereas nNOS activity remains unchanged. Depletion of either iNOS or nNOS did not prevent the decrease in oxygen consumption, suggesting that MPTP-induced respiratory defects and consequent ROS production run independently of iNOS activation. The interplay of these two MPTP-dependent consequences, respiration failure and NO^● production, could generate a positive feedback loop where inflammation and oxidative damage are prevalent and cause neurodegeneration. This misbalance generates an excess of ROS by electron leak that, in combination with RNS, is known to induce the expression of inflammatory cytokines and membrane oxidative damage. Melatonin that also accumulates in the mitochondria, harnesses the activity of the respiratory complexes including the inhibition of complex I induced by MPTP as a first action. Melatonin administration restored the coupling between the ETC and OXPHOS, rescued mitochondrial respiration and reduced ROS production, by reducing electron leak and directly scavenging free radicals. On the other hand, the indoleamine also inhibits iNOS activation, reducing NO^● production and consequently reducing inflammation that closes the cycle.</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

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 administration of melatonin increased its levels within the mitochondria of targeted tissues, SN and ST.

<p>Graph shows changes in the melatonin levels of mitochondria from 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; ^Φ<i>P</i> < 0.05, ^ΦΦΦ<i>P</i> < 0.001 and ^ΦΦΦΦ<i>P</i> < 0.0001 <i>vs</i>. equivalent group in SN.</p

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

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

OXPHOS analysis.

<p>Western blot (WB) analysis and densitometric quantification of OXPHOS: ND6 (complex I) (A), complex II (B), core II (complex III) (C), COX I (complex IV) (D), Cva (complex V) and Western blot (WB) analysis of OXPHOS (F) in the mitochondria of small intestine cells from non-irradiated rats (control) and irradiated rats treated with vehicle (IR) or melatonin gel (IR + aMT); n = 6 per group. Data are expressed as mean ± s.e.m. *<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control; and ##<i>P</i> < 0.01, ###<i>P</i> < 0.001 vs. IR.</p