SUMMARY Mitochondrial fission mediated by the GTPase dynamin-related protein-1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial Complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g. 50 μM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing Complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki>1.2 mM). Overall, results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit Complex I and modify mitochondrial ROS production may contribute to effects observed in disease models.

Succinate is an important metabolite at the cross-road of several metabolic pathways, also involved in the formation and elimination of reactive oxygen species. However, it is becoming increasingly apparent that its realm extends to epigenetics, tumorigenesis, signal transduction, endo- and paracrine modulation and inflammation. Here we review the pathways encompassing succinate as a metabolite or a signal and how these may interact in normal and pathological conditions.(1).

␣-Ketoglutarate dehydrogenase (␣-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that ␣-KGDH is able to generate H 2 O 2 and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated ␣-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H 2 O 2 after addition of ␣-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD ϩ ). NAD ϩ , which proved to be a powerful inhibitor of ␣-KGDH-mediated H 2 O 2 formation, switched the H 2 O 2 forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H 2 O 2 formation by ␣-KGDH, and for this, neither ␣-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD ϩ ratio determined the rate of H 2 O 2 production. The higher the NADH/NAD ϩ ratio the higher the rate of H 2 O 2 production. H 2 O 2 production as well as the catalytic function of the enzyme was activated by Ca 2ϩ . In synaptosomes, using ␣-ketoglutarate as respiratory substrate, the rate of H 2 O 2 production increased by 2.5-fold, and aconitase activity decreased, indicating that ␣-KGDH can generate H 2 O 2 in in situ mitochondria. Given the NADH/NAD ϩ ratio as a key regulator of H 2 O 2 production by ␣-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus ␣-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

In this study we addressed the function of the Krebs cycle to determine which enzyme(s) limits the availability of reduced nicotinamide adenine dinucleotide (NADH) for the respiratory chain under H 2 O 2 -induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H 2 O 2 proved to be aconitase, being completely blocked at 50 M H 2 O 2 . ␣-Ketoglutarate dehydrogenase (␣-KGDH) was also inhibited but only at higher H 2 O 2 concentrations (Ն100 M), and only partial inactivation was achieved. The rotenone-induced increase in reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] fluorescence reflecting the amount of NADH available for the respiratory chain was also diminished by H 2 O 2 , and the effect exerted at small concentrations (Յ50 M) of the oxidant was completely prevented by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNUinsensitive decline by H 2 O 2 in the rotenone-induced NAD(P)H fluorescence correlated with inhibition of ␣-ketoglutarate dehydrogenase. Decrease in the glutamate content of nerve terminals was induced by H 2 O 2 at concentrations inhibiting aconitase. It is concluded that (1) aconitase is the most sensitive enzyme in the Krebs cycle to inhibition by H 2 O 2 , (2) at small H 2 O 2 concentrations (Յ50 M) when aconitase is inactivated, glutamate fuels the Krebs cycle and NADH generation is unaltered, (3) at higher H 2 O 2 concentrations (Ն100 M) inhibition of ␣-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H 2 O 2 -induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of ␣-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.

Alpha-ketoglutarate dehydrogenase (a-KGDH) is a highly regulated enzyme, which could determine the metabolic flux through the Krebs cycle. It catalyses the conversion of a-ketoglutarate to succinylCoA and produces NADH directly providing electrons for the respiratory chain. a-KGDH is sensitive to reactive oxygen species (ROS) and inhibition of this enzyme could be critical in the metabolic deficiency induced by oxidative stress. Aconitase in the Krebs cycle is more vulnerable than a-KGDH to ROS but as long as a-KGDH is functional NADH generation in the Krebs cycle is maintained. NADH supply to the respiratory chain is limited only when a-KGDH is also inhibited by ROS. In addition being a key target, a-KGDH is able to generate ROS during its catalytic function, which is regulated by the NADH/NAD C ratio. The pathological relevance of these two features of a-KGDH is discussed in this review, particularly in relation to neurodegeneration, as an impaired function of this enzyme has been found to be characteristic for several neurodegenerative diseases.

Oxidative stress and partial deficiencies of mitochondrial complex I appear to be key factors in the pathogenesis of Parkinson's disease. They are interconnected; complex I inhibition results in an enhanced production of reactive oxygen species (ROS), which in turn will inhibit complex I. Partial inhibition of complex I in nerve terminals is sufficient for in situ mitochondria to generate more ROS. H2O2 plays a major role in inhibiting complex I as well as a key metabolic enzyme, alpha-ketoglutarate dehydrogenase. The vicious cycle resulting from partial inhibition of complex I and/or an inherently higher ROS production in dopaminergic neurons leads over time to excessive oxidative stress and ATP deficit that eventually will result in cell death in the nigro-striatal pathway.

In this study reactive oxygen species (ROS) generated in the respiratory chain were measured and the quantitative relationship between inhibition of the respiratory chain complexes and ROS formation was investigated in isolated nerve terminals. We addressed to what extent complex I, III and IV, respectively, should be inhibited to cause ROS generation. For inhibition of complex I, III and IV, rotenone, antimycin and cyanide were used, respectively, and ROS formation was followed by measuring the activity of aconitase enzyme. ROS formation was not detected until complex III was inhibited by up to 71 ± 4%, above that threshold inhibition, decrease in aconitase activity indicated an enhanced ROS generation. Similarly, threshold inhibition of complex IV caused an accelerated ROS production. By contrast, inactivation of complex I to a small extent (16 ± 2%) resulted in a significant increase in ROS formation, and no clear threshold inhibition could be determined. However, the magnitude of ROS generated at complex I when it is completely inhibited is smaller than that observed when complex III or complex IV was fully inactivated. Our findings may add a novel aspect to the pathology of Parkinson's disease, showing that a moderate level of complex I inhibition characteristic in Parkinson's disease leads to significant ROS formation. The amount of ROS generated by complex I inhibition is sufficient to inhibit in situ the activity of endogenous aconitase.

Mitochondrial membrane potential (⌬⌿ m ) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H 2 O 2 (0.1-1 mM) caused only a minor decrease in ⌬⌿ m . When complex I of the respiratory chain was inhibited by rotenone (2 M), ⌬⌿ m was unaltered, but on subsequent addition of H 2 O 2 , ⌬⌿ m started to decrease and collapsed during incubation with 0.5 mM H 2 O 2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H 2 O 2 . H 2 O 2 also induced a marked reduction in ⌬⌿ m when added after oligomycin (10 M), an inhibitor of F 0 F 1 -ATPase. H 2 O 2 (0.1 or 0.5 mM) inhibited ␣-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine ⌬⌿ m in the presence of H 2 O 2 : (a) The NADH level reduced owing to inhibition of ␣-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of ⌬⌿ m when the F 0 F 1 -ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H 2 O 2 prevents maintenance of ⌬⌿ m by F 0 F 1 -ATPase. The results indicate that to maintain ⌬⌿ m in the nerve terminal during H 2 O 2 -induced oxidative stress, both complex I and F 0 F 1 -ATPase must be functional. Collapse of ⌬⌿ m could be a critical event in neuronal injury in ischemia or Parkinson's disease when H 2 O 2 is generated in excess and complex I of the respiratory chain is simultaneously impaired. Key Words: Oxidative stress-Hydrogen peroxide -Mitochondrial membrane potential-Mitochondrial ATPase -␣-Ketoglutarate dehydrogenase -Parkinson's disease.