A LON-ClpP Proteolytic Axis Degrades Complex I to Extinguish ROS Production in Depolarized Mitochondria

Mitochondrial dysfunction is implicated in numerous neurodegenerative disorders and in Parkinson’s disease (PD) in particular. PINK1 and Parkin gene mutations are causes of autosomal recessive PD, and these respective proteins function cooperatively to degrade depolarized mitochondria (mitophagy). It is widely assumed that impaired mitophagy causes PD, as toxic reactive oxygen species (ROS)-producing mitochondria accumulate and progressively drive neurodegeneration. Instead, we report that a LON-ClpP proteolytic quality control axis extinguishes ROS in depolarized mitochondria by degrading the complex I ROS-generating domain. Complex I deficiency has also been identified in PD brain, and our study provides a compelling non-genetic mechanistic rationale to explain this observation: intact complex I depletes if mitochondrial bioenergetic capacity is robustly attenuated

PINK1 disables the anti-fission machinery to segregate damaged mitochondria for mitophagy

Mitochondrial fission is essential for the degradation of damaged mitochondria. It is currently unknown how the dynamin-related protein 1 (DRP1)-associated fission machinery is selectively targeted to segregate damaged mitochondria. We show that PTEN-induced putative kinase (PINK1) serves as a pro-fission signal, independently of Parkin. Normally, the scaffold protein AKAP1 recruits protein kinase A (PKA) to the outer mitochondrial membrane to phospho-inhibit DRP1. We reveal that after damage, PINK1 triggers PKA displacement from A-kinase anchoring protein 1. By ejecting PKA, PINK1 ensures the requisite fission of damaged mitochondria for organelle degradation. We propose that PINK1 functions as a master mitophagy regulator by activating Parkin and DRP1 in response to damage. We confirm that PINK1 mutations causing Parkinson disease interfere with the orchestration of selective fission and mitophagy by PINK1

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies

Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury

Targeting mitochondria with small molecules:The preparation of MitoB and MitoP as exomarkers of mitochondrial hydrogen peroxide

Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP-conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow temporal control, and be applied in a reversible dose-dependent fashion. An example is the detection and quantification of hydrogen peroxide in mitochondria of whole living organisms by MitoB. Hydrogen peroxide produced within the mitochondrial matrix is involved in signalling and implicated in the oxidative damage associated with aging and a wide range of age-associated conditions including cardiovascular disease, neurodegeneration, and cancer. MitoB accumulates in mitochondria and is converted into the exomarker, MitoP, by hydrogen peroxide in the mitochondrial matrix. The hydrogen peroxide concentration is determined from the ratio of MitoP to MitoB after a period of incubation, and this ratio is determined by mass spectrometry using d15-MitoP and d15-MitoB as standard. Here we describe the synthesis of MitoB and MitoP and the deuterated standards necessary for this method of quantification