?-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease.
ABSTRACT: Protein aggregation causes ?-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric ?-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric ?-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate ?-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of ?-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.
Project description:Misfolded ?-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded ?-synuclein is limited. Using brains of mice lacking ?-, ?-, and ?-synuclein, we report that extracellular monomeric ?-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit ?, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of ?-, ?-, and ?-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded ?-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that ?-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT:Misfolded ?-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded ?-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric ?-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of ?-, ?-, and ?-synuclein knock-out mice and monomeric ?-synuclein, this current study shows that ?-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric ?-synuclein to enhance ATP synthase efficiency under physiological conditions may be of importance when ?-synuclein undergoes the misfolding and aggregation reported in PD.
Project description:Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.
Project description:Mitochondrial electron transport is essential for oxidative phosphorylation (OXPHOS). Electron transport chain (ETC) activity generates an electrochemical gradient that is used by the ATP synthase to make ATP. ATP synthase is organized into supramolecular units called synthasomes that increase the efficiency of ATP production, while within ATP synthase is the cyclophilin D (CypD) regulated mitochondrial permeability transition pore (PTP). We investigated whether synthasomes are dynamic structures that respond to metabolic demands and whether CypD regulates this dynamic. Isolated heart mitochondria from wild-type (WT) and CypD knockout (KO) mice were treated to either stimulate OXPHOS or open the PTP. The presence and dynamics of mitochondrial synthasomes were investigated by native electrophoresis, immunoprecipitation, and sucrose density centrifugation. We show that stimulation of OXPHOS, inhibition of the PTP, or deletion of CypD increased high order synthasome assembly. In contrast, OXPHOS inhibition or PTP opening increased synthasome disassembly in WT, but not in CypD KO heart mitochondria. CypD activity also correlated with synthasome assembly in other tissues, such as liver and brain. We conclude that CypD not only regulates the PTP, but also regulates the dynamics of synthasome assembly depending on the bioenergetic state of the mitochondria.
Project description:The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.
Project description:The permeability transition pore (PTP) is a Ca2+-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase.
Project description:Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.
Project description:The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ?0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.
Project description:Aims:Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice are not protected from ischaemic injury. We aimed to investigate whether adaptive alterations occur in cell death signalling pathways in the hearts of global MCU-KO mice. Methods and results:First, we examined whether cell death may occur via an upregulation in necroptosis in MCU-KO mice. However, our results show that neither RIP1 inhibition nor RIP3 knockout afford protection against ischaemia-reperfusion injury in MCU-KO as in wildtype (WT) hearts, indicating that the lack of protection cannot be explained by upregulation of necroptosis. Instead, we have identified alterations in cyclophilin D (CypD) signalling in MCU-KO hearts. In the presence of a calcium ionophore, MCU-KO mitochondria take up calcium and do undergo PTP opening. Furthermore, PTP opening in MCU-KO mitochondria has a lower calcium retention capacity (CRC), suggesting that the calcium sensitivity of PTP is higher. Phosphoproteomics identified an increase in phosphorylation of CypD-S42 in MCU-KO. We investigated the interaction of CypD with the putative PTP component ATP synthase and identified an approximately 50% increase in this interaction in MCU-KO cardiac mitochondria. Mutation of the novel CypD phosphorylation site S42 to a phosphomimic reduced CRC, increased CypD-ATP synthase interaction by approximately 50%, and increased cell death in comparison to a phospho-resistant mutant. Conclusion:Taken together these data suggest that MCU-KO mitochondria exhibit an increase in phosphorylation of CypD-S42 which decreases PTP calcium sensitivity thus allowing activation of PTP in the absence of an MCU-mediated increase in matrix calcium.
Project description:Reactive oxygen species (ROS) and reactive nitrogen species (RNS) targeting mitochondria are major causative factors in disease pathogenesis. The mitochondrial permeability transition pore (PTP) is a mega-channel modulated by calcium and ROS/RNS modifications and it has been described to play a crucial role in many pathophysiological events since prolonged channel opening causes cell death. The recent identification that dimers of ATP synthase form the PTP and the fact that posttranslational modifications caused by ROS/RNS also affect cellular bioenergetics through the modulation of ATP synthase catalysis reveal a dual function of these modifications in the cells. Here, we describe mitochondria as a major site of production and as a target of ROS/RNS and discuss the pathophysiological conditions in which oxidative and nitrosative modifications modulate the catalytic and pore-forming activities of ATP synthase.