mPTP Activation Research Articles

The Role of Mitochondrial Permeability Transition in Bone Metabolism, Bone Healing, and Bone Diseases.

Bone is a dynamic organ with an active metabolism and high sensitivity to mitochondrial dysfunction. The mitochondrial permeability transition pore (mPTP) is a low-selectivity channel situated in the inner mitochondrial membrane (IMM), permitting the exchange of molecules of up to 1.5 kDa in and out of the IMM. Recent studies have highlighted the critical role of the mPTP in bone tissue, but there is currently a lack of reviews concerning this topic. This review discusses the structure and function of the mPTP and its impact on bone-related cells and bone-related pathological states. The mPTP activity is reduced during the osteogenic differentiation of mesenchymal stem cells (MSCs), while its desensitisation may underlie the mechanism of enhanced resistance to apoptosis in neoplastic osteoblastic cells. mPTP over-opening triggers mitochondrial swelling, regulated cell death, and inflammatory response. In particular, mPTP over-opening is involved in dexamethasone-induced osteoblast dysfunction and bisphosphonate-induced osteoclast apoptosis. In vivo, the mPTP plays a significant role in maintaining bone homeostasis, with many bone disorders linked to its excessive opening. Genetic deletion or pharmacological inhibition of the over-opening of mPTP has shown potential in enhancing bone injury recovery and alleviating bone diseases. Here, we review the findings on the relationship of the mPTP and bone at both the cellular and disease levels, highlighting novel avenues for pharmacological approaches targeting mitochondrial function to promote bone healing and manage bone-related disorders.

Effects of Anesthesia with Pentobarbital/Ketamine on Mitochondrial Permeability Transition Pore Opening and Ischemic Brain Damage.

The alteration of mitochondrial functions, especially the opening of the mitochondrial permeability transition pore (mPTP), has been proposed as a key mechanism in the development of lesions in cerebral ischemia, wherefore it is considered as an important target for drugs against ischemic injury. In this study, we aimed to investigate the effects of mitochondrial complex I inhibitors as possible regulators of mPTP using an in vitro brain ischemia model of the pentobarbital/ketamine (PBK)-anesthetized rats. We found that PBK anesthesia itself delayed Ca2+-induced mPTP opening and partially recovered the respiratory functions of mitochondria, isolated from rat brain cortex and cerebellum. In addition, PBK reduced cell death in rat brain slices of cerebral cortex and cerebellum. PBK inhibited the adenosine diphosphate (ADP)-stimulated respiration of isolated cortical and cerebellar mitochondria respiring with complex I-dependent substrates pyruvate and malate. Moreover, pentobarbital alone directly increased the resistance of isolated cortex mitochondria to Ca2+-induced activation of mPTP and inhibited complex I-dependent respiration and mitochondrial complex I activity. In contrast, ketamine had no direct effect on functions of isolated normal cortex and cerebellum mitochondria. Altogether, this suggests that modulation of mitochondrial complex I activity by pentobarbital during PBK anesthesia may increase the resistance of mitochondria to mPTP opening, which is considered the key event in brain cell necrosis during ischemia.

Mitochondrial permeability transition dictates mitochondrial maturation upon switch in cellular identity of hematopoietic precursors

The mitochondrial permeability transition pore (mPTP) is a supramolecular channel that regulates exchange of solutes across cristae membranes, with executive roles in mitochondrial function and cell death. The contribution of the mPTP to normal physiology remains debated, although evidence implicates the mPTP in mitochondrial inner membrane remodeling in differentiating progenitor cells. Here, we demonstrate that strict control over mPTP conductance shapes metabolic machinery as cells transit toward hematopoietic identity. Cells undergoing the endothelial-to-hematopoietic transition (EHT) tightly control chief regulatory elements of the mPTP. During EHT, maturing arterial endothelium restricts mPTP activity just prior to hematopoietic commitment. After transition in cellular identity, mPTP conductance is restored. In utero treatment with NIM811, a molecule that blocks sensitization of the mPTP to opening by Cyclophilin D (CypD), amplifies oxidative phosphorylation (OXPHOS) in hematopoietic precursors and increases hematopoiesis in the embryo. Additionally, differentiating pluripotent stem cells (PSCs) acquire greater organization of mitochondrial cristae and hematopoietic activity following knockdown of the CypD gene, Ppif. Conversely, knockdown of Opa1, a GTPase critical for proper cristae architecture, induces cristae irregularity and impairs hematopoiesis. These data elucidate a mechanism that regulates mitochondrial maturation in hematopoietic precursors and underscore a role for the mPTP in the acquisition of hematopoietic fate.

Cell-Free Hemoglobin Induces Vascular and Mitochondrial Dysfunction in the Pulmonary Endothelium in an Oxidation-State Dependent Manner

Background: Sepsis results in significant organ damage due in part to a breakdown in endothelial vascular function. Elevated levels of cell-free hemoglobin (CFH) drive endothelial dysfunction in the lungs. CFH generates reactive oxygen species (ROS) via the iron present in the heme moiety, which can be oxidized from ferrous (CFH2+) to ferric (CFH3+). Recent studies suggest mitochondrial dysfunction may be a key pathway leading to microvascular dysfunction. We hypothesize that CFH induces mitochondrial dysfunction and microvascular endothelial barrier disruption in the lung in an oxidative-state dependent manner. Methods: CFH was converted from CFH2+ to CFH3+ by UV irradiation (validated by spectrophotometry). Primary human lung microvascular endothelial cells (HLMVECs) were treated with 1.0 mg/mL CFH2+, CFH3+, or vehicle control. Barrier dysfunction was measured via electric cell-substrate impedance sensing (ECIS) and express permeability test (XPerT) at 24h. We quantified mitochondrial superoxide (MitoSOX Red; 5 uM) by flow cytometry (1, 6, 24h) and total cellular ROS production (CellROX Deep Red; 5 uM) by fluorescence microscopy (6 and 24 h). Mitochondrial morphology (electron density, roundness, area, circularity, and aspect ratio) was assessed by transmission electron micrographs at 6h and quantified by a blinded reviewer (ImageJ). Activation of the mitochondrial permeability transition pore (mPTP) was assessed by flow cytometry using calcein-AM with cobalt chloride. DNA was purified from patient plasma and the copy number of mitochondrial genes ND4L and CYB was quantified by digital droplet PCR. Results: In HLMVECs, CFH generated excess ROS (CellROX and MitoSOX) at 6h compared to vehicle (522 vs 265 MFI, p < 0.01). Morphology analysis demonstrated increased electron density and aspect ratio and decreased circularity and roundness. mPTP activity was increased by CFH3+ (1295 vs 2197 MFI, p < 0.01) compared to vehicle or CFH2+. Barrier dysfunction caused by CFH3+ measured by ECIS peaked at 6h (-1270 vs 262 max TER drop, p < 0.0001), while XPerT confirmed dysfunction at 24h (9.41e+06 vs 6.53e+05 total fluorescent area, p < 0.01). Quantification of mtDNA copy number in patient plasma revealed that circulating mt-ND4L copies correlate with CFH (r = 0.44, p < 0.01). Conclusions: The results demonstrate significant perturbations to mitochondrial function and network dynamics caused by oxidized CFH (3+). The novel findings point towards the ferric (3+) form of hemoglobin as the primary driver of endothelial dysfunction, and the underlying mechanism is directly related to disruption of the mitochondrial network. The association of mitochondrial and vascular biomarkers demonstrates an urgent need to better understand how mitochondrial dysfunction can be prevented to limit vascular damage. This work was funded by the National Institutes of Health NHLBI 5R35HL150783 and 1F31HL167471. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alpha-synuclein interaction with mitochondria is the final mechanism of ferroptotic death induced by erastin in SH-SY5Y cells

Ferroptosis has been characterized as a form of iron-dependent regulated cell death accompanied by an accumulation of reactive oxygen species and lipid oxidation products along with typical morphological alterations in mitochondria. Ferroptosis is activated by diverse triggers and inhibited by ferrostatin-1 and liproxstatin-1, apart from iron chelators and several antioxidants, and the process is implicated in multiple pathological conditions. There are, however, certain ambiguities about ferroptosis, especially regarding the final executioner of cell death subsequent to the accumulation of ROS. This study uses a typical inducer of ferroptosis such as erastin on SH-SY5Y cells, and shows clearly that ferroptotic death of cells is accompanied by the loss of mitochondrial membrane potential and intracellular ATP content along with an accumulation of oxidative stress markers. All these are prevented by ferrostatin-1 and liproxstatin-1. Additionally, cyclosporine A prevents mitochondrial alterations and cell death induced by erastin implying the crucial role of mitochondrial permeability transition pore (mPTP) activation in ferroptotic death. Furthermore, an accumulation of α-synuclein occurs during erastin induced ferroptosis which can be inhibited by ferrostatin-1 and liproxstatin-1. When the knock-down of α-synuclein expression is performed by specific siRNA treatment of SH-SY5Y cells, the mitochondrial impairment and ferroptotic death of the cells induced by erastin are markedly prevented. Thus, α-synuclein through the involvement of mPTP appears to be the key executioner protein of ferroptosis induced by erastin, but it needs to be verified if it is a generalized mechanism of ferroptosis by using other inducers and cell lines.

Quercetin protects cardiomyoblasts against hypertonic cytotoxicity by abolishing intracellular Ca2+ elevations and mitochondrial depolarisation

Background and AimOsmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. Experimental procedureProtective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. Key resultsQuercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. Conclusions and implicationsQuercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.

Hyperglycemia triggers RyR2-dependent alterations of mitochondrial calcium homeostasis in response to cardiac ischemia-reperfusion: Key role of DRP1 activation

Hyperglycemia increases the heart sensitivity to ischemia-reperfusion (IR), but the underlying cellular mechanisms remain unclear. Mitochondrial dynamics (the processes that govern mitochondrial morphology and their interactions with other organelles, such as the reticulum), has emerged as a key factor in the heart vulnerability to IR. However, it is unknown whether mitochondrial dynamics contributes to hyperglycemia deleterious effect during IR. We hypothesized that (i) the higher heart vulnerability to IR in hyperglycemic conditions could be explained by hyperglycemia effect on the complex interplay between mitochondrial dynamics, Ca2+ homeostasis, and reactive oxygen species (ROS) production; and (ii) the activation of DRP1, a key regulator of mitochondrial dynamics, could play a central role. Using transmission electron microscopy and proteomic analysis, we showed that the interactions between sarcoplasmic reticulum and mitochondria and mitochondrial fission were increased during IR in isolated rat hearts perfused with a hyperglycemic buffer compared with hearts perfused with a normoglycemic buffer. In isolated mitochondria and cardiomyocytes, hyperglycemia increased mitochondrial ROS production and Ca2+ uptake. This was associated with higher RyR2 instability. These results could contribute to explain the early mPTP activation in mitochondria from isolated hearts perfused with a hyperglycemic buffer and in hearts from streptozotocin-treated rats (to increase the blood glucose). DRP1 inhibition by Mdivi-1 during the hyperglycemic phase and before IR induction, normalized Ca2+ homeostasis, ROS production, mPTP activation, and reduced the heart sensitivity to IR in streptozotocin-treated rats. In conclusion, hyperglycemia-dependent DRP1 activation results in higher reticulum-mitochondria calcium exchange that contribute to the higher heart vulnerability to IR.

Caspase-3 cleaved tau impairs mitochondrial function through the opening of the mitochondrial permeability transition pore

Mitochondrial dysfunction is a significant factor in the development of Alzheimer's disease (AD). Previous studies have demonstrated that the expression of tau cleaved at Asp421 by caspase-3 leads to mitochondrial abnormalities and bioenergetic impairment. However, the underlying mechanism behind these alterations and their impact on neuronal function remains unknown. To investigate the mechanism behind mitochondrial dysfunction caused by this tau form, we used transient transfection and pharmacological approaches in immortalized cortical neurons and mouse primary hippocampal neurons. We assessed mitochondrial morphology and bioenergetics function after expression of full-length tau and caspase-3-cleaved tau. We also evaluated the mitochondrial permeability transition pore (mPTP) opening and its conformation as a possible mechanism to explain mitochondrial impairment induced by caspase-3 cleaved tau. Our studies showed that pharmacological inhibition of mPTP by cyclosporine A (CsA) prevented all mitochondrial length and bioenergetics abnormalities in neuronal cells expressing caspase-3 cleaved tau. Neuronal cells expressing caspase-3-cleaved tau showed sustained mPTP opening which is mostly dependent on cyclophilin D (CypD) protein expression. Moreover, the impairment of mitochondrial length and bioenergetics induced by caspase-3-cleaved tau were prevented in hippocampal neurons obtained from CypD knock-out mice. Interestingly, previous studies using these mice showed a prevention of mPTP opening and a reduction of mitochondrial failure and neurodegeneration induced by AD. Therefore, our findings showed that caspase-3-cleaved tau negatively impacts mitochondrial bioenergetics through mPTP activation, highlighting the importance of this channel and its regulatory protein, CypD, in the neuronal damage induced by tau pathology in AD.

The Reduction in the Mitochondrial Membrane Potential in Aging: The Role of the Mitochondrial Permeability Transition Pore.

It is widely reported that the mitochondrial membrane potential, ∆Ψm, is reduced in aging animals. It was recently suggested that the lower ∆Ψm in aged animals modulates mitochondrial bioenergetics and that this effect is a major cause of aging since artificially increased ∆Ψm in C. elegans increased lifespan. Here, I critically review studies that reported reduction in ∆Ψm in aged animals, including worms, and conclude that many of these observations are best interpreted as evidence that the fraction of depolarized mitochondria is increased in aged cells because of the enhanced activation of the mitochondrial permeability transition pore, mPTP. Activation of the voltage-gated mPTP depolarizes the mitochondria, inhibits oxidative phosphorylation, releases large amounts of calcium and mROS, and depletes cellular NAD+, thus accelerating degenerative diseases and aging. Since the inhibition of mPTP was shown to restore ∆Ψm and to retard aging, the reported lifespan extension by artificially generated ∆Ψm in C. elegans is best explained by inhibition of the voltage-gated mPTP. Similarly, the reported activation of the mitochondrial unfolded protein response by reduction in ∆Ψm and the reported preservation of ∆Ψm in dietary restriction treatment in C. elegans are best explained as resulting from activation or inhibition of the voltage-gated mPTP, respectively.

Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition.

The mitochondrial permeability transition pore (mPTP) is a large, weakly selective pore that opens in the mitochondrial inner membrane in response to the pathological increase in matrix Ca2+ concentration. mPTP activation has been implicated as a key factor contributing to stress-induced necrotic and apoptotic cell death. The molecular identity of the mPTP is not completely understood. Both ATP synthase and adenine nucleotide translocase (ANT) have been described as important components of the mPTP. Using a refractive index (RI) imaging approach, we recently demonstrated that the removal of either ATP synthase or ANT eliminates the Ca2+-induced mPTP in experiments with intact cells. These results suggest that mPTP formation relies on the interaction between ATP synthase and ANT protein complexes. To gain further insight into this process, we used RI imaging to investigate mPTP properties in cells with a genetically eliminated C subunit of ATP synthase. These cells also lack ATP6, ATP8, 6.8PL subunits and DAPIT but, importantly, have a vestigial ATP synthase complex with assembled F1 and peripheral stalk domains. We found that these cells can still undergo mPTP activation, which can be blocked by the ANT inhibitor bongkrekic acid. These results suggest that ANT can form the pore independently from the C subunit but still requires the presence of other components of ATP synthase.

GLUT inhibitor WZB117 induces cytotoxicity with increased production of amyloid-beta peptide in SH-SY5Y cells preventable by beta-hydroxybutyrate: implications in Alzheimer's disease.

Inhibitors of glucose transporters are being explored as potential anti-cancer drugs. Decreased cerebral glucose utilization with reduced levels of several glucose transporters is also an important pathogenic signature of neurodegeneration of Alzheimer's disease, but its exact role in the pathogenesis of this disease is not established. We explored in an experimental model if inhibitors of glucose transporters could lead to altered amyloid-beta homeostasis, mitochondrial dysfunction, and neuronal death, which are relevant in the pathogenesis of Alzheimer's disease. SH-SY5Y cells (human neuroblastoma cell line) were exposed to an inhibitor (WZB117) of several types of glucose transporters. We examined the effects of glucose hypometabolism on SH-SY5Y cells in terms of mitochondrial functions, production of reactive oxygen species, amyloid-beta homeostasis, and neural cell death. The effect of β-hydroxybutyrate in ameliorating the effects of WZB117 on SH-SY5Y cells was also examined. We observed that exposure of SH-SY5Y cells to WZB117 caused mitochondrial dysfunction, increased production of reactive oxygen species, loss of cell viability, increased expression of BACE 1, and intracellular accumulation of amyloid β peptide (Aβ42). All the effects of WZB117 could be markedly prevented by co-treatment with β-hydroxybutyrate. Cyclosporine A, a blocker of mitochondrial permeability transition pore (mPTP) activation, could not prevent cell death caused by WZB117. Results in this neuroblastoma model have implications for the pathogenesis of Alzheimer's disease and warrant further explorations of WZB117 in primary cultures of neurons and experimental animal models.

Curious case of a leaky mitochondria.

The mitochondrial permeability transition pore (mPTP) leads to cell death upon its activation. The past several decades have seen many studies regarding the modulation of the mPTP, but little is known about the structure. There has been a growing body of evidence that the ATP synthase c-subunit houses the mPTP leak channel. Experimental evidence has shown that ATP Synthase forms voltage-gated and Ca2+-activated channels, consistent to what is known about mPTP activation. Previous experimental and molecular dynamics simulation studies demonstrate that the ATP synthase c-subunit is occupied by lipid and/or detergent molecules, claiming that a leak channel cannot be formed and that ATP synthase does not play a role in mPTP formation. These studies have sparked much controversy regarding the structural identity of the mPTP leak channel. In this work, we investigated whether the ATP synthase c-subunit forms a leak channel upon introducing voltage. We performed molecular dynamics simulations on the ATP synthase c-subunit under an electric field, ranging from −180 mV to +180 mV. Our results show significant expansion of the c-subunit, and lipids exiting as water leaks into the pore. Additionally, we identified a dipole on the c-subunit monomer that aids in voltage-sensing. We also applied voltage to a c-subunit high and low-conductance mutants, where we observe similar results, except for decreased water leakage in the low-conductance mutant. Initially, we observed a bottleneck at a key glutamic acid residue that is responsible for proton transport in the c-subunit. To overcome this bottleneck, we introduced a series of deprotonations on these glutamic acid residues to promote water accessibility, resulting in a full leak channel formation. With our results, we seek to answer a long-standing question about the gating mechanism of ATP synthase c-subunit leak channel and the structural identity of the mPTP.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

RationaleSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. ObjectivesWe examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. MethodsWe used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. ResultsSARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. ConclusionsCoronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Mitochondrial calcium buffering depends upon temperature and is associated with hypothermic neuroprotection against hypoxia-ischemia injury.

Hypothermia (HT) is a standard of care in the management of hypoxic-ischemic brain injury (HI). However, therapeutic mechanisms of HT are not well understood. We found that at the temperature of 32°C, isolated brain mitochondria exhibited significantly greater resistance to an opening of calcium-induced permeability transition pore (mPTP), compared to 37°C. Mitochondrial calcium buffering capacity (mCBC) was linearly and inversely dependent upon temperature (25°C—37°C). Importantly, at 37°C cyclosporine A did not increase mCBC, but significantly increased mCBC at lower temperature. Because mPTP contributes to reperfusion injury, we hypothesized that HT protects brain by improvement of mitochondrial tolerance to mPTP activation. Immediately after HI-insult, isolated brain mitochondria demonstrated very poor mCBC. At 30 minutes of reperfusion, in mice recovered under normothermia (NT) or HT, mCBC significantly improved. However, at four hours of reperfusion, only NT mice exhibited secondary decline of mCBC. HT-mice maintained their recovered mCBC and this was associated with significant neuroprotection. Direct inverted dependence of mCBC upon temperature in vitro and significantly increased mitochondrial resistance to mPTP activation after therapeutic HT ex vivo suggest that hypothermia-driven inhibition of calcium-induced mitochondrial mPTP activation mechanistically contributes to the neuroprotection associated with hypothermia.

Temporal effect of melatonin posttreatment on anoxia/reoxygenation injury in H9c2 cells

Melatonin has been proven to reduce myocardial ischemia-reperfusion (MI/R) injury. However, in most studies, melatonin was administered before MI/R, thus, the results lack clinical significance in patients with acute myocardial infarction. We hypothesize that melatonin posttreatment at different times has different curative effects. Administered of Melatonin (150 μM) at different times after the onset of reoxygenation (t = -15, 0, 5, 10, 15, and 30 min). Cellular apoptosis, oxidative stress, and mitochondrial function were assessed. Mitophagy-related protein levels,mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) activity were also measured. A/R injury upregulated mitophagy, which was associated with increased cellular apoptosis, oxidative stress, and mitochondrial dysfunction. Melatonin posttreatment (t = -15, 0, 5, 10, 15, and 30 min) significantly inhibited excessive mitophagy after A/R injury, reduced cellular apoptosis and oxidative stress, restored mitochondrial function and MMP, and restrained mPTP opening. The therapeutic time window in which melatonin posttreatment protected H9c2 cells against A/R injury was large (from -15 to 30 min after the onset of reperfusion), but the earlier the melatonin administration was, the better its protective effect was. This mechanism is likely due to a reduction in mPTP activity and MMP collapse, which lead to the inhibition of mitophagy.

Molecular mechanisms and consequences of mitochondrial permeability transition.

Mitochondrial permeability transition (mPT) is a phenomenon that abruptly causes the flux of low molecular weight solutes (molecular weight up to 1,500) across the generally impermeable inner mitochondrial membrane. The mPT is mediated by the so-called mitochondrial permeability transition pore (mPTP), a supramolecular entity assembled at the interface of the inner and outer mitochondrial membranes. In contrast to mitochondrial outer membrane permeabilization, which mostly activates apoptosis, mPT can trigger different cellular responses, from the physiological regulation of mitophagy to the activation of apoptosis or necrosis. Although there are several molecular candidates for the mPTP, its molecular nature remains contentious. This lack of molecular data was a significant setback that prevented mechanistic insight into the mPTP, pharmacological targeting and the generation of informative animal models. In recent years, experimental evidence has highlighted mitochondrial F1Fo ATP synthase as a participant in mPTP formation, although a molecular model for its transition to the mPTP is still lacking. Recently, the resolution of the F1Fo ATP synthase structure by cryogenic electron microscopy led to a model for mPTP gating. The elusive molecular nature of the mPTP is now being clarified, marking a turning point for understanding mitochondrial biology and its pathophysiological ramifications. This Review provides an up-to-date reference for the understanding of the mammalian mPTP and its cellular functions. We review current insights into the molecular mechanisms of mPT and validated observations - from studies in vivo or in artificial membranes - on mPTP activity and functions. We end with a discussion of the contribution of the mPTP to human disease. Throughout the Review, we highlight the multiple unanswered questions and, when applicable, we also provide alternative interpretations of the recent discoveries.

Etifoxine Restores Mitochondrial Oxidative Phosphorylation and Improves Cognitive Recovery Following Traumatic Brain Injury.

The opening of the mitochondrial permeability transition pore (mPTP) has emerged as a pivotal event following traumatic brain injury (TBI). Evidence showing the impact of the translocator protein (TSPO) over mPTP activity has prompted several studies exploring the effect of TSPO ligands, including etifoxine, on the outcome of traumatic brain injury (TBI). Mitochondrial respiration was assessed by respirometry in isolated rat brain mitochondria (RBM) by measurements of oxidative phosphorylation capacity (OXPHOS). The addition of calcium to RBM was used to induce mitochondrial injury and resulted in significant OXPHOS reduction that could be reversed by preincubation of RBM with etifoxine. Sensorimotor and cognitive functions were assessed following controlled cortical impact and compared in vehicle and etifoxine-treated animals. There was no difference between the vehicle and etifoxine groups for sensorimotor functions as assessed by rotarod. In contrast, etifoxine resulted in a significant improvement of cognitive functions expressed by faster recovery in Morris water maze testing. The present findings show a significant neuroprotective effect of etifoxine in TBI through restoration of oxidative phosphorylation capacity associated with improved behavioral and cognitive outcomes. Since etifoxine is a registered drug used in common clinical practice, implementation in a phase II study may represent a reasonable step forward.

Abstract 937: The PP2A activation using a small molecule agonist triggers apoptosis by releasing mitochondrial permeability transition pores in multi-drug resistant leukemic B cells

Abstract The Bcl-2 inhibitor venetoclax (VEN) shows clinical activity in Chronic Lymphocytic Leukemia (CLL), and the response is potentiated when combined with the BTK inhibitor ibrutinib (IBR) (Portell et al. 2019; Tam et al. 2018). However, many VEN or VEN+IBR responses are incomplete and drug resistance develops in most cases. We previously noted that CLL cells exposed to microenvironmental agonists ex vivo exhibit resistance to VEN+IBR due to upregulation of multiple anti-apoptotic proteins (Jayappa et al. 2017). Here, we find in the circulation of CLL patients that activated (CD69Pos) leukemic B cells recently emigrated from the lymph node exhibit resistance to inhibitors of Bcl-2, Mcl-1, or Bcl-xL due to impaired activation of Bax/Bak proteins. Our molecular analysis suggests that multiple upregulated anti-apoptotic proteins (Mcl-1, Bcl-xL, and Bcl-2) swap for pro-apoptotic proteins (e.g. Bim) in these cells when subjected to single-drug treatments, leading to defective Bax/Bak activation resulting in resistance to drug induced apoptosis. A large-scale cell-based screen using small molecule agonist of Protein Phosphatase 2A (PP2A) (SMAP, TRC-382) revealed that blood cancer cell lines are highly sensitive to SMAP mediated activation of PP2A, a serine/threonine phosphatase known to regulate cell survival. A further pharmacologically optimized SMAP compound (DT061) showed activity even in leukemia cell lines or patient-derived CD69Pos CLL cells resistant to inhibitors of Bcl-2, Mcl-1, or Bcl-xL, suggesting PP2A activation can overcome apoptosis resistance. The PP2A activator DT061 induced apoptosis in these cells in the absence of Bax/Bak activation and was equally effective in an isogenic Bax/Bak double knockout cells, suggesting PP2A activation overcomes multi-drug resistance via induction of Bax/Bak-independent apoptosis. We next examined various Bax/Bak-independent apoptosis mechanisms using inhibitors, and found that inhibitors of mitochondrial permeability transition pores (mPTP) (NIM811 and cyclopsorin-A) blocked the DT061-induced apoptosis in CLL cells. Using the CalceinAM/CoCl2 assay, we noted that DT061 triggers mPTP opening in primary CLL cells, which was blocked by mPTP inhibitors, suggesting DT061 induces apoptosis via mPTP activation. In summary, microenvironmentally activated cancer cells displaying resistance to apoptosis due to defective Bax/Bak activation are found de novo in CLL patients. Reactivation of the tumor suppressive serine/threonine phosphatase by a series of small molecules (SMAPs) overcomes this resistance by inducing mPTP dependent apoptosis. Collectively, these findings demonstrate the existence of an anti-apoptotic multi-drug resistant pool of cancer cells in CLL patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Citation Format: Kallesh D. Jayappa, Caroline Farrington, Vicki L. Gordon, Shekhar Saha, Christopher Morris, Krista M. Isaac, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Goutham Narla, Michael J. Weber. The PP2A activation using a small molecule agonist triggers apoptosis by releasing mitochondrial permeability transition pores in multi-drug resistant leukemic B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 937.

The Mitochondrial Permeability Transition: Nexus of Aging, Disease and Longevity.

The activity of the mitochondrial permeability transition pore, mPTP, a highly regulated multi-component mega-channel, is enhanced in aging and in aging-driven degenerative diseases. mPTP activity accelerates aging by releasing large amounts of cell-damaging reactive oxygen species, Ca2+ and NAD+. The various pathways that control the channel activity, directly or indirectly, can therefore either inhibit or accelerate aging or retard or enhance the progression of aging-driven degenerative diseases and determine lifespan and healthspan. Autophagy, a catabolic process that removes and digests damaged proteins and organelles, protects the cell against aging and disease. However, the protective effect of autophagy depends on mTORC2/SKG1 inhibition of mPTP. Autophagy is inhibited in aging cells. Mitophagy, a specialized form of autophagy, which retards aging by removing mitochondrial fragments with activated mPTP, is also inhibited in aging cells, and this inhibition leads to increased mPTP activation, which is a major contributor to neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. The increased activity of mPTP in aging turns autophagy/mitophagy into a destructive process leading to cell aging and death. Several drugs and lifestyle modifications that enhance healthspan and lifespan enhance autophagy and inhibit the activation of mPTP. Therefore, elucidating the intricate connections between pathways that activate and inhibit mPTP, in the context of aging and degenerative diseases, could enhance the discovery of new drugs and lifestyle modifications that slow aging and degenerative disease.

Electrophysiological properties of the mitochondrial permeability transition pores: Channel diversity and disease implication

The mitochondrial permeability transition pore (mPTP) is a channel that, when open, is responsible for a dramatic increase in the permeability of the mitochondrial inner membrane, a process known as the mitochondrial permeability transition (mPT). mPTP activation during Ca2+ dyshomeostasis and oxidative stress disrupts normal mitochondrial function and induces cell death. mPTP opening has been implicated as a critical event in many diseases, including hypoxic injuries, neurodegeneration, and diabetes. Discoveries of recent years indicate that mPTP demonstrates very complicated behavior and regulation, and depending on specific induction or stress conditions, it can function as a high-conductance pore, a small channel, or a non-specific membrane leak. The focus of this review is to summarize the literature on the electrophysiological properties of the mPTP and to evaluate the evidence that it has multiple molecular identities. This review also provides perspective on how an electrophysiological approach can be used to quantitatively investigate the biophysical properties of the mPTP under physiological, pharmacological, pathophysiological, and disease conditions.