Voltage-dependent Anion Channel Research Articles

Appraisal of the Possible Role of PPARγ Upregulation by CLA of Probiotic Pediococcus pentosaceus GS4 in Colon Cancer Mitigation.

The prevalence of colon cancer (CC) is increasing at the endemic scale, which is accompanied by subsequent morbidity and mortality. Although there have been noteworthy achievements in the therapeutic strategies in recent years, the treatment of patients with CC remains a formidable task. The current study focused on to study role of biohydrogenation-derived conjugated linoleic acid (CLA) of probiotic Pediococcus pentosaceus GS4 (CLAGS4) against CC, which induced peroxisome proliferator-activated receptor gamma (PPARγ) expression in human CC HCT-116 cells. Pre-treatment with PPARγ antagonist bisphenol A diglycidyl ether has significantly reduced the inhibitory efficacy of enhanced cell viability of HCT-116 cells, suggesting the PPARγ-dependent cell death. The cancer cells treated with CLA/CLAGS4 demonstrated the reduced level of Prostaglandin E2 PGE2 in association with reduced COX-2 and 5-LOX expressions. Moreover, these consequences were found to be associated with PPARγ-dependent. Furthermore, delineation of mitochondrial dependent apoptosis with the help of molecular docking LigPlot analysis showed that CLA can bind with hexokinase-II (hHK-II) (highly expressed in cancer cells) and that this association underlies voltage dependent anionic channel to open, thereby causing mitochondrial membrane depolarization, a condition that initiates intrinsic apoptotic events. Apoptosis was further confirmed by annexin V staining and elevation of caspase 1p10 expression. Taken all together, it is deduced that, mechanistically, the upregulation of PPARγ by CLAGS4 of P. pentosaceus GS4 can alter cancer cell metabolism in association with triggering apoptosis in CC.

Cytosolic Release of Mitochondrial DNA and Associated cGAS Signaling Mediates Radiation-Induced Hematopoietic Injury of Mice.

Mitochondrion is an important organelle of eukaryotic cells and a critical target of ionizing radiation (IR) outside the nucleus. The biological significance and mechanism of the non-target effect originating from mitochondria have received much attention in the field of radiation biology and protection. In this study, we investigated the effect, role, and radioprotective significance of cytosolic mitochondrial DNA (mtDNA) and its associated cGAS signaling on hematopoietic injury induced by IR in vitro culture cells and in vivo total body irradiated mice in this study. The results demonstrated that γ-ray exposure increases the release of mtDNA into the cytosol to activate cGAS signaling pathway, and the voltage-dependent anion channel (VDAC) may contribute to IR-induced mtDNA release. VDAC1 inhibitor DIDS and cGAS synthetase inhibitor can alleviate bone marrow injury and ameliorate hematopoietic suppression induced by IR via protecting hematopoietic stem cells and adjusting subtype distribution of bone marrow cells, such as attenuating the increase of the F4/80+ macrophage proportion in bone marrow cells. The present study provides a new mechanistic explanation for the radiation non-target effect and an alternative technical strategy for the prevention and treatment of hematopoietic acute radiation syndrome.

TMT-based proteomics profile reveals changes of the entorhinal cortex in a kainic acid model of epilepsy in mice

Experimental modeling and clinical neuroimaging of patients has shown that certain seizures are capable of causing neuronal death. Research into cell death after seizures has identified the induction of the molecular machinery of apoptosis. Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults, which is characterized by substantial pathological abnormalities in the temporal lobe, including the hippocampus and entorhinal cortex (EC). Although decades of studies have revealed numerous molecular abnormalities in the hippocampus that are linked to TLE, the biochemical mechanisms associated with TLE in EC remain unclear.In this study, we explored these early phenotypical alterations in the EC 5 days after mice were given a systemic injection of kainic acid (KA) to induce status epilepticus (KA-SE). we used the Tandem Mass Tag (TMT) combined with LC-MS/MS approach to identify distinct proteins in the EC in a mouse model of KA-SE model.According to the findings, 355 differentially abundant proteins including 199 upregulated and 156 downregulated differentially abundant proteins were discovered. The first-ranked biological process according to Gene Ontology (GO) analysis was “negative control of extrinsic apoptotic signaling”. “Apoptosis” was the most significantly enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway. Compared with those in control mice, BCL2L1, NTRK2 and MAPK10 abundance levels were reduced in KA mice. MAPK10 and NTRK2 act as upstream regulators to regulate BCL2L1, and BCL2L1 Inhibits cell death by blocking the voltage- dependent anion channel (VDAC) and preventing the release of the caspase activator, CYC1, from the mitochondrial membrane. However, ITPR1 was increased at the mRNA and protein levels in KA mice. Furthermore, there was no significant difference in ACTB, TUBA1A and TUBA4A levels between the two groups. Our results offer clues to help identify biomarkers for the development of pharmacological therapies targeted at epilepsy.

Voltage dependent anion channel and its interaction with N-acetyl-L-Cysteine (NAC) under oxidative stress on planar lipid bilayer

Mitochondria are the major source of Hydrogen Peroxide (H2O2), a reactive oxygen species, in the cells. The reactive oxygen species generated by the mitochondria oxidize major proteins including Voltage Dependent Anion Channel (VDAC). We were interested to know how the effect of H2O2 is countered by antioxidants present around the mitochondria. N-Acetyl-l-Cysteine (NAC) is a naturally existing antioxidant in the cells. Keeping this in view, the modulatory effect of antioxidant NAC on H2O2 oxidized VDAC has been investigated through in vitro electrophysiological studies. First, the effect of H2O2 and NAC was studied on independently incorporated single-channel VDAC. It was observed that NAC suppresses VDAC conductance with a half-maximal inhibitory concentration (IC50) of ∼1.04 μM. In contrast, H2O2 enhances VDAC conductance. Later, oxidative stress was induced by H2O2 on VDAC increased conductance with half-maximal effective concentration (EC50) of ∼302 nM. An application of 1 μM NAC on H2O2 treated (300 nM) VDAC reversed the effect of oxidation. In the next step, NAC and H2O2 were added in reverse order. When oxidative stress was induced using H2O2, reduction in conductance by NAC was 4.5 ± 0.404 nS. The change in conductance is nearly 6.3%. However, if antioxidant NAC was incubated first followed by H2O2 treatment, the conductance of VDAC was 3.09 ± 0.27 nS. The change in conductance is near 33%. Both H2O2 and NAC also affected various conducting states of VDAC. In-silico studies indicated the binding of NAC at Lysine and Glutamic acid of VDAC. Hence, NAC was found to be effective in protection of VDAC against H2O2-induced oxidative stress due to its strong binding.

Inhibition of VDAC1 Rescues Aβ 1-42-Induced Mitochondrial Dysfunction and Ferroptosis via Activation of AMPK and Wnt/β-Catenin Pathways.

Beta-amyloid (Aβ) accumulation in the brains of Alzheimer's disease (AD) patients leads to mitochondrial dysfunction and ferroptosis in neurons. Voltage-dependent anion channel 1 (VDAC1) is a major protein in the mitochondrial outer membrane. It has been reported that VDAC1 associated with mitochondrial dysfunction and ferroptosis. However, the mechanism by which VDAC1 regulates mitochondrial dysfunction and ferroptosis of neurons in AD remains unclear. This study is aimed at investigating the mechanism of action of VDAC1 in mitochondrial dysfunction and ferroptosis in neurons of the AD model. In this study, we determined cell viability after treatment with Aβ 1-42 via the MTT assay. The SOD, MDA, ROS, and MMP production was measured via the SOD kit, MDA kit, DCFDA staining, and JC-1 staining. The memory abilities of mice were detected via the Morris water maze test. The expression of AMPK/mTOR, Wnt/β-catenin, and GPX4 regulated by VDAC1 was detected via western blotting. Our present study showed that PC12 cells had decreased cell viability, increased LDH release, and decreased GPX4 expression after Aβ 1-42 treatment. Meanwhile, Aβ 1-42 induced MMP and SOD downregulation and increased MDA and ROS generation in PC12 cells. In addition, the expression of VDAC1 is increased in the brain tissue of AD mice and Aβ 1-42-treated PC12 cells. Further investigation of the role of VDAC1 in regulating AD found that all effects induced by Aβ 1-42 were reversed by inhibition of VDAC1. Additionally, inhibition of VDAC1 activates the AMPK/mTOR and Wnt/β-catenin pathways. Taken together, these findings demonstrate that inhibition of VDAC1 alleviates mitochondrial dysfunction and ferroptosis in AD neurons by activating AMPK/mTOR and Wnt/β-catenin.

Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-β production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

Site-specific phosphorylation of VDAC1 decreases its susceptibility to oligomerization.

The voltage-dependent anion channel 1 (VDAC1) is the primary pathway for metabolite and ion flux across the outer mitochondrial membrane. Oligomerization of VDAC1 that results in the formation of a mega-pore is a critical step in the release of cytochrome c and mitochondrial DNA that triggers apoptosis and inflammasome activation, respectively. However, the underlying mechanism that leads to mega-pore formation is unresolved. Previously, we identified phosphorylation of VDAC1 at S35 to be associated with cardioprotection. Here we report a novel finding that S35 phosphorylation regulates VDAC1 oligomerization and, subsequently, mega-pore formation. In 90 min recordings of channel activity of recombinant VDAC1 in planar lipid bilayers, wild-type (WT) VDAC1 exhibited cumulative current increases due to multiple channel insertions. In 50% of the recordings, a dramatic increase in cumulative channel conductance was observed (>1.8 µS). This was accompanied by a progressive shift at baseline, suggestive of high local ionic accumulation at the bilayer surface. These distinct biophysical characteristics were attributed to mega-pore formation. The probability of similar cumulative large conductances with associated baseline shifts was reduced in the phosphomimetic S35E VDAC1 (20% vs 50% in WT), while it was increased in a non-phosphorylatable mutant S35A (90% vs 50% in WT). In VDAC1 knock-out H9c2 cells expressing WT or mutant (S35A/S35E) VDAC1, 400 µM H2O2 induced the formation of VDAC1 oligomers. However, compared to WT, the formation of trimers and higher order oligomers was significantly decreased in cells expressing the S35E mutant, while it was significantly increased in cells expressing the S35A mutant. These effects are consistent with the corresponding biophysical characteristics observed in the bilayer experiments. Our results suggest that phosphorylation of S35 decreases, while dephosphorylation increases, the susceptibility of VDAC1 to oligomerization and mega-pore formation.

The single residue K12 governs highly sensitive voltage gating of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) has emerged as a key player in mitochondrial bioenergetics. As the main pathway for calcium and mitochondrial metabolites, such as ATP and ADP, to cross the mitochondrial outer membrane, VDAC is central to maintenance of mitochondrial homeostasis. It is thus unsurprising that VDAC is implicated in nearly every mitochondria-related pathology, including cardiovascular disease, cancers, and neurodegeneration. Despite decades of research, the relatively uncomplicated structure of VDAC—a β-barrel with an internal broken α-helical belt—has hampered efforts to elucidate its functional mechanisms. Unlike other β-barrel channels, such as bacterial porins and toxins, VDAC transitions to low-conducting states, or “gates”, at relatively low voltages, less than 30 mV. In VDAC's gated state, metabolite transport is suppressed and calcium transport enhanced; thus, gating is one means by which VDAC may regulate fluxes across the mitochondrial membrane. In this talk, I will describe computational and experimental efforts that have led to identification of K12 as a key residue that is responsible for the exquisite sensitivity to voltage of its gating process [JACS 2022, 144(32) 14564-14577, DOI:10.1021/jacs.2c03316]. K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the β-barrel and augment the likelihood of gating. These results are confirmed by single channel electrophysiology of various K12 mutants, which show a dramatic reduction of the frequency of voltage-induced gating transitions. The crystallographic structure of the K12E mutant is similar to the wild type; however, 60 µs of atomistic MD simulations using the K12E mutant structure show restricted motion of the mutated residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We conclude that β-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions.

Establishing the role of the mitochondrial voltage-dependent anion channel (VDAC) in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas Disease, a neglected tropical disease that affects millions globally. As it transitions from insect vector to human host, T. cruzi faces homeostatic challenges, including oxidative stress, fluctuations in osmolarity, and nutrient availability. Ion channels regulate the parasite's ability to respond to environmental stressors. Our group has previously identified a mechanosensitive channel located in the contractile vacuole that, when knocked out, leads to reduced infectivity, abnormal morphology, and impaired motility. Transcriptomic analysis showed that Voltage-Dependent Anion Channel (VDAC) was significantly downregulated in the knockouts, suggesting a functional link between the two proteins. VDAC is a highly expressed outer mitochondrial membrane protein that, in other eukaryotes, regulates cell death, mitochondrial dynamics and metabolism. In protozoans, VDAC has been shown to share similar functions, with additional roles in tRNA and protein import. However, the function of VDAC in T. cruzi remains uncharacterized. The genome of T. cruzi contains five copies of VDAC sharing 99% homology. Four of them are organized in tandem, suggesting a gene expansion event as their most probable origin. To elucidate the function of this channel in the parasites, we targeted a conserved region using CRISPR/Cas9 strategies to generate a VDAC-deficient cell line. The mutant strain exhibited abnormal morphology and significant growth defects relative to the wild type, suggesting that VDAC is critical for parasite replication. Fluorometric analysis indicates that VDAC downregulation results in mitochondrial depolarization and reduced calcium uptake supporting its role in maintenance of mitochondrial functions.

VDAC interactions with lipids and their effect on isoform specificity.

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel, the most abundant protein of the mitochondrial outer membrane, allowing passage of ions and metabolites (such as ATP, ADP, NADH⋯) in and out of the mitochondria. Besides its transport function, VDAC is also implicated in mitochondrial regulation with roles in apoptosis, calcium homeostasis or neurodegeneration. However, its role in those different pathways remains to be understood at the molecular level. VDAC possesses 3 isoforms in mammals (VDAC1, 2 and 3), sharing a high sequence identity, the same architecture and an overlapping tissue distribution. They display very similar transport properties such as conductance, selectivity and voltage-induced closure while their isoform specificity originates from their different interaction with partner proteins. This may determine their unique role in mitochondrial regulation, where each isoform seems to have different functions and a different set of partners: VDAC1 is implicated in neurodegeneration (Alzheimer and Parkinson disease), VDAC2 in apoptosis and calcium homeostasis, and VDAC3 probably in spermatogenesis and oxidative stress. Using a combination of biochemistry, molecular dynamics and electrophysiology, we investigated the lipid organization around each VDAC isoform and their impact on VDAC function. Our data suggest that the three isoforms have different affinities for lipids, and that could contribute to the selection of interacting partners.

Defining the roles and regulation of the mitochondrial VDAC isoforms one molecule at a time.

The voltage-dependent anion channel (VDAC) is recognized as the key metabolite conduit in the mitochondrial outer membrane and regulator for fluxes of water-soluble metabolites, ions, and polypeptides. The uniqueness of VDAC arises from its position at the interface between the mitochondria and the cytosol making VDAC an important cellular communication hub. VDAC has three isoforms in mammals: VDAC1, 2, and 3 which share high sequence similarity, indicative of a similar pore-forming structure. All three VDAC isoforms form similar highly conductive, anion-selective, voltage-gated channels when reconstituted into planar lipid membranes implying that they can concurrently facilitate the transport of the negatively charged respiratory metabolites. However, cellular model systems and isoform-specific knock-out mice demonstrate distinctive functional roles for each VDAC isoform. We explored the biophysical basis for these differences by reconstituting human, mouse, and zebrafish VDAC isoforms in planar lipid bilayers and performing single channel electrophysiology. We have uncovered that interactions with cytosolic regulators, such as the Parkinson's disease associated alpha-synuclein, vary in their on-rates by two-orders of magnitude between each isoform. Furthermore, each isoform varies in their ability translocate synuclein across the lipid bilayer. Exploring the calcium transport properties of each isoform, we show that VDAC3 has the highest calcium permeability. We demonstrate that VDAC2 can dynamically switch between channel properties of VDAC1 and VDAC3, and explored the structural basis for its unique plasticity using mutational and structural analysis and molecular dynamics simulations. We find that both VDAC2's N-terminal extension and cysteine residues contribute to, but are not sufficient to explain VDAC2's striking behavior. Our results suggest that tuning of beta-barrel and loop flexibility between the isoforms could define their biophysical properties and help to explain distinct physiological roles.

The role of mitochondrial-associated-membranes in Ca-dependent arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological and genetic approaches were employed to disrupt or promote MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, and increased RyR2-VDAC interactions. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. When treated with colchicine to induce a partial disruption of MAMs, arrhythmogenic Ca waves were exacerbated in intact Cnull cells. In permeabilized cells, colchicine also increased the frequency of Ca waves and reduced mitochondrial Ca uptake. Manipulation of MAMs by genetically targeting Mitofusin 2 also affected the frequency of Ca waves. Our results suggest that SR-mitochondria tethering or MAMs shape intracellular Ca signaling to impact arrhythmogenesis. Targeting this signaling microdomain may represent a novel strategy to reduce Ca waves and Ca-dependent arrhythmias.

Role of VDAC isoforms in bioenergetics and metabolism.

Voltage-dependent anion channel (VDAC) is a beta-barrel channel on the mitochondrial outer membrane (MOM) known to transport water-soluble metabolites such as ATP, ADP, and pyruvate. It is central to the regulation of cellular metabolism and serves as a cellular hub given its location at the interface between cytosol and mitochondria. There are three mammalian isoforms of VDAC: VDAC1, 2, and 3. They show high sequence similarity and form similar highly conductive voltage-gated channels in vitro. However, cellular and mice knockout (KO) studies of VDAC isoforms suggest a unique physiological role for each isoform. While VDAC1 KO only leads to mild bioenergetic defects, VDAC3 KO results in male infertility and VDAC2 KO causes developmental impairment in mice. To investigate the role of VDAC isoforms in cellular function, we generated HeLa cells with CRISPR-Cas9 KO of each VDAC isoform. We found isoform-specific differences in mitochondria respiration such as basal and maximal respiration rates for the different isoform KO cells using the Seahorse Metabolic Flux Analyzer. VDAC1 and VDAC3 KO cells showed a significant loss in respiration compared to WT while there was no significant difference detected for VDAC2 KO cells. Interestingly, only VDAC3 KO resulted in complete loss of spare respiratory capacity which could be attributed to defects in the fuel uptake pathway as the VDAC3 KO cells show increased glutamine dependence and loss of flexibility to utilize other metabolites compared to WT HeLa cells. We also detected isoform-dependent differences in calcium signal in mitochondrial intermembrane space (IMS) using IMS targeted calcium sensor (IMS-RGeco1). These results suggest different metabolite and calcium transport functionality for the VDAC isoforms which may contribute to their distinct physiological functions in vivo through isoform-specific regulation of metabolism and calcium signaling.

Small molecules targeting the NADH-binding pocket of VDAC enhance T cell cytotoxicity and selectively inhibit cancer cell proliferation.

Adoptive cell therapy involves administration of cytotoxic immune cells. Upon activation of naïve T cells, CD8+ (cytotoxic) T cells increase both mitochondrial metabolism and glycolysis. Upregulated mitochondrial metabolism is essential for the activation of effector T cells. Voltage dependent anion channels (VDAC 1/2/3) regulate the flux of most metabolites entering and exiting mitochondria. VDAC opening increases mitochondrial metabolism. NADH binding to a VDAC pocket, conserved in all isoforms, closes the channel. The small molecules SC01 and SC18 bind to the VDAC-NADH binding pocket. We hypothesize that SC01 and SC18 stabilize VDAC in an open state enhancing T cell oxidative metabolism, survival and cytotoxicity. We used melanoma epitope gp100 reactive mouse splenic CD8+ T cells, HepG2 and SNU-449 human hepatocarcinoma cancer cells, and B16 mouse melanoma cells. We determined T cell activation (CD25 expression), proliferation (Ki67), cytokine secretion (IFNγ, TNFα), cytolytic activity (granzyme-B), and expression of memory markers CD44 and CD62L, by FACS. We assessed mitochondrial membrane potential (ΔΨm) and NADH by confocal microscopy. Cell proliferation and cell death were determined by cell count and propidium iodide uptake, respectively. VDAC isoform distribution in T cells was ∼54%, 24% and 22% for VDAC 1, 2 and 3, respectively. SC18 moderately decreased T cell ΔΨm and NADH. SC01 and SC18 did not affect proliferation or activation of splenic CD8+ T cells exposed to cognate antigen (gp100). By contrast, SC18 inhibited cell proliferation in SNU-449, HepG2, and B16 cells. SC18 also increased the secretion of IFNγ, TNF-α, and granzyme-B. Memory markers determined by CD44 and CD62L co-expression were unchanged. These data suggest a two-sided antitumor property of SC18, that enhances T cell cytotoxicity and selectively inhibits cancer cell proliferation.

Abstract TMP109: Labeling And Isolating Cell Specific Neuronal Mitochondria And Their Functional Analysis In Mice Post Stroke

Introduction: In vivo neuronal mitochondria dysfunction post stroke has not been well investigated. Methods: Using Mito-tag mice in combine with local infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by neuronal promoters, we generated reporter mice expressing a hemagglutinin (HA)-tagged EGFP that localizes to mitochondrial outer membranes in synaptophysin (SYN) and calmodulin-kinase II (CaMKII) glutamatergic neurons localized to cerebral cortex. These mice were subjected to permanent middle cerebral artery occlusion and sacrificed 14 days post stroke. Fresh brain tissues were excised from the ischemic core (IC), ischemic boundary zone (IBZ) as well as from the homologous contralateral hemisphere, and the crude mitochondrial fractions (CMF) were extracted. Neuronal mitochondria were then isolated from the CMF by anti-HA magnetic beads (Thermo Scientific) and their specificity and oxidative phosphorylation (OXPHOS) complexes were confirmed by means of Western-blot. Seahorse assay was employed to measure oxygen consumption rate (OCR) of the isolated mitochondria as an indicator of mitochondrial function. Results: The bead pulled components were co-precipitated with GFP and enriched with mitochondrial markers, e.g. voltage-dependent anion channel, cytochrome C, and COX IV, but lacked the Golgi protein RCAS1 as well as endoplasmic reticulum markers: Heme-oxygenase 1 and Calnexin, indicating that specific neuronal mitochondria have been selectively isolated. Western-blot data showed that OXPHOS components in SYN + and CAMKII + neuronal mitochondria were significantly downregulated in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function measured by OCR (p<0.05, respectively, for both neuron types). These data suggesting dysfunction of neuronal mitochondria post stroke. Conclusions: We, for the first time, measure mitochondrial function in 2 types of neurons post stroke. By introducing specific neuronal promoter controlled Cre recombinase to Mito-tag mouse, mitochondria from individual neuronal linage cells can be efficiently labeled and isolated to investigate functional change post stroke.

Causes and consequences of endogenous hypoxia on growth and metabolism of developing maize kernels.

Maize (Zea mays) kernels are the largest cereal grains, and their endosperm is severely oxygen deficient during grain fill. The causes, dynamics, and mechanisms of acclimation to hypoxia are minimally understood. Here, we demonstrate that hypoxia develops in the small, growing endosperm, but not the nucellus, and becomes the standard state, regardless of diverse structural and genetic perturbations in modern maize (B73, popcorn, sweet corn), mutants (sweet4c, glossy6, waxy), and non-domesticated wild relatives (teosintes and Tripsacum species). We also uncovered an interconnected void space at the chalazal pericarp, providing superior oxygen supply to the placental tissues and basal endosperm transfer layer. Modeling indicated a very high diffusion resistance inside the endosperm, which, together with internal oxygen consumption, could generate steep oxygen gradients at the endosperm surface. Manipulation of oxygen supply induced reciprocal shifts in gene expression implicated in controlling mitochondrial functions (23.6 kDa Heat-Shock Protein, Voltage-Dependent Anion Channel 2) and multiple signaling pathways (core hypoxia genes, cyclic nucleotide metabolism, ethylene synthesis). Metabolite profiling revealed oxygen-dependent shifts in mitochondrial pathways, ascorbate metabolism, starch synthesis, and auxin degradation. Long-term elevated oxygen supply enhanced the rate of kernel development. Altogether, evidence here supports a mechanistic framework for the establishment of and acclimation to hypoxia in the maize endosperm.

Inhibition of mitochondrial VDAC1 oligomerization alleviates apoptosis and necroptosis of retinal neurons following OGD/R injury

Ischemia-reperfusion (I/R) injury is a common pathological mechanism in many retinal diseases, which can lead to cell death via mitochondrial dysfunction. Voltage-dependent anion channel 1 (VDAC1), which is mainly located in the outer mitochondrial membrane, is the gatekeeper of mitochondria. The permeability of mitochondrial membrane can be regulated by controlling the oligomerization of VDAC1. However, the functional mechanism of VDAC1 in retinal I/R injury was unclear. Our results demonstrate that oxygen-glucose deprivation and re-oxygenation (OGD/R) injury leads to apoptosis, necroptosis, and mitochondrial dysfunction of R28 cells. The OGD/R injury increases the levels of VDAC1 oligomerization. Inhibition of VDAC1 oligomerization by VBIT-12 rescued mitochondrial dysfunction by OGD/R and also reduced apoptosis/necroptosis of R28 cells. In vivo, the use of VBIT-12 significantly reduced aHIOP-induced neuronal death (apoptosis/necroptosis) in the rat retina. Our findings indicate that VDAC1 oligomers may open and enlarge mitochondrial membrane pores during OGD/R injury, leading to the release of death-related factors in mitochondria, resulting in apoptosis and necroptosis. This study provides a potential therapeutic strategy against ocular diseases caused by I/R injury.

Chaihu Shugan San promotes gastric motility in rats with functional dyspepsia by regulating Drp-1-mediated ICC mitophagy

Context Chaihu Shugan San (CHSGS) was effective in the treatment of functional dyspepsia (FD). Objective To investigate the mechanism of CHSGS in FD through dynamin-related protein 1 (Drp-1)-mediated interstitial cells of cajal (ICC) mitophagy. Materials and methods Forty Sprague-Dawley (SD) rats were randomly divided into control, model, mdivi-1, mdivi-1 + CHSGS and CHSGS groups. Tail-clamping stimulation was used to establish the FD model. Mdivi-1 + CHSGS and CHSGS groups were given CHSGS aqueous solution (4.8 g/kg) by gavage twice a day. Mdivi-1 (25 mg/kg) was injected intraperitoneally once every other week for 4 w. Mitochondrial damage was observed by corresponding kits and related protein expressions were assessed by Immunofluorescence and (or) Western Blot. Results Compared with the mean value of the control group, superoxide dismutase (SOD) and citrate synthase (CS) in the model group were decreased by 11% and 35%; malondialdehyde (MDA) and reactive oxygen species (ROS) were increased by 1.2- and 2.8-times; ckit fluorescence and protein expressions were decreased by 85% and 51%, co-localization expression of LC3 and voltage dependent anion channel 1 (VDAC1), Drp-1 and translocase of the outer mitochondrial membrane 20 (Tom20) were increased by 10.1- and 5.4-times; protein expressions of Drp-1, Beclin-1, and LC3 were increased by 0.5-, 1.4-, and 2.5-times whereas p62 was decreased by 43%. After mdivi-1 and (or) CHSGS intervention, the above situation has been improved. Discussion and conclusion CHSGS could improve mitochondrial damage and promote gastric motility in FD rats by regulating Drp-1-mediated ICC mitophagy.

The ferroptosis in haemocytes of Pacific oyster Crassostrea gigas upon erastin treatment

Ferroptosis is an iron and oxidative dependent form of cell death usually mediated by redox related molecules in vertebrates. In the present study, a glutathione peroxidase 4 (GPX4) and a solute carrier family 7 member 11 (SLC7A11, xCT) homologues were identified from the oyster Crassostrea gigas (designed as CgGPX4 and CgxCT), which contained a GSHPx domain and an AA_permease domain, respectively. The mRNA transcripts of CgGPX4 and CgxCT were expressed in all the examined tissues, including gill, gonad, adductor muscle, labial palp, mantle, hepatopancreas and haemocytes, with the highest expression in haemocytes. After erastin treatment, the rate of cell malformation and cell death increased significantly in haemocytes, and the mitochondrial atrophy, crest loss and fracture were observed in haemocytes. While the amount of Fe2+ and Malondialdehyde (MDA) increased significantly, the mRNA expressions of CgGPX4, CgxCT and voltage-dependent anion channel 2 (CgVDAC2) in haemocytes decreased significantly after erastin treatment. These results indicated that erastin was able to induce the ferroptosis of oyster haemocytes.

Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.