Nuclear Translocation Of AIF Research Articles

Neuroprotective Effects of Ethanol Extract Polyscias guilfoylei (EEPG) Against Glutamate Induced Neurotoxicity in HT22 Cells.

Oxidative stress induced by glutamate is a significant contributor to neuronal cell damage and can lead to neurodegenerative diseases such as Alzheimer's, Huntington's, and ischemic brain injury. At the cellular level, oxidative stress increases Ca2+ ion influx and reactive oxygen species (ROS), which activate the MAPK signaling pathway. Additionally, the generation of ROS causes mitochondrial dysfunction, triggering apoptosis by promoting the translocation of AIF to the nucleus from the mitochondria. The neuroprotective potential of Polyscias guilfoylei has not yet been reported. Therefore, in this study, the ethanol extract of Polyscias guilfoylei (EEPG) was examined for its protective effect against oxidative cell damage caused by glutamate in neuronal cells. EEPG treatment increased the viability of HT22 cells exposed to high concentrations of glutamate. Cellular Ca2+ ion influx and ROS generation decreased with EEPG treatment in glutamate-treated HT22 cells. EEPG treatment inhibited MAPK activation and AIF nuclear translocation. In an in vivo study, EEPG attenuated brain cell death in an ischemic brain injury rat model. This study demonstrates the potential therapeutic effects of Polyscias guilfoylei in the treatment of ischemic brain injury.

Piceid Octanoate Protects Retinal Cells against Oxidative Damage by Regulating the Sirtuin 1/Poly-ADP-Ribose Polymerase 1 Axis In Vitro and in rd10 Mice.

Retinitis pigmentosa is a common cause of inherited blindness in adults, which in many cases is associated with an increase in the formation of reactive oxygen species (ROS) that induces DNA damage, triggering Poly-ADP-Ribose Polymerase 1 (PARP1) activation and leading to parthanatos-mediated cell death. Previous studies have shown that resveratrol (RSV) is a promising molecule that can mitigate PARP1 overactivity, but its low bioavailability is a limitation for medical use. This study examined the impact of a synthesized new acylated RSV prodrug, piceid octanoate (PIC-OCT), in the 661W cell line against H2O2 oxidative stress and in rd10 mice. PIC-OCT possesses a better ADME profile than RSV. In response to H2O2, 661W cells pretreated with PIC-OCT preserved cell viability in more than 38% of cells by significantly promoting SIRT1 nuclear translocation, preserving NAD+/NADH ratio, and suppressing intracellular ROS formation. These effects result from expressing antioxidant genes, maintaining mitochondrial function, reducing PARP1 nuclear expression, and preventing AIF nuclear translocation. In rd10 mice, PIC-OCT inhibited PAR-polymer formation, increased SIRT1 expression, significantly reduced TUNEL-positive cells in the retinal outer nuclear layer, preserved ERGs, and enhanced light chamber activity (all p values < 0.05). Our findings corroborate that PIC-OCT protects photoreceptors by modulating the SIRT1/PARP1 axis in models of retinal degeneration.

α-Viniferin-Induced Apoptosis through Downregulation of SIRT1 in Non-Small Cell Lung Cancer Cells.

α-Viniferin, a natural stilbene compound found in plants and a polymer of resveratrol, had demonstrated potential anti-cancer and anti-inflammatory effects. However, the specific mechanisms underlying its anti-cancer activity were not yet fully understood and required further investigation. This study evaluated the effectiveness of α-viniferin and ε-viniferin using MTT assay. Results showed that α-viniferin was more effective than ε-viniferin in reducing the viability of NCI-H460 cells, a type of non-small cell lung cancer. Annexin V/7AAD assay results provided further evidence that the decrease in cell viability observed in response to α-viniferin treatment was due to the induction of apoptosis in NCI-H460 cells. The present findings indicated that treatment with α-viniferin could stimulate apoptosis in cells by cleaving caspase 3 and PARP. Moreover, the treatment reduced the expression of SIRT1, vimentin, and phosphorylated AKT, and also induced AIF nuclear translocation. Furthermore, this research provided additional evidence for the effectiveness of α-viniferin as an anti-tumor agent in nude mice with NCI-H460 cell xenografts. As demonstrated by the TUNEL assay results, α-viniferin promoted apoptosis in NCI-H460 cells in nude mice.

Amentoflavone induces caspase-dependent/-independent apoptosis and dysregulates cyclin-dependent kinase-mediated cell cycle in colorectal cancer in vitro and in vivo.

Colorectal cancer (CRC) is recognized as the third most common malignancy and the second most deadly in highly developed countries. Although the treatment of CRC has improved in the past decade, the mortality rate of CRC is still increasing. Amentoflavone, one of the flavonoids detected in medical plants, is reported to possess potential anticancer properties in various cancers. However, its role in CRC has not been studied. This study aimed to investigate the role and underlying mechanism of amentoflavone on CRC in vitro and in vivo. We identified the cytotoxicity, apoptosis effect, cell cycle alteration, DNA damage induction and tumor progression inhibition of amentoflavone in HT-29 model by using MTT assay, flow cytometry, immunofluorescence (IF) staining, Western blotting and animal experiments. Amentoflavone induced cytotoxicity is caused by triggering G1 arrest, DNA damage and apoptosis in HT-29 cells. The expression of cyclin D1, CDK4 and CDK6 was decreased by amentoflavone; in contrast, the phosphorylation of ATM and CHK2 and the expression of p21 and p27 were increased. The apoptosis induction of amentoflavone in CRC is not only caspase-dependent but also increases EndoG and AIF nuclear translocation in a caspase-independent manner. Importantly, the apoptosis induction of amentoflavone is not affected by the activity of p53 in CRC. Amentoflavone suppressed the progression of CRC by initiating G1 arrest and ATM/CHK2-mediated DNA damage-responsive, caspase-dependent/independent apoptotic effects. We uncovered a novel tumor-inhibitory role of amentoflavone in CRC that is not associated with p53 activity, which may serve as a potential treatment for CRC.

Resveratrol Preconditioning Downregulates PARP1 Protein to Alleviate PARP1-Mediated Cell Death Following Cerebral Ischemia.

Stroke remains a leading cause of mortality; however, available therapeutics are limited. The study of ischemic tolerance, in paradigms such as resveratrol preconditioning (RPC), provides promise for the development of novel prophylactic therapies. The heavily oxidative environment following stroke promotes poly-ADP-ribose polymerase 1 (PARP1)-overactivation and parthanatos, both of which are major contributors to neuronal injury. In this study, we tested the hypothesis that RPC instills ischemic tolerance through decreasing PARP1 overexpression and parthanatos following in vitro and in vivo cerebral ischemia. To test this hypothesis, we utilized rat primary neuronal cultures (PNCs) and middle cerebral artery occlusion (MCAO) in the rat as in vitro and in vivo models, respectively. RPC was administered 2days preceding ischemic insults. RPC protected PNCs against oxygen and glucose deprivation (OGD)-induced neuronal loss, as well as increases in total PARP1 protein, implying protection against PARP1-overactivation. Twelve hours following OGD, we observed reductions in NAD+/NADH as well as an increase in AIF nuclear translocation, but RPC ameliorated NAD+/NADH loss and blocked AIF nuclear translocation. MCAO in the rat induced AIF nuclear translocation in the ischemic penumbra after 24h, which was ameliorated with RPC. We tested the hypothesis that RPC's neuroprotection was instilled through long-term downregulation of nuclear PARP1 protein. RPC downregulated nuclear PARP1 protein for at least 6days in PNCs, likely contributing to RPC's ischemic tolerance. This study describes a novel mechanism by which RPC instills prophylaxis against ischemia-induced PARP1 overexpression and parthanatos, through a long-term reduction of nuclear PARP1 protein.

Cyclophilin A contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis

Shikonin induces glioma cell death via necroptosis, a caspase-independent programmed cell death pathway that is chiefly regulated by receptor-interacting serine/threonine protein kinase1 (RIP1) and 3 (RIP3). Chromatinolysis is considered as one of the key events leading to cell death during necroptosis. It is usually accompanied with nuclear translocation of AIF and formation of γ-H2AX. Cyclophilin A (CypA) is reported to participate in the nuclear translocation of AIF during apoptosis. However, it remains unclear whether CypA contributes to necroptosis and regulation of chromatinolysis. In this study, our results revealed for the first time that shikonin promoted time-dependent CypA activation, which contributed to nuclear translocation of AIF and γ-H2AX formation. In vitro studies showed that knockdown of CypA by siRNA or inhibition of CypA by its specific inhibitor, cyclosporine A (CsA), not only significantly mitigated shikonin-induced glioma cell death, but also prevented chromatinolysis. Mechanistically, activated CypA targeted mitochondria and triggered mitochondrial superoxide overproduction, which then promoted AIF translocation from mitochondria into the nucleus by depolarizing the mitochondria and intensified the formation of γ-H2AX by promoting intracellular accumulation of ROS. Additionally, the CypA in the nucleus can form DNA degradation complexes with AIF and γ-H2AX, which also promote the execution of chromatinolysis. Thus, we demonstrate that CypA contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis.

Regulation of mitochondrial fusion and mitophagy by intra-tumoral delivery of membrane-fused mitochondria or Midiv-1 enhances sensitivity to doxorubicin in triple-negative breast cancer

Increasing mitochondrial fusion by intra-tumoral grafting of membrane-fused mitochondria created with Pep-1 conjugation (P-Mito) contributes to breast cancer treatment, but it needs to be validated. Using mitochondrial division inhibitor-1 (Mdivi-1, Mdi) to disturb mitochondrial dynamics, we showed that the antitumor action of P-Mito in a mouse model of triple-negative breast cancer depends upon mitochondrial fusion and that Mdi treatment alone is ineffective. P-Mito significantly enhanced Doxorubicin (Dox) sensitivity by inducing mitochondrial fusion and mitophagy, and the same efficiency was also achieved with Mdi by inhibiting mitophagy. Cell death was induced via the p53 pathway and AIF nuclear translocation in the case of P-Mito, versus the caspase-dependent pathway for Mdi. Notably, both mitochondrial treatments reduced oxidative stress and blood vessel density of xenograft tumors, especially P-Mito, which was accompanied by inhibition of nuclear factor kappa-B activation. Furthermore, through enrichment analysis, four microRNAs in serum microvesicles induced by P-Mito caused expression of predicted targets via the PI3K-Akt pathway, and significantly impacted regulation of nuclear processes and myeloid cell differentiation. Clustering of gene-sets implicated a major steroid catabolic network. This study showed diverse roles of mitochondria in breast cancer and revealed effective adjuvant therapy targeting mitochondrial fusion and mitophagy.

Baicalein-ameliorated cerebral ischemia-reperfusion injury dependent on calpain 1/AIF pathway.

Cerebral ischemia reperfusion (CIR) has become the leading cause of death and disability. Baicalein is a natural bioactive ingredient extracted from Scutellaria baicalensis Georgi and has neuroprotective activity. In our work, baicalein was found to reduce neurological deficits, brain water content, infarct area, and neuronal death of rats induced by middle cerebral artery occlusion/reperfusion. In vitro, oxygen-glucose deprivation/reperfusion induced inordinate ROS production and apoptosis that could be reversed by baicalein. Our study revealed for the first time that baicalein has the potential to bind and inhibit the activity of calpain 1, thereby inhibiting AIF nuclear translocation. These findings demonstrated that baicalein protected against CIR injury via inhibiting AIF nuclear translocation by inhibiting calpain 1 activity.

Cannabinoids Induce Cell Death in Leukemic Cells through Parthanatos and PARP-Related Metabolic Disruptions

BACKGROUNDWe have previously described the antitumor effect of the cannabinoid WIN-55,212-2 (WIN-55) and a set of cannabinoid derivatives (CNB) specific for CB2 in multiple myeloma (Barbado et al, 2018). In AML, we also observed a potent and selective antileukemic effect, affecting signaling and metabolic pathways essential for the viability of tumor cells. Among them, we found an increased stress on the endoplasmic reticulum, mitochondrial damage, and alteration of the metabolism of ceramides, although none of these events turned out to be the main trigger of cell death, since the inhibition of each of them did not prevent the antileukemic effect of CNB.On the other hand, disruption of the mecanisms of DNA repair have been identified as a key oncogeneic event in different solid tumors, and some studies have also suggested that it might be involved in leukemogenesis. More specifically, PARP1 is involved in DNA damage repair. Other functions include the regulation of glycolysis enzymes through the addition of Poly ADP-Ribose (PAR) and the execution of Parthanatos, that occurs whenever PARP-1 becomes over-activated in response to extreme damage inducing nuclear translocation of AIF and depletion of NAD +. OBJECTIVESIn this study we set out to identify the ultimate mechanism that justifies the aforementioned pleiotropic effect of CNB on the metabolism of leukemic cells and their viability. METHODSCell viability was determined by MTT and flow cytometry. The mRNA and / or protein expression profile of AML samples or healthy progenitor cells were studied by qPCR and / or Western blot. Glycolytic flux was studied with the XF Glycolytic Rate Assay (Seahorse Biosciences). NAD + levels and glycolytic enzyme activity were measured using quantification kits. Parylation of different enzymes were confirmed by Co-IP using the corresponding antibodies.Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60-Luciferase cell line. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. Also we tested the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. RESULTSPretreatment of leukemic cells with Olaparib, a PARP1 inhibitor, reversed WIN-55 induced apoptosis by almost 100%. WIN-55 affected the activity of most glycolysis enzymes, with a marked drop of the activity of GAPDH and pyruvate kinase which was reversed by Olaparib pretreatment. Also G6PDH activity was markedly affected upon culture with WIN-55. Co-IP confirmed parylation of these enzymes which was reversed with Olaparib.ECAR data detected by Seahorse also confirmed the drop in glycolytic capacity produced by WIN-55 in leukemic cells which again was reversed upon culture with Olaparib. In addition, the addition of nicotinamide mononucleotide (NAM), a precursor of NAD +, reversed the loss of viability produced by WIN-55. Next, we confirmed that PARP1 levels were significantly higher in leukemic cell lines and in a series of 40 AML patients as compared to healthy hematopoietic stem cells (HSC).Finally, we observed a translocation of AIF to the nucleus, confirming that WIN-55 produces PARTHANATOS.In a murine model we confirmed treatment with WIN-55,212-2 significantly prolonged survival in AML xenograft mice, with disappearance of the leukemic clone in a significant proportion of cases. By contrast, cannabinoids did not affect the viability of hematopoietic stem cells (HSC) in vivo, resulting in a lack of myeloid toxicity in healthy treated mice. CONCLUSIONSWIN-55 exerts a selective antileukemic effect through the overactivation of PARP1, affecting the levels of parylation in enzymes involved in glycolysis and pentose phosphate pathways, leading to the translocation of AIF to the nucleus and to depletion of NAD +, which were reversed through PARP1 inhibition. These effects are not observed in normal HSC. These data are confirmed in murine models in which we confirmed the antileukemic effect of WIN-55 withouth hampering normal hematopoiesis. [Display omitted] DisclosuresPérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

14,15-EET Reduced Brain Injury from Cerebral Ischemia and Reperfusion via Suppressing Neuronal Parthanatos

To investigate the effect of 14,15-EET on the parthanatos in neurons induced by cerebral ischemia and reperfusion, middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen glucose deprivation/reoxygenation (OGD/R) were used to simulate cerebral ischemia reperfusion in vivo and in vitro, respectively. TTC staining and the Tunel method were used to detect cerebral infarct volume and neuronal apoptosis. Western blot and immunofluorescence were used to detect poly (ADP-ribose) polymerase-1 (PARP-1) activation and AIF nuclear translocation. The production of reactive oxygen species (ROS) and the expression of antioxidant genes were detected by Mito SOX, DCFH-DA and qPCR methods. MCAO/R increased cerebral infarct volume and neuronal apoptosis in mice, while 14,15-EET pretreatment increased cerebral infarct volume and neuronal apoptosis. OGD/R induced reactive oxygen species generation, PARP-1 cleavage, and AIF nuclear translocation in cortical neurons. 14,15-EET pretreatment could enhance the antioxidant gene expression of glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) in cortical neurons after ischemia and reperfusion. 14,15-EET inhibits the neuronal parthanatos induced by MCAO/R through upregulation of the expression of antioxidant genes and by reducing the generation of reactive oxygen species. This study advances the EET neuroprotection theory and provides a scientific basis for targeted clinical drugs that reduce neuronal parthanatos following cerebral ischemia and reperfusion.

Retraction Note to: Differential modulatory effects of GSK-3b and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis) in melanoma

GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis. Our data demonstrate a complex partnership between GSK-3β and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.

Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

Oxaliplatin induces the PARP1-mediated parthanatos in oral squamous cell carcinoma by increasing production of ROS.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors worldwide, and its prognosis is still not optimistic. Oxaliplatin is a type of platinum chemotherapeutic agent, but its treatment effects on OSCC and molecular mechanisms have not been fully elucidated. Parthanatos, a unique form of cell death, plays an important role in a variety of physiological and pathological processes. This study aims to investigate whether oxaliplatin inhibits OSCC by inducing parthanatos. Our results showed that oxaliplatin inhibited the proliferation and migration of OSCC cells in vitro, and also inhibited the tumorigenesis in vivo. Further experiments proved that oxaliplatin induced parthanatos in OSCC cells, characterized by depolarization of the mitochondrial membrane potential, up-regulation of PARP1, AIF and MIF in the nucleus, as well as the nuclear translocation of AIF. Meanwhile, PARP1 inhibitor rucaparib and siRNA against PARP1 attenuated oxaliplatin-induced parthanatos in OSCC cells. In addition, we found that oxaliplatin caused oxidative stress in OSCC cells, and antioxidant NAC not only relieved oxaliplatin-induced overproduction of reactive oxygen species (ROS) but also reversed parthanatos caused by oxaliplatin. In conclusion, our results indicate that oxaliplatin inhibits OSCC by activating PARP1-mediated parthanatos through increasing the production of ROS.

Combined treatment with auranofin and trametinib induces synergistic apoptosis in breast cancer cells

ABSTRACT Auranofin is a gold complex used as an anti-rheumatic agent and may act as a potent anticancer drug against breast tumors. Trametinib is a specific mitogen-activated protein kinase inhibitor, approved for the treatment of metastatic melanoma. The aim of this study was to examine the synergistic effects of auranofin and trametinib on apoptosis in MCF-7 human breast cancer cells. The combination treatment inhibited cancer cell proliferation and induced cell cycle arrest at the sub-G1 phase and apoptosis via poly (ADP-ribose) polymerase cleavage and caspase-3/7 activation. It is noteworthy that this treatment significantly increased p38 mitogen-activated protein kinase (MAPK) phosphorylation to induce mitochondrial stress, subsequently promoting cancer cell apoptosis through release of apoptosis-inducing factor. Further data demonstrated that combined treatment significantly induced increase in nuclear translocation of AIF. These results indicated that activation of the p38 MAPK signaling pathway and mitochondrial apoptosis may contribute to the synergistic consequences in MCF-7 cells. Collectively, our data demonstrated that combined treatment with auranofin and trametinib exhibited synergistic breast cancer cell death and this combination might be utilized as a novel therapeutic strategy for breast cancer.

NPD1 rapidly targets mitochondria-mediated apoptosis after acute injection protecting brain against ischemic injury

Mitochondria-related cell death pathways play a major role in ischemic brain injury. Thus, mitochondrial “protective” molecules could be considered for new therapeutic regimens. We recently reported that acute administration of docosahexaenoic acid (DHA) triglyceride lipid emulsion, immediately after hypoxic-ischemic (HI) insult, markedly attenuated brain infarct size. This was associated with an early change of DHA-derived specialized pro-resolving mediator (SPM) profiles. Specifically, DHA treatment induced a 50% increase of neuroprotectin D1 (NPD1) levels in ischemic brain. Based on these findings, we questioned if direct administration of NPD1 after HI injury also affords neuroprotection, and if so, by what mechanisms.Using HI insult to mimic ischemic stroke in neonatal mice, we observed that acute intraperitoneal injection of NPD1 immediately after HI injury prevented the expansion of the ischemic core by ~40% and improved coordination and motor abilities compared to the control group. At 7 days after HI injury, NPD1 treatment decreased ipsilateral hemisphere atrophy and preserved motor functions in wire-holding and bridge-crossing tests compared to control littermates. Brain mitochondria, isolated at 4 h after reperfusion from mice treated with NPD1, showed an increase in the capacity to buffer calcium after HI injury, as result of the preservation of mitochondrial membranes. Further, NPD1 induced a reduction of mitochondrial BAX translocation and oligomerization, attenuated cytochrome C release and decreased AIF nuclear translocation. To confirm whether NPD1 acts as BAX inhibitor, we evaluated NPD1 action co-administrated with a pro-apoptotic agent, staurosporine, using mouse embryonic fibroblasts as in vitro model of apoptosis. NPD1 exposure markedly decreased mitochondria-mediated apoptosis, blocking BAX translocation from cytosol to mitochondria and subsequently reducing caspase-3 activation. Our findings provide novel evidence that the neuroprotective action of NPD1 is elicited rapidly in the first few hours after ischemic injury and is associated with both preserved mitochondrial membrane structure and reduced BAX mitochondrial translocation and activation.

Neuroprotective effects of Dendropanax morbifera leaves on glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells

Ethnopharmacological relevanceDendropanax morbifera (DM) has long been used as a traditional herbal medicine for migraines. Glutamate toxicity and oxidative stress have emerged as the possible triggers implicated in migraine pathogenesis. Aim of the studyWe aimed to examine the neuroprotective effects of DM leaves (DML) on glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells. Materials and methodsMolecular authentication of DML was assessed using DNA barcoding analysis. Four different solvent extracts of DML were prepared and subjected to antioxidant activity and phytochemical assays. Neuroprotective effects of DML extracts were evaluated using relevant biochemical and imaging assays that measure cell viability/death, ROS generation, Ca2+ levels, mitochondrial dysfunction, and AIF nuclear translocation. ResultsThe sequences of matK, rbcL, atpF-H, and psbK-I in DML were identical with those in voucher specimens, confirming that DML was indeed D. morbifera. The ethyl acetate extract of DML (DMLE) showed the highest flavonoid and phenolic content, and prominent DPPH/superoxide radical scavenging and reducing power activities. In the HT22 cell model, glutamate was shown to be the causative agent for apoptotic cell death via elevation of intracellular ROS and Ca2+ levels, induction of mitochondrial depolarization and membrane permeabilization, and translocation of AIF to the nucleus. Of note, N-acetyl-L-cysteine and necrostatin-1, but not z-VAD-fmk, completely prevented glutamate-induced cell death, implying that oxidative stress and AIF translocation were pivotal in glutamate cytotoxicity. DMLE significantly recovered glutamate-induced apoptotic cell death in a concentration-dependent manner. It completely inhibited intracellular/mitochondrial ROS generation, the elevation of Ca2+ levels, and mitochondrial dysfunction induced by glutamate during early exposure within 8 h. It significantly reversed subsequent AIF nuclear translocation after 12 h of treatment. Antioxidant activities of DMLE may be the protective mechanism that regulates homeostatic balance of ROS and Ca2+ as well as maintains mitochondrial function. ConclusionsDMLE shows significant neuroprotective effects against glutamate-induced oxidative neuronal cell death. Therefore, DM could be a potential therapeutic candidate for neurological disorders propagated by glutamate toxicity.

MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis.

Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

The effect of butylphthalide on cell necroptosis in cerebral ischemia reperfusion injury model mice

Objective To explore the protective effect and mechanism of butylphthalide on cerebral ischemia reperfusion injury in mice. Methods The cerebral ischemia reperfusion injury model was established by middle cerebral ischemia occlusion(MCAO). Thirty mice were divided into sham group, ischemia reperfusion group(I/R group) and butylphthalide group(NBP group) with 10 in each group.Neurological defect score and brain infarction volume were detected by TTC to evaluate the treatment effects of butylphthalide.Western blot was used to detect expression of RIP, RIP3 and AIF.Immunocoprecipitation(IP) was used to detect the interaction of AIF and RIP3.Immunofluorescence(IF) was used to detect the nuclear translocation and co-localization of AIF and RIP3. Results Compared with the I/R group, NBP treatment reduced the neurological defect score(I/R: (2.60±0.22), NBP: (1.90±0.23), t=2.18, P<0.05) and brain infarction volume(I/R: (38.32±2.22)%, NBP: (25.23±2.70)%, t=3.74, P<0.01) .I/R elevated the expression of RIP1 and RIP3 whereas NBP significantly inhibited the expression of these two proteins (RIP1(I/R: 0.99±0.24, NBP: 0.47±0.10, t=2.71, P<0.05); RIP3(I/R: 0.52±0.17, NBP: 0.15±0.04, t=2.87, P<0.05)). I/R and NBP had no significant effects on the expression of AIF, but the IP results showed that I/R increased the interaction between AIF and RIP3 compared with the sham group.NBP alleviated the interaction between AIF and RIP3.IF results showed that the colocalization and nuclear expression of AIF and RIP3 increased after I/R whereas NBP treatment decreased the effect induced by I/R. Conclusion Butylphthalide alleviated cerebral ischemia reperfusion injury in mice through inhibiting the cell necroptosis.The mechanisms may correlate with decreasing the expression of RIP1 and RIP3 and alleviating the interaction and nuclear translocation of AIF and RIP3. Key words: Butylphthalide; Ischemia reperfusion; Necroptosis; Apoptosis inducing factor; Mice

Regulated Necrosis Orchestrates Microglial Cell Death in Manganese-Induced Toxicity

Microglia, the brain resident immune cells, play prominent roles in immune surveillance, tissue repair and neural regeneration. Despite these pro-survival actions, the relevance of these cells in the progression of several neuropathologies has been established. In the context of manganese (Mn) overexposure, it has been proposed that microglial activation contributes to enhance the neurotoxicity. However, the occurrence of a direct cytotoxic effect of Mn on microglial cells remains controversial. In the present work, we investigated the potential vulnerability of immortalized mouse microglial cells (BV-2) toward Mn2+, focusing on the signaling pathways involved in cell death. Evidence obtained showed that Mn2+ induces a decrease in cell viability which is associated with reactive oxygen species (ROS) generation. In this report we demonstrated, for the first time, that Mn2+ triggers regulated necrosis (RN) in BV-2 cells involving two central mechanisms: parthanatos and lysosomal disruption. The occurrence of parthanatos is supported by several cellular and molecular events: (i) DNA damage; (ii) AIF translocation from mitochondria to the nucleus; (iii) mitochondrial membrane permeabilization; and (iv) PARP1-dependent cell death. On the other hand, Mn2+ induces lysosomal membrane permeabilization (LMP) and cathepsin D (CatD) release into the cytosol supporting the lysosomal disruption. Pre-incubation with CatB and D inhibitors partially prevented the Mn2+-induced cell viability decrease. Altogether these events point to lysosomes as players in the execution of RN. In summary, our results suggest that microglial cells could be direct targets of Mn2+ damage. In this scenario, Mn2+ triggers cell death involving RN pathways.

Bisphenol A exhibits cytotoxic or genotoxic potential via oxidative stress-associated mitochondrial apoptotic pathway in murine macrophages

Bisphenol A (BPA) is primarily used in production of polycarbonate plastics and epoxy resins including plastic containers. BPA is an endocrine disruptor and supposes to induce asthma and cancer. However, so far only a few evidences have shown the BPA-induced toxic effect and its related mechanism in macrophages. BPA demonstrated cytotoxic effect on RAW264.7 macrophages in a concentration and time-dependent manner. BPA induces necrosis, apoptosis, and genotoxicity in a concentration-dependent manner. Phosphorylation of cytochrome C (cyto C) and p53 was due to mitochondrial disruption via BCL2 and BCL-XL downregulation and BAX, BID, and BAD upregulation. Both caspase-dependent, including caspase-9, caspase-3, and PARP-1 cleavage, and caspase-independent, such as nuclear translocation of AIF, pathways were activated by BPA. Furthermore, generation of reactive oxygen species (ROS) and reduction of antioxidative enzyme activities were induced by BPA. Parallel trends were observed in the effect of BPA on cytotoxicity, apoptosis, genotoxicity, p53 phosphorylation, BCL2 family expression exchange, caspase-dependent and independent apoptotic pathways, and ROS generation in RAW264.7 macrophages. Finally, BPA-exhibited cytotoxicity, apoptosis, and genotoxicity could be inhibited by N-acetylcysteine. These results indicated that the toxic effect of BPA was functioning via oxidative stress-associated mitochondrial apoptotic pathway in macrophages.