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Role of apoptosis inducing factor (AIF) for hippocampal neuronal cell death following global cerebral ischemia in mice
Abstract
The molecular mechanisms of neuronal cell death following circulatory arrest are still not fully understood. In the current study we investigated the role of apoptosis-inducing factor (AIF), a major caspase-independent mitochondrial cell death protein, for neuronal cell death following global cerebral ischemia (GCI). C57/Bl6 or low AIF expressing Harlequin mutant mice (AIF(low)) and their wild-type littermates were subjected to 10 min of GCI. DNA damage, nuclear pathology, and localization of AIF were investigated 6, 24, and 72 h after GCI by TUNEL and DAPI staining, and immunohistochemistry, respectively. Cell death of hippocampal CA1 neurons following GCI was associated with nuclear translocation of AIF, nuclear pyknosis, and DNA fragmentation, i.e. ∼80% of all TUNEL-positive neurons had nuclear AIF staining. In AIF(low) mice neuronal cell loss was reduced by 60% (p<0.02). The current experiments suggest that AIF-mediated signaling represents a novel mechanism of neuronal cell death following GCI.
... We found increased number of cleaved caspase-3-positive neurons and higher increase of TUNEL-positive cells in cultures exposed to OGD. In the range of concentrations used, CHF5074 significantly reduced the number of caspase-3-positive cells and, even to a greater extent, the percentage of TUNEL positive neurons, suggesting its efficacy in reversing both caspase-dependent and caspase-independent apoptosis in brain ischemia [25,28]. ...
... Importantly, CHF5074 application was able to normalize all the metabolic parameters investigated, demonstrating that this compound exerts profound neuroprotective effects against ischemic damage. Moreover, by evaluating the cleaved caspase-3 as a marker of caspase-3 activation and TUNEL staining as a marker of caspase-dependent and caspase-independent DNA fragmentation in brain ischemia [28,35], we demonstrated anti-apoptotic activity of CHF5074 in primary hippocampal cells previously exposed to OGD. Significant neuroprotection was achieved by CHF5074 even at the lower concentrations tested, within the concentration range of 1-3 M compatible with the brain level of CHF5074 detected in treated mice [28]. ...
... Moreover, by evaluating the cleaved caspase-3 as a marker of caspase-3 activation and TUNEL staining as a marker of caspase-dependent and caspase-independent DNA fragmentation in brain ischemia [28,35], we demonstrated anti-apoptotic activity of CHF5074 in primary hippocampal cells previously exposed to OGD. Significant neuroprotection was achieved by CHF5074 even at the lower concentrations tested, within the concentration range of 1-3 M compatible with the brain level of CHF5074 detected in treated mice [28]. ...
... PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the 1 3 could produce neuroprotective effects in rodent ischemia models [2][3][4][5]. ...
... # P < 0.05, ## P < 0.01 and ### P < 0.001, versus OGD/R (n = 3) [19,20]. After ischemia-reperfusion, the expression of AIF in the hippocampus was increased along with an increase in neuro-apoptosis, which is contrary to the case in which the AIF gene was knocked out [4]. This study shows that soluble epoxide hydrolase gene deletion suppresses caspase3 activation in cerebral ischemia-reperfusion. Furthermore, cytochrome C release into the cytoplasm and AIF nuclear translocation are also inhibited. ...
Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia-reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia-reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen-glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia-reperfusion by 14,15-EET.
... As previously described, 17 the filament perforation model used in the current study leads to significant neuronal damage in the hippocampus which is thought to be responsible for long-term neurocognitive and behavioral deficits in SAH survivors. Three days after SAH, the animals (n ¼ 8 mice per group) were transcardially perfused with 4% paraformaldehyde under deep chloralhydrate anesthesia. ...
... Pyknotic and viable neurons were then imaged in the CA1, CA2, and CA3 regions of the hippocampus, at 200 Â magnification and quantified using a standard analysis software program (Olympus DP-Soft, analySIS, Olympus, Hamburg, Germany) as described previously. 17 ...
Subarachnoid hemorrhage is a stroke subtype with particularly bad outcome. Recent findings suggest that constrictions of pial arterioles occurring early after hemorrhage may be responsible for cerebral ischemia and – subsequently – unfavorable outcome after subarachnoid hemorrhage. Since we recently hypothesized that the lack of nitric oxide may cause post-hemorrhagic microvasospasms, our aim was to investigate whether inhaled nitric oxide, a treatment paradigm selectively delivering nitric oxide to ischemic microvessels, is able to dilate post-hemorrhagic microvasospasms; thereby improving outcome after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to experimental SAH. Three hours after subarachnoid hemorrhage pial artery spasms were quantified by intravital microscopy, then mice received inhaled nitric oxide or vehicle. For induction of large artery spasms mice received an intracisternal injection of autologous blood. Inhaled nitric oxide significantly reduced number and sever
... In these models AIF translocation from the mitochondria to the nucleus was associated with chromatin condensation and large-scale DNA fragmentation in dying neurons. [6][7][8][9] Inhibition of mitochondrial AIF release to the nucleus or reduction of AIF expression, such as in Hq mice, is neuroprotective and significantly reduces brain damage in the respective mouse models of acute brain damage. [9][10][11][12] Despite the findings on the crucial role of AIF in mitochondrial pathways of caspase-independent neuronal death, attempts for direct inhibition of AIF to achieve neuroprotective effects have not been reported and pharmacological inhibitors of AIF are not available. ...
... [6][7][8][9] Inhibition of mitochondrial AIF release to the nucleus or reduction of AIF expression, such as in Hq mice, is neuroprotective and significantly reduces brain damage in the respective mouse models of acute brain damage. [9][10][11][12] Despite the findings on the crucial role of AIF in mitochondrial pathways of caspase-independent neuronal death, attempts for direct inhibition of AIF to achieve neuroprotective effects have not been reported and pharmacological inhibitors of AIF are not available. ...
Delayed neuronal cell death largely contributes to the progressive infarct development and associated functional impairments after cerebral ischemia or brain trauma. Previous studies exposed a key role for the interaction of the mitochondrial protein apoptosis-inducing factor (AIF) and cytosolic cyclophilin A (CypA) in pathways of programmed cell death in neurons in vitro and in vivo. These studies suggested that pro-apoptotic activities of AIF, such as its translocation to the nucleus and subsequent DNA degradation, depend on the physical interaction of AIF with CypA. Hence, this protein complex may represent a new pharmacological target for inhibiting the lethal action of AIF on the brain tissue. In this study, we show that the AIF amino-acid residues 370-394 mediate the protein complex formation of AIF with CypA. The synthetic AIF(370-394) peptide inhibited AIF/CypA complex formation in vitro by binding CypA with a KD of 12 μM. Further, the peptide exerted pronounced neuroprotective effects in a model of glutamate-induced oxidative stress in cultured HT-22 cells. In this model system of AIF-dependent cell death, the AIF(370-394) peptide preserved mitochondrial integrity, as detected by measurements of the mitochondrial membrane potential and quantification of mitochondrial fragmentation. Further, the AIF(370-394) peptide inhibited perinuclear accumulation of fragmented mitochondria, mitochondrial release of AIF to the nucleus and glutamate-induced cell death to a similar extent as CypA-siRNA. These data indicate that the targeting of the AIF-CypA axis is an effective strategy of neuroprotection.
... Permeabilization results in the release of mitochondrial apotogenic proteins, triggering the cell death cascade via caspase-or apoptosisinducing factor (AIF)-dependent mechanisms. In particular, AIF-dependent cell death is known to be involved in cerebrovascular and neurodegenerative diseases [42,43]. Nuclear translocation of AIF is a major contributor to Glu-induced oxidative cell death in HT22 cells [6,17,44]. ...
Dendropanax morbifera leaves (DML) have long been used as traditional medicine to treat diverse symptoms in Korea. Ethyl acetate-soluble extracts of DML (DMLE) rescued HT22 mouse hippocampal neuronal cells from glutamate (Glu)-induced oxidative cell death; however, the protective compounds and mechanisms remain unknown. Here, we aimed to identify the neuroprotective ingredients and mechanisms of DMLE in the Glu-HT22 cell model. Five antioxidant compounds were isolated from DMLE and characterized as chlorogenic acid, hyperoside, isoquercitrin, quercetin, and rutin by spectroscopic methods. Isoquercitrin and quercetin significantly inhibited Glu-induced oxidative cell death by restoring intracellular reactive oxygen species (ROS) levels and mitochondrial superoxide generation, Ca2+ dysregulation, mitochondrial dysfunction, and nuclear translocation of apoptosis-inducing factor. These two compounds significantly increased the expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the presence or absence of Glu treatment. Combinatorial treatment of the five compounds based on the equivalent concentrations in DMLE showed that significant protection was found only in the cells cotreated with isoquercitrin and quercetin, both of whom showed prominent synergism, as assessed by drug–drug interaction analysis. These findings suggest that isoquercitrin and quercetin are the active principles representing the protective effects of DMLE, and these effects were mediated by the Nrf2/HO-1 pathway.
... AIF is an ancient mitochondrial flavoprotein implicated in the mitochondrial respiratory chain complex I of healthy cells [3,4]. However, upon apoptotic stimuli such as oxidative stress and DNA damage, AIF in its apoptotic form, known as AIF(Δ1-121), is released from mitochondria and reaches the nuclei, where induces chromatin condensation and cell death by a caspases-independent mechanism [5][6][7]. Microinjection of AIF into caspase-deficient cells induces cell death [8]. Inhibition of either AIF mitochondrial release or nuclear translocation [9,10] or also reduction of its expression levels, such as in Hq mice [11], leads to a significant neuroprotection in a variety of rodent models of acute brain injury induced by cerebral hypoxia/ ischemia (HI), arrest-induced brain damage, epileptic seizures or even brain trauma [7][8][9][10][11]. ...
Background
The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370–394)) provides a significant neuroprotection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical compounds targeting this complex are missing.
Methods
We designed and synthesized a set of mono and bicyclic AIF(370–394) analogs containing both disulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments.
Results
We identified a stapled peptide analogue of AIF(370–394) with a ten-fold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires β-turn/β-fold structures and shares with the parent molecule the same binding region on CypA.
Conclusions
Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic approaches in neurodegenerative diseases.
General significance
Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conformational constraints on the protein−ligand interaction, and their utility for drug design.
... Several studies have demonstrated that HMGB1 can be transferred from the nucleus of the neuron to the cytoplasm or released extracellularly by stimulation such as brain ischemia (Qiu et al., 2008;Zhang et al., 2016;Chen et al., 2019) and subarachnoid hemorrhage (Sun et al., 2014). Under severe cerebral ischemic conditions, neuronal cells undergo apoptosis or necrosis by the induction of a number of pathways (Thal et al., 2011). As a result, HMGB1 could be released from necrotic neurons and interact with microglia as an extracellular cytokine (Frasch and Nygard, 2017). ...
Neonatal hypoxic-ischemic (HI) encephalopathy is a severe disease for which there is currently no curative treatment. Recent evidence suggests that high-mobility group box 1 (HMGB1) protein can promote neuroinflammation after stroke in adult rodents, but its role in perinatal hypoxic-ischemic brain damage (HIBD) remains largely uninvestigated. In the present work, the potential role of HMGB1 in the pathogenesis of HIBD was explored. A HIBD model was established in postpartum day 7 rat pups. HMGB1 expression, the cellular distribution of HMGB1, and microglial activation were all evaluated. Glycyrrhizin (GL), an inhibitor of HMGB1, was used to investigate whether the inhibition of HMGB1 modulated microglial M1/M2 polarization or attenuated brain damage after HI. HAPI microglial cells and primary neurons were cultured in vitro and an oxygen-glucose deprivation model was established to evaluate the effects of different microglial-conditioned media on neurons using GL and recombinant HMGB1. Results showed that the expression of HMGB1 was increased in both the ipsilateral cortex and peripheral blood 72 h after HI. Immunofluorescence analyses showed that HMGB1 in the cortex was primarily expressed in neurons. This increase in cortical HMGB1 expression 72 h after HI was characterized by increased co-expression with microglia, rather than neurons or astrocytes. The expression of both M1 and M2 microglia was upregulated 72 h after HI. The administration of GL significantly suppressed M1 microglial polarization and promoted M2 microglial polarization. Meanwhile, GL pretreatment significantly alleviated brain edema and cerebral infarction. In vitro experimentation showed that HMGB1-induced M1-conditioned media aggravated neuronal damage, but this effect was neutralized by GL. These findings suggest that HMGB1 may result in an imbalance of M1/M2 microglial polarization in the cortex and thus cause neuronal injury. Pharmacological blockade of HMGB1 signaling may attenuate this imbalanced polarization of microglia and thus could be used as a therapeutic strategy against brain injury in HIBD.
... Since AIF could also act as a free radical scavenger, by displaying NADH oxidoreductase and peroxide scavenging activities (80,81), this increased AIF expression in SN neurons of certain cases could be a compensatory mechanism against the increased oxidative stress and free radical production caused by inflammation and/or PHI. High AIF levels could, however, render neurons more vulnerable to death, given that downregulation of AIF significantly reduces brain damage (82)(83)(84). Interestingly, in the adult rat, SN neurons have been reported to undergo death through AIF after inflammation caused by either intranigral or systemic lipopolysaccharide administration (85,86). ...
Our previous postmortem studies on neonates with neuropathological injury of perinatal hypoxia/ischemia (PHI) showed a dramatic reduction of tyrosine hydroxylase expression (dopamine synthesis enzyme) in substantia nigra (SN) neurons, with reduction of their cellular size. In order to investigate if the above observations represent an early stage of SN degeneration, we immunohistochemically studied the expression of cleaved caspase-3 (CCP3), apoptosis inducing factor (AIF), and DNA fragmentation by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin 3′-end-labeling (TUNEL) technique in the SN of 22 autopsied neonates (corrected age ranging from 34 to 46.5 gestational weeks), in relation to the severity/duration of PHI injury, as estimated by neuropathological criteria. No CCP3-immunoreactive neurons and a limited number of apoptotic TUNEL-positive neurons with pyknotic characteristics were found in the SN. Nuclear AIF staining was revealed only in few SN neurons, indicating the presence of early signs of AIF-mediated degeneration. By contrast, motor neurons of the oculomotor nucleus showed higher cytoplasmic AIF expression and nuclear translocation, possibly attributed to the combined effect of developmental processes and increased oxidative stress induced by antemortem and postmortem factors. Our study indicates the activation of AIF, but not CCP3, in the SN and oculomotor nucleus of the human neonate in the developmentally critical perinatal period.
... Thal et al. investigated the role of AIF in C57/Bl6 or low AIF expressing Harlequin mutant mice (AIF low ) and their wild-types. Their report shows that hippocampal CA1 neurons cell death, following GCI, was associated with nuclear translocation of AIF [54]. In our study, the nuclear levels of AIF were reduced by AQTT, which suggest that they also attenuate apoptosis through caspase independent pathway. ...
The present study has been aimed to explore the different secondary messengers of the inflammatory pathway NF-κB, kinases (JNK, P38MAPK, GSK3β/βcatenin), apoptosis pathway (Caspase-3 and AIF), and neuronal survival pathway (BDNF) in order to understand the neuroprotective mechanism of aqueous extract of Tribulus terrestris (AQTT). In primary cortical neurons, the ischemic condition was induced through oxygen–glucose deprivation (OGD). Anti-inflammatory activity of AQTT was evaluated in formalin induced inflammation model and carrageenan-induced paw edema test. The bilateral common carotid artery occlusion model was employed for whole animal studies. Treatment of AQTT (100 mg/kg) significantly reduced the inflammation induced by formalin and carrageenan. The neuroprotective mechanism of AQTT (50 and 100 mg/kg) was assessed by pre- and post-administration. The results indicate down regulation of kinases and NFkB, suggesting possible anti-inflammatory activity of AQTT. Additionally, AQTT down regulated both caspase dependent and independent apoptotic pathways suggesting its possible anti-apoptotic activity. The treatment of AQTT also reduced GSK3β levels and increased p-Ser9 GSK3β levels; stabilizing the unphosphorylated form of β-catenin and its translocation into the nucleus suggesting role of AQTT in neuronal survival and GSK3β mediated anti-inflammatory property. In comparison to pretreatment, post treatment of AQTT had lesser effects indicating tribulusterine standardized AQTT may have prophylactic effect. This study can be concluded with the thesis that AQTT has neuroprotective effect through alternating neuroinflammation, apoptosis, and promoting neuron survival. Being that it produced better effect with pretreatment, exploring this with thrombolytic drugs will be beneficial. For the first time AQTT has been reported for this indication.
... AIF is considered to be a major contributor to neuron loss induced by cerebral ischemia and trauma in both neonates and adults, because these conditions cause the release of AIF from mitochondria and its translocation to nuclei, where it is detected immediately after insult, only in damaged areas 19,20,25,26 . Harlequin mice, which have an 80% reduction in the expression of both AIF isoforms (AIF1 and AIF2) 22 , exhibit a significant reduction in brain injury. ...
Abstract Apoptosis-inducing factor (AIF) may contribute to neuronal cell death, and its influence is particularly prominent in the immature brain after hypoxia–ischemia (HI). A brain-specific AIF splice-isoform (AIF2) has recently been discovered, but has not yet been characterized at the genetic level. The aim of this study was to determine the functional and regulatory profile of AIF2 under physiological conditions and after HI in mice. We generated AIF2 knockout (KO) mice by removing the AIF2-specific exon and found that the relative expression of Aif1 mRNA increased in Aif2 KO mice and that this increase became even more pronounced as Aif2 KO mice aged compared to their wild-type (WT) littermates. Mitochondrial morphology and function, reproductive function, and behavior showed no differences between WT and Aif2 KO mice. However, lack of AIF2 enhanced brain injury in neonatal mice after HI compared to WT controls, and this effect was linked to increased oxidative stress but not to caspase-dependent or -independent apoptosis pathways. These results indicate that AIF2 deficiency exacerbates free radical production and HI-induced neonatal brain injury.
... Genetic or pharmacological inhibition of PARP-1 and (in some cases) PARP-2 reduces AIF nuclear translocation and neuroprotection in models of stroke (92,141,191,194,403,433), excitotoxic stress (374,379,448,702,704,793), Parkinson's disease (348,449,781), and traumatic brain injury (649). Furthermore, genetic or pharmacological modulation of AIF also offers neuroprotection in models of stroke, excitotoxicity, and traumatic brain injury (109,182,635,672,713,803). In addition, increased AIF activation and nuclear translocation is observed in relevant nervous system regions of patients with Parkinson's disease (84), Alzheimer's disease (385,781), and ALS (625). ...
Neuronal cell death occurs extensively during development and pathology, where it is especially important because of the limited capacity of adult neurons to proliferate or be replaced. The concept of cell death used to be simple as there were just two or three types, so we just had to work out which type was involved in our particular pathology and then block it. However, we now know that there are at least a dozen ways for neurons to die, that blocking a particular mechanism of cell death may not prevent the cell from dying, and that non-neuronal cells also contribute to neuronal death. We review here the mechanisms of neuronal death by intrinsic and extrinsic apoptosis, oncosis, necroptosis, parthanatos, ferroptosis, sarmoptosis, autophagic cell death, autosis, autolysis, paraptosis, pyroptosis, phagoptosis, and mitochondrial permeability transition. We next explore the mechanisms of neuronal death during development, and those induced by axotomy, aberrant cell-cycle reentry, glutamate (excitoxicity and oxytosis), loss of connected neurons, aggregated proteins and the unfolded protein response, oxidants, inflammation, and microglia. We then reassess which forms of cell death occur in stroke and Alzheimer’s disease, two of the most important pathologies involving neuronal cell death. We also discuss why it has been so difficult to pinpoint the type of neuronal death involved, if and why the mechanism of neuronal death matters, the molecular overlap and interplay between death subroutines, and the therapeutic implications of these multiple overlapping forms of neuronal death.
... The CypA/AIF(Δ1-121) complex is considered a very promising target for compounds with neuroprotective activity [8][9][10][11][12][13][14][15][16][17][18] . Therefore, understanding the molecular mechanism underpinning the CypA/AIF(Δ1-121) recognition may have a broad impact on the development of novel molecular therapies for neurodegeneration. ...
The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370–394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases.
... In contrast, Thal et al. (Plesnila) found significant levels of AIF also in the hippocampus after GCI This was accompanied by an improved neuroscore (n = 7 for vehicle, n = 7 for LOXBlock-1). c LOXBlock-1 still reduced neuronal cell death when given 1 h after onset of ischemia (n = 11 for vehicle, n = 12 for LOXBlock-1) [44]. We instead detected mostly activated caspase-3, as has been commonly noted in the literature. ...
Global ischemia following cardiac arrest is characterized by high mortality and significant neurological deficits in long-term survivors. Its mechanisms of neuronal cell death have only partially been elucidated. 12/15-lipoxygenase (12/15-LOX) is a major contributor to delayed neuronal cell death and vascular injury in experimental stroke, but a possible role in brain injury following global ischemia has to date not been investigated. Using a mouse bilateral occlusion model of transient global ischemia which produced surprisingly widespread injury to cortex, striatum, and hippocampus, we show here that 12/15-LOX is increased in a time-dependent manner in the vasculature and neurons of both cortex and hippocampus. Furthermore, 12/15-LOX co-localized with apoptosis-inducing factor (AIF), a mediator of non-caspase-related apoptosis in the cortex. In contrast, caspase-3 activation was more prevalent in the hippocampus. 12/15-lipoxygenase knockout mice were protected against global cerebral ischemia compared to wild-type mice, accompanied by reduced neurologic impairment. The lipoxygenase inhibitor LOXBlock-1 similarly reduced neuronal cell death both when pre-administered and when given at a therapeutically relevant time point 1 h after onset of ischemia. These findings suggest a pivotal role for 12/15-LOX in both caspase-dependent and caspase-independent apoptotic pathways following global cerebral ischemia and suggest a novel therapeutic approach to reduce brain injury following cardiac arrest.
... However, necrotic cells often also become Annexin-V-positive before the plasma membrane is ruptured 29 . The simultaneous detection of PI positivity and DNA hypoploidy (a measure of apoptotic DNA degradation) by flow cytometry is another apoptosis/necrosis detection technique 20 , but DNA degradation by Endonuclease G (EndoG) or Apoptosis Inducing Factor (AIF) during necrotic cell death has been reported as well 30,31 . Thus, these flow cytometric approaches should be backed up with other techniques to confirm the apoptotic phenotype. ...
Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is ∼2 h.
... Nonetheless, a large amount of experimental evidence convincingly links PARP1 hyperactivation to the calpain-independent and PAR-dependent release of AIF from mitochondria and neuronal death in vivo (Wang et al., 2009(Wang et al., , 2011. Thus, rodents pretreated with chemical inhibitors of PARP1, Parp1 À / À mice as well as Harlequin mice (Klein et al., 2002), reportedly resist a wide panel of neurotoxic insults including multiple chemicals (Mandir et al., 1999(Mandir et al., , 2000Wang et al., 2009), excitotoxic neurotransmitters (Andrabi et al., 2011), irradiation (Osato et al., 2010), trauma (Piao et al., 2012;Sarnaik et al., 2010), retinal detachment (Hisatomi et al., 2008), as well as perinatal (Hagberg et al., 2004;Zhu et al., 2003Zhu et al., , 2007b, and adult cerebral ischemia (Culmsee et al., 2005;Zhu et al., 2007a;Thal et al., 2011;Li et al., 2010). ...
Besides producing the majority of intracellular adenosine triphosphate and participating in many anabolic circuitries that are required for the survival of eukaryotic cells, mitochondria play a central role in various signaling pathways that actively provoke cell death, including distinct forms of apoptosis and regulated necrosis. Here, we discuss the lethal mechanisms activated by mitochondria in response to adverse conditions or developmental cues, placing special emphasis on the signal transduction cascades that control the cell fate at mitochondrial membranes.
... 41 Such neurotoxic triggers include, but presumably are not limited to methylnitronitrosoguanidine (MNNG), 38-40 N-methyl-d-aspartate (NMDA), 56,57 glutamate, 58 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 59 irradiation, 60 trauma, 61,62 retinal detachment, 63 and perinatal 64-66 as well as adult cerebral ischemia. [67][68][69] Additional studies using RIP3 À/À , and Bax À/À /RIP3 À/À double knockout mice, and chemical inhibition or genetic ablation of PARP1 or AIF are needed to define the roles that PARP1-and AIF-mediated necroptosis might have in the death of uninfected bystander retinal cells, and how the processes of apoptosis and necroptosis interact during the evolution of cytomegalovirus retinitis. Supplementary Material includes TUNEL assay and H&E staining in uninfected control eyes ( Supplementary Fig. S1) and Western blot of other BH3 domain proapoptotic proteins ( Supplementary Fig. S2). ...
Purpose:
Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells.
Methods:
BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3.
Results:
Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes.
Conclusions:
During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway.
... Excitotoxic stimuli such as glutamate overstimulation (Zhang and Bhavnani, 2006) triggers a form of cell death that differs from that caused by classical inducers of apoptosis, oxidative stress, or the withdrawal of trophic factors in that excitotoxic cell death shows virtually no caspase-3 activity and is accompanied by an increase in cytoplasmic calcium (Diwakarla et al., 2009). Injuries to the brain such as trauma or stroke often trigger an efflux of glutamate leading to nuclear fragmentation that was shown to be AIF-and PARP-1dependent in mouse models of focal and global brain ischemia (Eliasson et al., 1997;Thal et al., 2011). Similar AIF-and PARP-1dependent signaling occurs in neurodegenerative disease models for Parkinson's disease and retinitis pigmentosa, in programmed cell death that occurs as embryonic stem cell differentiate into neuroepithelial cells, and in response to the DNA alkylating agent MMNG (Andrabi et al., 2008;Cimadamore et al., 2009;Murakami et al., 2008;Perier et al., 2010;Yamashima et al., 2001;Yu et al., 2002). ...
Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4orf3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4orf3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins.
... This said, PARP1 and AIF have convincingly been shown to contribute to the (most-often non-apoptotic) demise of neurons exposed to a variety of cytotoxic stimuli in vivo. Such neurotoxic triggers include Page 13 of 38 A c c e p t e d M a n u s c r i p t 13 (but presumably are not limited to) MNNG [104], N-methyl-D-aspartate (NMDA) [104,110], glutamate [111], 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [112], irradiation [113], trauma [114,115], retinal detachment [116], and perinatal [117][118][119] as well as adult cerebral ischemia [102,[120][121][122]. Of note, most of these findings have been obtained with chemical inhibitors of PARP1, with Parp1 -/mice or with so-called Harlequin mice, which express lower levels of AIF owing to a hypomorphic, X-linked recessive mutation in Aifm1 [123]. ...
It is now clear that apoptosis does not constitute the sole genetically-encoded form of cell death. Rather, cells can spontaneously undertake or exogenously be driven into a cell death subroutine that manifests with necrotic features, yet can be inhibited by pharmacological and genetic interventions. As regulated necrosis (RN) plays a major role in both physiological scenarios (e.g., embryonic development) and pathological settings (e.g., ischemic disorders), consistent efforts have been made throughout the last decade toward the characterization of the molecular mechanisms that underlie this cell death modality. Contrarily to initial beliefs, RN does not invariably result from the activation of a receptor interacting protein kinase 3 (RIPK3)-dependent signaling pathway, but may be ignited by distinct molecular networks. Nowadays, various types of RN have been characterized, including (but not limited to) necroptosis, mitochondrial permeability transition (MPT)-dependent RN and parthanatos. Of note, the inhibition of only one of these modules generally exerts limited cytoprotective effects in vivo, underscoring the degree of interconnectivity that characterizes RN. Here, we review the signaling pathways, pathophysiological relevance and therapeutic implications of the major molecular cascades that underlie RN.
... Hallmarks of this pathway are breakdown of mitochondrial membrane potential, release of inner membrane space proteins from mitochondria, inhibition of the respiratory chain and ATP depletion. Depending on sensitivity to caspase-inhibitors or the activation of apoptosis-inducing-factor (AIF) (Thal et al., 2011) or endoneuclease G (Nielsen et al., 2009), this form of cell death may occur in caspase-dependent or-independent ways. ...
... Both caspase-dependent and independent pathways critically contribute to neuronal death in ischemic stroke (Pallast et al., 2010;Van Hoecke et al., 2005). We next examined the effects of NBO, minocycline and their combination on the cleavage (or activation) of caspase-3 and -9 as well as the induction of AIF, a major caspase-independent mitochondrial cell death protein (Thal et al., 2011), to determine whether inhibition of caspase-dependent and independent death pathways accounts for their neuroprotective effects. Compared to its low basal level in the contralateral tissue, AIF protein was significantly increased in the ischemic tissue of the control group (air plus vehicle) (Fig. 5A). ...
... We, thus, sought to determine whether natural aging, or reproductive senescence, also leads to dysregul ation of Dkk1 in the CA1 hippocampal region, and whether E2's ability to attenuate ischemic induction of Dkk1 is also lost in aged, reproductively senescent female rats. Additionally , since we and others have reported that aged rats are more susceptibl e to damage from GCI [43,[48][49][50][51], we sought to determine whether induction of the neurodegen erative factor Dkk1 was associate d with neuronal cell death by examining co-localizat ion of Dkk1 with TUNEL, a marker of DNA damage and apoptosis [52][53][54]. To address these questions, we subjected young (3-month-old) and aged (24-month-old) Fisher 344 rats to GCI one week following bilateral ovariectom y and examined immunosta ining of Dkk1 and TUNEL in hippocampal CA1 sections. ...
Surgically menopausal women incur a 2-5 fold increased risk for dementia and mortality from neurological diseases, but the mechanisms underlying these increased risks remain unclear. Previously, we demonstrated that after global cerebral ischemia (GCI), 17β-estradiol (E2 or estrogen) suppresses hippocampal elevation of the Wnt antagonist Dickkopf-1 (Dkk1), a neurodegenerative factor. We, thus, hypothesized that prolonged loss of E2 may lead to dysregulation of neural Dkk1 and Wnt/β-Catenin signaling, which could contribute to an increased risk of neurodegeneration. To test this hypothesis, we examined the effect of short-term (1 week - STED) and long-term E2 deprivation (10 weeks - LTED) via ovariectomy upon basal and E2-regulated Dkk1 levels and Wnt/β-Catenin signaling in the hippocampal CA1 region following GCI. In STED rats, E2 exerted robust neuroprotection against GCI, suppressed post-ischemic elevation of Dkk1, and enhanced pro-survival Wnt/β-Catenin signaling, effects that were lost in LTED rats. Intriguingly, LTED rats displayed modest basal changes in Dkk1 and survivin expression. Further work showed that c-Jun N-Terminal Kinase (JNK) mediated GCI-induced changes in Dkk1 and survivin, and JNK inhibition afforded neuroprotection in LTED rats. Finally, we extended our findings to natural aging, as 24-month-old, reproductively senescent female rats also displayed a modest increase in basal Dkk1 in the CA1, which consistently co-localized with the apoptotic marker TUNEL after GCI and coincided with a loss of E2 neuroprotection. As a whole, this study supports the "critical period hypothesis" and further suggests that perimenopausal estradiol replacement may prevent neurodegenerative changes in the hippocampus by maintaining favorable Wnt/β-Catenin signaling.
... Activation of caspase-3 and cleavage of its downstream substrate PARP-1 as well as DNA fragmentation are considered as classical hallmarks of apoptosis [75,15]. Several molecules like calpains, cathepsins and AIF which participate in caspase-independent forms of programmed cell death and necrosis exhibit significant crosstalk which can result in apoptosis, necrosis or intermediate forms of cell death [89,10,14,12,13]. Several of these proteases and signaling events are modified by hypothermia and resulting in protection against brain injury and ischemia. ...
Highlights
► We define clinical therapeutic hypothermia used in treating brain injury. ► We examine the clinical history and applications of therapeutic hypothermia. ► We examine molecular events in brain injury potentially suppressed by hypothermia. ► We evaluate future use, refinements and perspectives of therapeutic hypothermia.
... In the same model, the AIF hypomorphic mutation and caspase inhibition showed additive neuroprotective effects ( Zhu et al., 2006). More recently, in an adult mouse model of global ischemia, significant attenuation in neuronal loss in the hippocampus was observed in Hq animals compared to wild type ( Thal et al., 2011). Fewer studies have examined the role of AIF in TBI models. ...
Objectives:
The 10-O-(N N-dimethylaminoethyl)-ginkgolide B methane-sulfonate (XQ-1H) is an effective novel drug for the treatment of ischemic cerebrovascular disease derived from Ginkgolide B, a traditional Chinese medicine, has been widely used in the treatment of cardiovascular and cerebrovascular diseases. However, whether XQ-1H exerts neuroprotective effect via regulating neuronal apoptosis and the underlying mechanism remain to be elucidated.
Materials and methods:
This study was aimed to investigate the neuroprotective effect of XQ-1H in rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and the oxygen glucose deprivation/reoxygenation (OGD/R) induced neuronal apoptosis on pheochromocytoma (PC-12) cells.
Results:
The results showed that administration of XQ-1H at different dosage (7.8, 15.6, 31.2 mg/kg) reduced the brain infarct and edema, attenuated the neuro-behavioral dysfunction, and improved cell morphology in brain tissue after MCAO/R in rats. Moreover, incubation with XQ-1H (1 µM, 3 µM, 10 µM, 50 µM, 100 µM) could increase the cell viability, and showed no toxic effect to PC-12 cells. XQ-1H at following 1 µM, 10 µM, 100 µM decreased the lactate dehydrogenase (LDH) activity and suppressed the cell apoptosis in PC-12 cells exposed to OGD/R. In addition, XQ-1H treatment could significantly inhibit caspase-3 activation both in vivo and in vitro, reciprocally modulate the expression of apoptosis related proteins, bcl-2, and bax via activating PI3K/Akt signaling pathway. For mechanism verification, LY294002, the inhibitor of PI3K/Akt pathway was introduced the expressions of bcl-2 and phosphorylated Akt were down-regulated, the expression of bax was up-regulated, indicating that XQ-1H could alleviate the cell apoptosis through activating the PI3K/Akt pathway.
Conclusions:
Our findings demonstrated that XQ-1H treatment could provide a neuroprotective effect against ischemic stroke induced by cerebral ischemia/reperfusion injury in vivo and in vitro through regulating neuronal survival and inhibiting apoptosis. The findings of the study confirmed that XQ-1H could be develop as a potential drug for treatment of cerebral ischemic stroke.
The data on the molecular mechanisms of normal and pathological apoptosis are summarized. Three phases of apoptosis are distinguished: signal, effector, and degradation. The signal phase includes the extrinsic (caspase-dependent) and extrinsic (mitochondrial) pathways. Molecular markers of extrinsic and extrinsic apoptotic pathways play an important role in the diagnostics and treatment of immune, bronchopulmonary, excretory, and cardiovascular system pathologies, oncology, and senescence. This review considers the initiator caspases-8 and -9 and the effector caspase-3 as the molecular markers of the caspase-dependent apoptosis. The main molecular markers of the mitochondrial (or caspase-independent) apoptosis are p53, p21, and p16 proteins, which respond to DNA damage and are involved in cellular senescence, as well as chaperon prohibitin and flavoprotein apoptosis-inducing factor.
Objective: Hypothermia is a neuroprotective mechanism that has been validated for use in alleviating neonatal hypoxic-ischemic (HI) brain injury. Nevertheless, it is unclear whether poly (ADP-ribose) (PAR) signaling is involved in hypothermia-induced neuroprotection. In this study, we investigated whether mild hypothermia rescues oxygen glucose deprivation (OGD)-induced cell death by modifying PAR-relative protein expression, such as AIF, PARP-1, and PAR polymer, in primary-cultured hippocampal neurons.
Methods: We analyzed neuronal morphology and related protein expression of PAR signaling after OGD followed by mild hypothermia in primary-cultured newborn hippocampal neurons.
Results: Hypothermic treatment resulted in improved neuronal viability and alleviated DNA damage. Results from the protein assay showed that hypothermia attenuated nuclear translocation of apoptosis-inducing factor (AIF), inhibited overactivation of poly(ADP-ribose) polymerase-1 (PARP-1), and decreased production of PAR polymer induced by PARP-1 activation after OGD.
Conclusions: These results showed that mild hypothermia partially protects immature hippocampal neurons against OGD injury in part by interfering with the PAR signaling pathway.
Objective:
To explore the effects of mild hypothermia combined with ifenprodil on the survival of neuronal and translocation of apoptosis inducing factor (AIF) following global cerebral ischemia-reperfusion to understand the mechanism of combination in cerebral resuscitation.
Methods:
Eighty male SD rats were randomly divided into 5 groups of sham (I), model (II), ifenprodil (III), mild hypothermia (IV) and ifenprodil plus mild hypothermia (V) (n = 16 each). Group I completed all procedures except for ventricular fibrillation (VF) and cardio pulmonary resuscitation (CPR). For groups II and V, the model of global cerebral ischemia-reperfusion was established and VF induced with transoesophageal cardiac pacing; groups III and V received by an intraperitoneal injection of ifenprodil immediately after reperfusion and other groups had an equal volume of distilled water. Rectal temperature was cooled down to (32 ± 1)°C in groups IV and V by rubbing body surface with ethanol in 10 min after reperfusion and maintained 4 hours continuously while other groups at (37 ± 1)°C. In hippocampal CA1 region at 24 hours after reperfusion, the pathomorphological changes and quantity of pyramidal cells were detected with hematoxylin and eosin staining, nuclear translocation of AIF was shown with immunofluorescence technique and the nuclear expression level of AIF was measured with Western blot.
Results:
Compared with group I (75.0 ± 3.2), the number of pyramidal cells decreased in other groups (P < 0.05); compared with group II (36.0 ± 1.2), the number increased in group III (46.8 ± 1.3), IV (49.0 ± 2.7) and V (61.3 ± 2.60) (P < 0.05). In particular, cell count increased significantly in group V (P < 0.05). Compared to group I, the translocation of AIF form mitochondria to nucleus was detected in other groups; compared with group I (0.022 ± 0.003), the expression level of AIF in the nucleus was higher in other groups (P < 0.05). Compared with group II (1.020 ± 0.029) , the expression levels of AIF in groups III (0.870 ± 0.016), IV (0.820 ± 0.050) and V (0.550 ± 0.050) were lower (P < 0.05). And it decreased significantly in group V (P < 0.05).
Conclusion:
Mild hypothermia plus ifenprodil may alleviate neuronal damage after global cerebral ischemia/reperfusion injury through mitigating its pro-apoptotic role after AIF translocation.
During the decade the importance of regulated necrotic cell death in several pathologies has been increasingly recognized. Intensified research has uncovered many pathways involved in regulated necrosis (RN) that has resulted in several neologisms. The best characterized form of RN is induced by receptor-interacting protein kinase-1 and -3, coined as necroptosis. Unfortunately, until now there are no positive and distinguishing markers available for any of these forms of regulated necrosis, let alone a single marker that identifies regulated necrosis from apoptosis. Here we present an overview of different methods that can be used to discriminate apoptosis from necrosis, more in particular necroptosis, both in vivo and in vitro. Methods for necrotic cell death detection are summarized, and potential pitfalls of these methods are briefly discussed. Basically, a sequential approach is proposed to determine the cell death modality by following a decision tree, allowing discrimination between apoptosis and different forms of RN. Finally, pharmacological and transgenic methods for dissecting subroutines in RN are mentioned
Given the current limitation of therapeutic approach for ischemic stroke, a leading cause of disability and mortality in the developed countries, to develop new therapeutic strategies for this devastating disease is urgently necessary. As a serine/threonine kinase, mammalian target of rapamycin (mTOR) activation can mediate broad biological activities that include protein synthesis, cytoskeleton organization, and cell survival. mTOR functions through mTORC1 and mTORC2 complexes and their multiple downstream substrates, such as eukaryotic initiation factor 4E-binding protein 1, p70 ribosomal S6 kinase, sterol regulatory element-binding protein 1, hypoxia inducible factor-1, and signal transducer and activator transcription 3, Yin Ying 1, Akt, protein kinase c-alpha, Rho GTPase, gucocorticoid-induced protein kinase 1, and etc. Specially, the role of mTOR in the central nervous system has attracting considerable attention. Based on the ability of mTOR to prevent neuronal apoptosis, inhibit autophagic cell death, promote neurogenesis, and improve angiogenesis, mTOR may acquire the capability of limiting the ischemic neuronal death and promoting the neurological recovery. Consequently, to regulate the activity of mTOR holds a potential as a novel therapeutic strategy for ischemic stroke.
In recent studies, acute ethanol administration appears to play a neuroprotective role during ischemic stroke. We sought to confirm these findings by identifying if ethanol-derived neuroprotection is associated with a reduction in apoptosis. Ethanol at 0.5 and 1.5g/kg doses was given by intraperitoneal injections to Sprague-Dawley rats after 2hours of middle cerebral artery (MCA) occlusion, followed by reperfusion. We quantified apoptotic cell death in each of the treatment groups with ELISA, and measured pro- and anti-apoptotic protein expression with Western blot analysis. Cell death was significantly increased in rats after ischemia and was subsequently significantly reduced by the administration of 1.5g/kg of ethanol. We found that the 1.5g/kg dose promoted the expression of pro-survival factors and decreased the expression of apoptotic proteins at 3hours after reperfusion. This effect was maintained at 24hours for Caspase-3 and apoptosis-inducing factor (AIF), although not for Bcl-2, Bcl-xL, and Bcl-2-associated X (Bax). Administration of 0.5g/kg of ethanol was not as effective in regulating protein expression as the 1.5g/kg dose. Our study suggests that administration of ethanol at a dose of 1.5g/kg after stroke -which provides rat blood alcohol levels equivalent to the legal driving limit-produces a differential protein profile, with increased expression of anti-apoptotic proteins and decrease in pro-apoptotic factors. This results in a significant reduction of neuronal apoptosis and is neuroprotective in ischemia-reperfusion injury.
Acute administration of ethanol is associated with neuroprotection in rat with transient cerebral ischemia. To investigate the molecular mechanism of ethanol-induced neuroprotection, we determined the effect of ethanol on expression levels of apoptotic proteins, including caspase-3, Bcl-2-associated X protein (Bax), and apoptosis-inducing factor (AIF). To assess overall cell viability following ethanol treatment, ADP/ATP ratio was measured.
Brain slice cultures were prepared using postnatal 10-day-old Sprage-Dawley rats. Brain slices were divided into control and hypoxia groups. Hypoxia groups include a non-treatment group and three treatment groups (10, 30, or 90 mM ethanol). Levels of caspase-3, Bax, and AIF were determined by western blot. ADP/ATP ratio was assessed using ADP/ATP assay kit.
Ethanol administration reduced ADP/ATP ratio in all three treatment groups (10, 30, and 90 mM). A reduction in caspase-3, BAX, and AIF expression was observed with all three treatment groups in conjunction with decreased ADP/ATP levels. The three treatment groups showed similar levels of reduction in ADP/ATP ratio and apoptotic protein expression.
Ethanol-induced neuroprotection involves inhibition of apoptotic pathways, including Bax, caspase-3, and AIF. Dose range of 10-90 mM ethanol provides similar level of protection compared to 10 mM ethanol.
Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.
Streptococcus pneumoniae is the major cause of bacterial meningitis and it damages the hippocampus by inducing neuronal apoptosis. The blocking of
caspases provides only partial protection in experimental meningitis, which suggests that there is an additional apoptotic
pathway. A trigger of this pathway is the bacterium itself, as exposure of microglia or neurons to live pneumococci induces
rapid apoptosis. In this study, apoptosis was not associated with the activation of caspases-1–10 and was not inhibited by
z-VAD-fmk, a broad-spectrum caspase inhibitor. Rather, apoptosis was attributed to damage to mitochondria, which was followed
by the release of apoptosis-inducing factor (AIF) from the mitochondria, large-scale DNA fragmentation, and hypodiploidy.
Furthermore, intracytoplasmatic microinjection of AIF-specific antiserum markedly impaired pneumococcus-induced apoptosis.
These findings indicate that AIF may play a central role in brain cell apoptosis and bacterial pathogenesis
Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.
Delayed neuronal cell death occurring hours after reperfusion is a hallmark of ischemic stroke and a primary target for neuroprotective strategies. In the present study, we investigated whether apoptosis-inducing factor (AIF), a caspase-independent proapoptotic protein, is responsible for neuronal cell death after glutamate toxicity and oxygen-glucose deprivation (OGD) in vitro and after experimental stroke in vivo. AIF translocated to the nucleus in which it colocalized with DNA fragmentation and nuclear apoptotic morphology after exposure to glutamate or OGD in cultured neurons or after transient middle cerebral artery occlusion (MCAo) in mice. Small inhibitory RNA-mediated downregulation of AIF reduced glutamate- and OGD-induced neuronal apoptosis by 37 and 60%, respectively (p < 0.01). Moreover, Harlequin mutant mice, which express AIF at low levels (approximately 20% of wild-type mice), displayed smaller infarct volumes (-43%; p < 0.03) and showed dramatically reduced cell death in the ischemic penumbra after 45 min of MCAo compared with wild-type littermates. Inhibition of poly(ADP-ribose) polymerase and Bid reduced nuclear AIF translocation. These results provide the first evidence for a causal role of AIF in ischemic neuronal cell death. Therefore, caspase-independent cell death signaling may provide a promising novel target for therapeutic interventions in cerebrovascular diseases.
Existing murine models of global cerebral ischemia are technically challenging thereby hampering the use of genetically engineered mice to study cardiac arrest-induced brain damage. We therefore investigated, if disconnecting the cerebral circulation from vertebral collateral blood flow by proximal occlusion of the basilar artery together with temporary bilateral common carotid artery occlusion (BCCAo) may be a more feasible approach. C57/Bl6 mice were anesthetized and the basilar artery was occluded through a ventral approach. Ten days later BCCAo was performed for 8-14min. Increasing durations of ischemia resulted in enhanced neuronal cell death in cortex, striatum, and hippocampus (22-63%) and increased neurological dysfunction and mortality (0-36%). Following 10min of BCCAo, the duration of global ischemia with the most favorable mortality/neuronal cell death ratio, hippocampal damage started 6h after the insult while cortical and striatal damage was delayed by at least 24h. No further loss of neuronal cells was observed later than 3 days. The proposed two-step approach resulted in complete cerebral ischemia and caused neuronal damage with high reproducibility and small variability. In combination with transgenic and knock-out mice this technically feasible model may help to extend our knowledge on the pathophysiology of cardiac arrest-induced brain damage.
Acute neurological conditions such as cerebrovascular diseases and trauma are associated with irreversible loss of neurons and glial cells. Severe or prolonged injury results in uncontrollable cell death within the core of lesions. Conversely, cells that are less severely damaged succumb in a relatively slow fashion, frequently via the intrinsic pathway of cell death, through the deterioration of mitochondrial functions. The permeabilization of mitochondrial membranes determines whether cells will succumb to or survive the injury, and represents a 'point of no return' in mitochondrial cell death. It is therefore an attractive target for the development of new neuroprotective interventions.
Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.
Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.
In both the nematode Caenorhabditis elegans and mammals, two proteins released from the mitochondrion - apoptosis inducing factor (AIF) and endonuclease G - cooperate in executing programmed cell death. Although both factors can kill cells in a caspase-independent fashion, new studies indicate that their translocation from mitochondria depends, in part, on caspase activation. Together, these data raise new questions about the functional hierarchy between caspases, AIF and mitochondrial membrane permeabilization.
Apoptosis-inducing factor (AIF) triggers apoptosis in a caspase-independent manner. Here we report for the first time involvement of AIF in neuronal death induced by cerebral ischemia. Unilateral cerebral hypoxia-ischemia (HI) was induced in 7-day-old rats by ligation of the left carotid artery and hypoxia (7.7% O2) for 55 min. AIF release from mitochondria and AIF translocation to nuclei was detected immediately after HI, and only in damaged areas, as judged by the concurrent loss of MAP-2. AIF release was detected earlier than that of cytochrome c. Cells with AIF-positive nuclei displayed nuclear condensation and signs of DNA damage. The number of AIF-positive nuclei showed a positive correlation with the infarct volume 72 h post-HI, and this was not changed by treating the animals with boc-Asp-fmk (BAF), a multicaspase inhibitor. BAF treatment reduced the activity of caspase-3, -2 and -9 (78, 73 and 33%, respectively), and prevented caspase-dependent fodrin cleavage in vivo, but did not affect AIF release from mitochondria or the frequency of positive nuclear AIF or DNA damage 72 h post-HI, indicating that these processes occurred in a caspase-independent fashion. In summary, AIF-mediated cell death may be an important mechanism of HI-induced neuronal loss in the immature brain.
Loss of mitochondrial membrane integrity and the resulting release of apoptogenic factors may play a critical role in mediating hippocampal neurodegeneration after transient global ischemia. In the present study, the authors have cloned and characterized the rat cDNA encoding apoptosis-inducing factor (AIF), an intramitochondrial protein that promotes cell death in a caspase-independent manner upon release into nonmitochondrial compartments. In contrast to the expression patterns of a number of apoptosis-regulatory gene products during brain development, the expression of AIF protein increases gradually with brain maturation and peaks in adulthood. In a rat model of transient global ischemia, AIF was found to translocate from mitochondria to the nucleus in the hippocampal CA1 neurons after ischemia and to manifest a DNA-degrading activity that mimicked the purified AIF protein and was inhibitable by AIF immunodepletion. The temporal profile of AIF translocation after ischemia (24 to 72 hours) coincided with the induction of large-scale DNA fragmentation at the size of 50 kbp, a well-characterized hallmark of AIF-like activity but preceded the formation of internucleosomal DNA fragmentation (72 hours), a DNA degradation associated with the terminal stage of cell death. Further, the nuclear translocation of AIF after ischemia was not blocked by inhibiting caspase-3/-7 activities, but, as shown in neuronal cultures that were challenged with transient oxygen-glucose deprivation, it can be prevented by intracellular delivery of the mitochondria-associated antiapoptotic protein Bcl-xL. The results presented here strongly suggest that mitochondrial release of AIF may be an important factor, in addition to the previously reported cytochrome c and Smac, which could contribute to the selective vulnerability of CA1 neurons to transient global ischemic injury.
Signaling cascades associated with apoptosis contribute to cell death after focal cerebral ischemia. Cytochrome c release from mitochondria and the subsequent activation of caspases 9 and 3 are critical steps. Recently, a novel mitochondrial protein, apoptosis-inducing factor (AIF), has been implicated in caspase-independent programmed cell death following its translocation to the nucleus. We, therefore, addressed the question whether AIF also plays a role in cell death after focal cerebral ischemia. We detected AIF relocation from mitochondria to nucleus in primary cultured rat neurons 4 and 8 hours after 4 hours of oxygen/glucose deprivation. In ischemic mouse brain, AIF was detected within the nucleus 1 hour after reperfusion after 45 minutes occlusion of the middle cerebral artery. AIF translocation preceded cell death, occurred before or at the time when cytochrome c was released from mitochondria, and was evident within cells showing apoptosis-related DNA fragmentation. From these findings, we infer that AIF may be involved in neuronal cell death after focal cerebral ischemia and that caspase-independent signaling pathways downstream of mitochondria may play a role in apoptotic-like cell death after experimental stroke.
Cerebral ischemia results in a rapid depletion of energy stores that triggers a complex cascade of cellular events such as cellular depolarization and Ca2+ influx, resulting in excitotoxic cell death. The critical determinant of severity of brain injury is the duration and severity of the ischemic insult and early restoration of CBF. Induced therapeutic hypothermia following CA is the only strategy that has demonstrated improvement in outcomes in prospective, randomized clinical trials. Although pharmacologic neuro-protection has been disappointing thus far in a variety of experimental animal models, further research efforts are directed at using some agents that demonstrate marginal or moderate efficacy in combination with hypothermia. Although the signal transduction pathways and intracellular molecular events during cerebral ischemia and reperfusion are complex, potential therapeutic neuroprotective strategies hold promise for the future.
Despite the constantly increasing use of genetically engineered mice in biomedical research, control of crucial physiological parameters such as blood pressure and arterial blood gases is difficult to achieve in temporarily anesthetized mice due to lack of techniques for reversible arterial cannulation. Here we report that arterial blood pressure and blood gases can be measured reliably in anaesthetized and artificially ventilated mice using non-invasive technology. C57Bl6 mice were anaesthetized by i.p. injection of midazolam, fentanyl, and medetomidin, intubated, and ventilated for 3h. End tidal pCO2 was monitored by micro-capnometry. Arterial blood pressure was measured non-invasively using a tail cuff. Non-invasive blood pressure (NIBP) correlated strongly with the invasive arterial blood pressure measured at the external carotid artery (r = 0.99, P < 0.001) and end tidal pCO2 values correlated very well with arterial blood pCO2 (r = 0.93, P < 0.001). The current results demonstrate that it is possible to reliably measure and control the most relevant physiological parameters in anesthetized mice. Thereby the current study may help to reduce animal numbers and perform mice experiments under more defined and controlled physiological conditions in the future.
Molecular characteriza-tion of mitochondrial apoptosis-inducing factor
- D P Aebersold
- J M Siderovski
- G Penninger
- Kroemer
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Involvement of apoptosis-inducing factor in neuronal death after hypoxiaischemia in the neonatal rat brain
- C Zhu
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- X Wang
- U Hallin
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