O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.
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ABSTRACT:
SUBMITTER: Shi J
PROVIDER: S-EPMC4585968 | biostudies-other | 2015 Sep
REPOSITORIES: biostudies-other
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Project description:O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling.
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Project description:Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (β2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by β2-AR but not β1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a β2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the β2-AR/PI3K/AKT cascade.
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Project description:BackgroundThis study investigated the potential effects of 3-iodothyronamine (T1AM) on myocardial ischemia reperfusion injury (MIRI) and the underlying molecular mechanisms.MethodsA total of 16 adult male Sprague-Dawley rats were randomly divided into 4 groups and administered the following: control [60% dimethyl sulfoxide (DMSO) and 40% saline, pH 7.4], T1AM (25 mg/kg), T1AM (50 mg/kg), or T1AM (100 mg/kg). The rectal temperatures of the rats were measured at different time points. A further 30 adult male Sprague-Dawley rats were randomized and divided into the following 3 groups (n=10 in each group): sham operation, ischemia/reperfusion (I/R), and I/R + T1AM. In the I/R and I/R + T1AM groups, the left anterior descending (LAD) coronary artery of the rats were occluded for 0.5 hour to induce myocardial ischemia, followed by reperfusion for 3 hours in the I/R group. The electrocardiography (ECG), cardiac function, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were examined in rats to evaluate the myocardial injury. The differences in the expression of apoptosis-related and Akt-FoxO1 signaling-related proteins were determined via Western blot.ResultsThis work verified that T1AM reduced the body temperature of rats in a dose-dependent manner. Additionally, T1AM improved cardiac function and decreased the infarction size caused by MIRI. T1AM reduced the expression of biochemical parameters and apoptosis of myocardial cells. In addition, after treatment with T1AM, the expression of Glut1, pFoxO1 and Akt were reduced, while the expression of FoxO1 and PPARα were increased significantly.ConclusionsPretreatment of cardiomyocytes with T1AM inhibited apoptosis and protected against ischemia reperfusion injury via the Akt/FoxO1 signaling pathway.
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Project description:BackgroundThis study aims to investigate the regulation of herbal polysaccharide, Coriolus versicolor polysaccharides (CVP), on neuronal apoptosis in a rat cerebral ischemia-reperfusion injury (CIRI) model. We also intend to explore the mechanisms and effectiveness of CVP in the treatment of neuronal apoptosis in CIRI rats, including neurological function, cerebral infarction volume, inflammatory factors, and the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway as well as its downstream protein cleaved-Caspase-3.MethodsA CIRI model was established in rats using the Longa method of middle cerebral artery occlusion. Neurological function scores and cerebral infarction volumes were measured in CIRI rats. Annexin V-fluorescein isothiocyanate (FITC) were used to measure neuronal apoptosis in CIRI rats. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in CIRI rats were determined using enzyme-linked immunosorbent assay (ELISA). A western blot assay was used to measure the protein expression levels of p38MAPK, phospho-p38MAPK (p-p38MAPK), Bcl-2, Bax and cleaved-Caspase-3 in brain tissue of CIRI rats.ResultsCVP can effectively improve the neurological function of rats after 6, 12, 24, and 48 h of CIRI. It can also improve the behavioral test, reduce the cerebral infarction volume and inhibit the apoptosis of nerve cells in CIRI rats. The protein expression levels of p-p38MAPK and cleaved-Caspase-3 exhibited a decreasing trend following CVP administration.ConclusionsCVP can significantly reduce the pathological characteristics of CIRI in rats and inhibit the apoptosis of nerve cells around the lesions. The mechanism of its effectiveness is related to inhibiting the activation of the p38MAPK signaling pathway.
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Project description:Berberine (BBR) has a neuroprotective effect against ischemic stroke, but its specific protective mechanism has not been clearly elaborated. This study explored the effect of BBR on the canopy FGF signaling regulator 2 (CNPY2) signaling pathway in the ischemic penumbra of rats. The model of cerebral ischemia-reperfusion injury (CIRI) was established by the thread embolization method, and BBR was gastrically perfused for 48 h or 24 h before operation and 6 h after operation. The rats were randomly divided into four groups: the Sham group, BBR group, CIRI group, and CIRI + BBR group. After 2 h of ischemia, followed by 24 h of reperfusion, we confirmed the neurologic dysfunction and apoptosis induced by CIRI in rats (p < 0.05). In the ischemic penumbra, the expression levels of CNPY2-regulated endoplasmic reticulum stress-induced apoptosis proteins (CNPY2, glucose-regulated protein 78 (GRP78), double-stranded RNA-activated protein kinase-like ER kinase (PERK), C/EBP homologous protein (CHOP), and Caspase-3) were significantly increased, but these levels were decreased after BBR treatment (p < 0.05). To further verify the inhibitory effect of BBR on CIRI-induced neuronal apoptosis, we added an endoplasmic reticulum-specific agonist and a PERK inhibitor to the treatment. BBR was shown to significantly inhibit the expression of apoptotic proteins induced by endoplasmic reticulum stress agonist, while the PERK inhibitor partially reversed the ability of BBR to inhibit apoptotic protein (p < 0.05). These results confirm that berberine may inhibit CIRI-induced neuronal apoptosis by downregulating the CNPY2 signaling pathway, thereby exerting a neuroprotective effect.
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Project description:Targeting neuronal apoptosis after intracerebral hemorrhage (ICH) may be an important therapeutic strategy for ICH patients. Emerging evidence indicates that C1q/TNF-Related Protein 9 (CTRP9), a newly discovered adiponectin receptor agonist, exerts neuroprotection in cerebrovascular disease. The aim of this study was to investigate the anti-apoptotic role of CTRP9 after experimental ICH and to explore the underlying molecular mechanisms. ICH was induced in mice via intrastriatal injection of bacterial collagenase. Recombinant CTRP9 (rCTRP9) was administrated intranasally at 1 h after ICH. To elucidate the underlying mechanisms, adiponectin receptor1 small interfering ribonucleic acid (AdipoR1 siRNA) and selective PI3 K inhibitor LY294002 were administered prior to rCTRP9 treatment. Western blots, neurofunctional assessments, immunofluorescence staining, and Fluoro-Jade C (FJC) staining experiments were performed. Administration of rCTRP9 significantly improved both short- and long-term neurofunctional behavior after ICH. RCTRP9 treatment significantly increased the expression of AdipoR1, PI3 K, p-Akt, and Bcl-2, while at the same time was found to decrease the expression of Bax in the brain, which was reversed by inhibition of AdipoR1 and PI3 K. The neuroprotective effect of rCTRP9 after ICH was mediated by attenuation of neuronal apoptosis via the AdipoR1/PI3K/Akt signaling pathway; therefore, rCTRP9 should be further evaluated as a potential therapeutic agent for ICH patients.
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Project description:Hyperglycemia, as well as diabetes mellitus, has been shown to trigger cardiac cell apoptosis. We have previously demonstrated that myricitrin prevents endothelial cell apoptosis. However, whether myricitrin can attenuate H9c2 cell apoptosis remains unknown. In this study, we established an experiment model in H9c2 cells exposed to high glucose. We tested the hypothesis that myricitrin may inhibit high glucose (HG)-induced cardiac cell apoptosis as determined by TUNEL staining. Furthermore, myricitrin promoted antioxidative enzyme production, suppressed high glucose-induced reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) in H9c2 cells. This agent significantly inhibited apoptotic protein expression, activated Akt and facilitated the transcription of NF-E2-related factor 2 (Nrf2)-mediated protein (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1) expression as determined by Western blotting. Significantly, an Akt inhibitor (LY294002) or HO-1 inhibitor (ZnPP) not only inhibited myricitrin-induced HO-1/NQO-1 upregulation but also alleviated its anti-apoptotic effects. In summary, these observations demonstrate that myricitrin activates Nrf2-mediated anti-oxidant signaling and attenuates H9c2 cell apoptosis induced by high glucose via activation of Akt signaling.
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Project description:Growing evidence suggests that the increased neuronal apoptosis is involved in n-hexane-induced neuropathy. We have recently reported that bone marrow-mesenchymal stem cells-derived conditioned medium (BMSC-CM) attenuated 2,5-hexanedione (HD, the active metabolite of n-hexane)-induced apoptosis in PC12 cells. Here, we explored the anti-apoptotic efficacy of BMSC in vivo. HD-treated rats received BMSC by tail vein injection 5 weeks after HD intoxication. We found that in grafted rats, BMSC significantly attenuated HD-induced neuronal apoptosis in the spinal cord, which was associated with elevation of nerve growth factor (NGF). Neutralization of NGF in BMSC-CM blocked the protection against HD-induced apoptosis in VSC4.1 cells, suggesting that NGF is essential for BMSC-afforded anti-apoptosis. Mechanistically, we found that the decreased activation of Akt induced by HD was significantly recovered in the spinal cord by BMSC and in VSC4.1 cells by BMSC-CM in a TrkA-dependent manner, leading to dissociation of Bad/Bcl-xL complex in mitochondria and release of anti-apoptotic Bcl-xL. The importance of Akt was further corroborated by showing the reduced anti-apoptotic potency of BMSC in HD-intoxicated VSC4.1 cells in the presence of Akt inhibitor, MK-2206. Thus, our findings show that BMSC attenuated HD-induced neuronal apoptosis in vivo through a NGF/Akt-dependent manner, providing a novel solution against n-hexane-induced neurotoxicity.
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Project description:One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2-p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2-p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.
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Project description:Acute cerebral ischaemia may lead to serious consequences, including brain injury caused by uncontrolled reperfusion, which occurs when circulation is re-established. The long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) plays an important role in the immune system. However, the potential roles and underlying molecular mechanisms of NEAT1 in cerebral ischaemia/reperfusion (I/R) injury remain unclear. The aim of the present study was to investigate the function of the lncRNA NEAT1 in cerebral I/R injury and its potential beneficial effects on neurons. In our study, oxygen-glucose deprivation (OGD)/reoxygenation (OGD/R) was induced in vitro to mimic cerebral I/R injury. Cholecystokinin-octopeptide (CCK-8) was used to measure cell viability, and flow cytometry was used to measure cell apoptosis. Real-time quantitative PCR (qRT-PCR) was used to measure the expression of phenotypic markers of classically activated (M1) and alternatively activated (M2) microglia, and western blotting was performed to detect the levels of proteins related to the AKT/STAT3 pathway. The expression of the lncRNA NEAT1 was significantly upregulated in patients with ischaemic stroke, and knockdown of the lncRNA NEAT1 alleviated OGD/R-induced apoptosis and increased neuronal viability. Furthermore, the lncRNA NEAT1 may inhibit microglial polarization towards the M1 phenotype to reduce the damage caused by OGD/R and reduce the activity of the AKT/STAT3 pathway. In conclusion, the lncRNA NEAT1 may be a potential target for new therapeutic interventions for cerebral I/R.
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