Ischemia induces different levels of hypoxia inducible factor-1α protein expression in interneurons and pyramidal neurons

Introduction Pyramidal (glutamatergic) neurons and interneurons are morphologically and functionally well defined in the central nervous system. Although it is known that glutamatergic neurons undergo immediate cell death whereas interneurons are insensitive or survive longer during cerebral ischemia, the protection mechanisms responsible for this interneuronal survival are not well understood. Hypoxia inducible factor-1 (HIF-1) plays an important role in protecting neurons from hypoxic/ischemic insults. Here, we studied the expression of HIF-1α, the regulatable subunit of HIF-1, in the different neuronal phenotypes under in vitro and in vivo ischemia. Results In a primary cortical culture, HIF-1α expression was observed in neuronal somata after hypoxia (1% oxygen) in the presence of 5 or 25 mM glucose but not under normoxia (21% oxygen). Interestingly, only certain MAP2-positive neurons containing round somata (interneuron-like morphology) co-localized with HIF-1α staining. Other neurons such as pyramidal-like neurons showed no expression of HIF-1α under either normoxia or hypoxia. The HIF-1α positive neurons were GAD65/67 positive, confirming that they were interneuron-type cells. The HIF-1α expressing GAD65/67-positive neurons also possessed high levels of glutathione. We further demonstrated that ischemia induced significant HIF-1α expression in interneurons but not in pyramidal neurons in a rat model of middle cerebral artery occlusion. Conclusion These results suggest that HIF-1α protein expression induced by ischemia is neuron-type specific and that this specificity may be related to the intracellular level of glutathione (GSH).This research was supported in part by a grant from the National Institutes of Health (R01NS058807) and a Kansas University Center for Research startup fund

Relative Contribution of Prolyl Hydroxylase-Dependent and -Independent Degradation of HIF-1alpha by Proteasomal Pathways in Cerebral Ischemia

Hypoxia inducible factor-1 (HIF-1) is a key regulator in hypoxia and can determine the fate of brain cells during ischemia. However, the mechanism of HIF-1 regulation is still not fully understood in ischemic brains. We tested a hypothesis that both the 26S and the 20S proteasomal pathways were involved in HIF-1α degradation under ischemic conditions. Using in vitro ischemic model (oxygen and glucose deprivation) and a mouse model of middle cerebral artery occlusion, we tested effects of inhibitors of proteasomes and prolyl hydroxylase (PHD) on HIF-1α stability and brain injury in cerebral ischemia. We observed that 30 and 60 min of oxygen-glucose deprivation significantly increased the 20S proteasomal activity. We demonstrated that proteasome inhibitors increased HIF-1α stabilization and cell viability and were more effective than PHD inhibitors in primary cultured cortical neurons exposed to oxygen and glucose deprivation. Furthermore, the administration of the proteasome inhibitor, epoxomicin, to mice resulted in smaller infarct size and brain edema than a PHD inhibitor. Our results indicate that 20S proteasomes are involved in HIF-1α degradation in ischemic neurons and that proteasomal inhibition provides more HIF-1α stabilization and neuroprotection than PHD inhibition in cerebral ischemia.R01NS05880

Hyperglycemia as a Risk Factor of Ischemic Stroke

Diabetes is considered a major risk factor for stroke and is associated with worsened stroke outcomes. Here, we discuss and summarize the mechanisms that have been associated with the increased risk of stroke due to the hyperglycemia in diabetes mellitus. In diabetic stroke models, hyperglycemia exaggerates the following damaging processes: acidosis, accumulation of reactive oxygen species/reactive nitrogen, inflammation and mitochondrial dysfunction. Understanding the mechanism of diabetes acting as a stroke risk factor will definitely assist to reveal issues related to drug metabolism and toxicity in diabetic stroke. In addition, it is suggested that future studies may focus on the mechanisms mediating blood-brain barrier and astrocytes dysfunction under hyperglycemic stroke.This research was supported in part by a grant from the National Institutes of Health (R01NS058807) and a Kansas University Center for Research startup fund

Hypoxia-inducible factor 1 contributes to N-acetylcysteine’s protection in stroke

Stroke is a leading cause of adult morbidity and mortality with very limited treatment options. Evidence from preclinical models of ischemic stroke has demonstrated that the antioxidant N-acetylcysteine (NAC) effectively protects the brain from ischemic injury. Here, we evaluated a new pathway through which NAC exerted its neuroprotection in a transient cerebral ischemia animal model. Our results demonstrated that pretreatment with NAC increased protein levels of hypoxia-inducible factor-1α (HIF-1α), the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3, in the ipsilateral hemispheres of rodents subjected to 90 min middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Interestingly, after NAC pretreatment and stroke, the contralateral hemisphere also demonstrated increased levels of HIF-1α, EPO, and GLUT-3, but to a lesser extent. Suppressing HIF-1 activity with two widely used pharmacological inhibitors, YC-1 and 2ME2, and specific knockout of neuronal HIF-1α abolished NAC’s neuroprotective effects. The results also showed that YC-1 and 2ME2 massively enlarged infarcts, indicating that their toxic effect was larger than just abolishing NAC’s neuroprotective effects. Furthermore, we determined the mechanism of NAC-mediated HIF-1α induction. We observed that NAC pretreatment upregulated heat-shock protein 90 (Hsp90) expression and increased the interaction of Hsp90 with HIF-1α in ischemic brains. The enhanced association of Hsp90 with HIF-1α increased HIF-1α stability. Moreover, Hsp90 inhibition attenuated NAC-induced HIF-1α protein accumulation and diminished NAC-induced neuroprotection in the MCAO model. These results strongly indicate that HIF-1 plays an important role in NAC-mediated neuroprotection and provide a new molecular mechanism involved in the antioxidant’s neuroprotection in ischemic stroke

Action of Pyrroloquinolinequinol As an Antioxidant Against Lipid Peroxidation in Solution

This is the publisher's version, also available electronically from: http://online.liebertpub.com/doi/pdfplus/10.1089/ars.1999.1.4-547.The activities of pyrroloquinoline quinone (PQQ), a coenzyme of methanol dehydrogenase and amine oxidase, and its reduced form pyrroloquinoline quinol (PQQH2 ) as an antioxidant have been measured in solution. PQQH2 was stable in the absence of oxygen but rapidly auto-oxidized to PQQ in the presence of oxygen in water. PQQH2 was stable in an aprotic solvent such as acetonitrile, even in air. PQQ did not exert appreciable antioxidant activity, whereas PQQH2 exerted higher reactivity than a-tocopherol toward galvinoxyl radical and peroxyl radical. PQQH2 acted as a potent antioxidant against the oxidation of methyl linoleate in acetonitrile induced by azo compound and produced a clear induction period, from which the apparent stoichiometric number was obtained as 1.1. PQQH2 reduced the a-tocopheroxyl radical and spared a-tocopherol in the oxidation of methyl linoleate. These results suggest that PQQH2 may act as a potent antioxidant, particularly in combination with a-tocopherol. Antiox. Redox Signal. 1, 547-554

Ischemic Tolerance in an In Vivo Model of Glutamate “Preconditioning”

This is the peer reviewed version of the following article: Badawi, Y., Pal, R., Hui, D., Michaelis, E. K. and Shi, H. (2015), Ischemic tolerance in an in vivo model of glutamate preconditioning. Journal of Neuroscience Research, 93: 623–632. doi:10.1002/jnr.23517, which has been published in final form at http://doi.org/10.1002/jnr.23517. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Ischemia initiates a complicated biochemical cascade of events that triggers neuronal death. In this study, we focused on glutamate –mediated neuronal tolerance to ischemia-reperfusion. We employed an animal model of life-long excess release of glutamate, the glutamate dehydrogenase 1 transgenic (Tg) mouse, as a model of in vivo “glutamate preconditioning”. Nine- and 22-month old Tg and wild type (wt) mice were subjected to 90 min of middle cerebral artery occlusion followed by 24 hr reperfusion. The Tg mice suffered significantly reduced infarction and edema volume, compared with their wt counterparts. We further analyzed proteasomal activity, level of ubiquitin immunostaining, and MAP2A expression to understand the mechanism of neuroprotection observed in the Tg Mice. We found that in the absence of ischemia, the Tg mice exhibited higher activity of the 20S and 26S proteasomes while there were no significant differences in the level of hippocampal ubiquitin immunostaining between wt and Tg mice. A surprising observation was that of a significant increase in MAP2A expression in neurons of the Tg hippocampus following ischemia-reperfusion, compared with that in wt hippocampus. The results suggest that increased proteasome activity and MAP2A synthesis and transport might account for the effectiveness of glutamate preconditioning against ischemia-reperfusion

The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function

This work is licensed under a Creative Commons Attribution 4.0 International License.Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1′s deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.Hedwig Miller Fund for Aging Research of the University of KansasU.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE-FG02-05ER15650)NIH R01 GM5749

Dual Effects of Chinese Herbal Medicines on Angiogenesis in Cancer and Ischemic Stroke Treatments: Role of HIF-1 Network

This work is licensed under a Creative Commons Attribution 4.0 International License.Hypoxia-inducible factor-1 (HIF-1)–induced angiogenesis has been involved in numerous pathological conditions, and it may be harmful or beneficial depending on the types of diseases. Exploration on angiogenesis has sparked hopes in providing novel therapeutic approaches on multiple diseases with high mortality rates, such as cancer and ischemic stroke. The HIF-1 pathway is considered to be a major regulator of angiogenesis. HIF-1 seems to be involved in the vascular formation process by synergistic correlations with other proangiogenic factors in cancer and cerebrovascular disease. The regulation of HIF-1–dependent angiogenesis is related to the modulation of HIF-1 bioactivity by regulating HIF-1α transcription or protein translation, HIF-1α DNA binding, HIF-1α and HIF-1α dimerization, and HIF-1 degradation. Traditional Chinese herbal medicines have a long history of clinical use in both cancer and stroke treatments in Asia. Growing evidence has demonstrated potential proangiogenic benefits of Chinese herbal medicines in ischemic stroke, whereas tumor angiogenesis could be inhibited by the active components in Chinese herbal medicines. The objective of this review is to provide comprehensive insight on the effects of Chinese herbal medicines on angiogenesis by regulating HIF-1 pathways in both cancer and ischemic stroke.National Natural Science Foundation of China (No. 81673627)Guangzhou Science Technology and Innovation Commission Research Projects (201805010005)Research Grant Council, HKSAR (Project code: RGC GRF 17152116)Commissioner for Innovation Technology, HKSAR (Project code: ITS/091/16FX

Development of Biomarkers Based on Diet-Dependent Metabolic Serotypes: Concerns and Approaches for Cohort and Gender Issues in Serum Metabolome Studies

This is the publisher's version, also available electronically from: http://online.liebertpub.com/doi/pdfplus/10.1089/omi.2004.8.209Mathematical models that reflect the effects of dietary restriction (DR) on the sera metabolome may have utility in understanding the mechanisms of DR and in applying this knowledge to human epidemiological studies. Previous studies demonstrated both the feasibility of identifying biomarkers through metabolome analysis and the validity of our approach in independent cohorts of 6-month-oId male and female ad libitum fed or DR rats. Cross-cohort studies showed that cohort-specific effects distorted the dataset The present study extends these observations across the entire sample set, thereby validating our markers independently of specific cohorts. Metabolites originally identified in males were examined in females and vice-versa. DR's effect on the metabolom e is partially gender-specific and is modulated by environmental factors. DR reduces inter-gender differences in the metabolome. Univariate statistical methods showed that 56/93 metabolites in the female samples and 39/93 metabolites in the male samples were significantly altered (using our previous cut-off criteria of p ^ 0.2) by DR. The metabolites modulated by DR present a wide spectrum of concentration, redox reactivity and hydrophilicity, suggesting that our serotype is broadly representative of the metabolome and that DR has broad effects on the metabolome. These studies, coupled with those in the preceding and following reports, also highlight the utility for consideration of the metabolome as a network of metabolites using appropriate data analysis approaches. The inter-cohort and inter-gender differences addressed herein suggest potential cautions, and potential approaches, for identification of multivariate biomarker profiles that reflect changes in physiological status, such as a metabolism that predisposes to increased risk of neoplasia

Development of Biomarkers Based on Diet-Dependent Metabolic Serotypes: Practical Issues in Development of Expert System-Based Classification Models in Metabolomic Studies

This is the publisher's official version, also available electronically from: http://online.liebertpub.com/doi/pdfplus/10.1089/omi.2004.8.197Dietary restriction (DR)-induced changes in the serum metabolome may be biomarkers for physiological status (e.g., relative risk of developing age-related diseases such as cancer). Megavariate analysis (unsupervised hierarchical cluster analysis IHCAJ; principal components analysis [PCAJ) of serum metabolites reproducibly distinguish DR from ad libitum fed rats. Component-based approaches (i.e., PCA) consistently perform as well as or better than distance-based metrics (i.e., HCA). We therefore tested the following: (A) Do identified subsets of serum metabolites contain sufficient information to construct mathematical models of class membership (i.e., expert systems)? (B) Do component-based metrics out-perform distance-based metrics? Testing was conducted using KNN (k-nearest neighbors, supervised HCA) and SIMCA (soft independent modeling of class analogy, supervised PCA). Models were built with single cohorts, combined cohorts or mixed samples from previously studied cohorts as training sets. Both algorithms over-fit models based on single cohort training sets. KNN models had >85% accuracy within training/test sets, but were unstable (i.e., values of k could not be accurately set in advance). SIMCA models had 100% accuracy within all training sets, 89% accuracy in test sets, did not appear to over-fit mixed cohort training sets, and did not require post-hoc modeling adjustments. These data indicate that (i) previously defined metabolites are robust enough to construct classification models (expert systems) with SIMCA that can predict unknowns by dietary category; (ii) component-based analyses outperformed distance-based metrics; (iii) use of over-fitting controls is essential; and (iv) subtle inter-cohort variability may be a critical issue for high data density biomarker studies that lack state markers