The Experts below are selected from a list of 38700 Experts worldwide ranked by ideXlab platform
Kohji Fukunaga - One of the best experts on this subject based on the ideXlab platform.
-
role of pericyte derived senp1 in neuronal injury after Brain Ischemia
CNS Neuroscience & Therapeutics, 2020Co-Authors: Meiling Sun, Kohji Fukunaga, Xiang Chen, Yi Xuan Yin, Yinping Gao, Li Zhang, Boqian Chen, Feng HanAbstract:Aims SUMOylation is a posttranslational modification related to multiple human diseases. SUMOylation can be reversed by classes of proteases known as the sentrin/SUMO-specific proteases (SENPs). In the present study, we investigate the potential role of SENP1 in pericytes in the Brain Ischemia. Methods Pericyte-specific deletion of senp1 mice (Cspg4-Cre; senp1f/f ) were used for Brain function and neuronal damage evaluation following Brain Ischemia. The cerebral blood vessels of diameter, velocity, and flux were performed in living mice by two-photon laser scanning microscopy (TPLSM). Biochemical analysis and immunohistochemistry methods were used to address the role and mechanism of pericyte-specific SENP1 in the pathological process of Brain Ischemia. A coculture model of HBVPs and HBMECs mimicked the BBB in vitro and was used to evaluate BBB integrity after glucose deprivation. Results Our results showed that senp1-specific deletion in pericytes did not affect the motor function and cognitive function of mice. However, the pericyte-specific deletion of senp1 aggravated the infarct size and motor deficit following focal Brain Ischemia. Consistently, the TPLSM data demonstrated that SENP1 deletion in pericytes accelerated thrombosis formation in Brain microvessels. We also found that pericyte-specific deletion of senp1 exaggerated the neuronal damage significantly following Brain Ischemia in mice. Moreover, SENP1 knockdown in pericytes could activate the apoptosis signaling and disrupt the barrier integrity in vitro coculture model. Conclusions Our findings revealed that targeting SENP1 in pericytes may represent a novel therapeutic strategy for neurovascular protection in stroke.
-
combined citicoline and docosahexaenoic acid treatment improves cognitive dysfunction following transient Brain Ischemia
Journal of Pharmacological Sciences, 2019Co-Authors: Eri Nakazaki, Yasushi Yabuki, Hisanao Izumi, Yasuharu Shinoda, Fumiko Watanabe, Yukihiro Hishida, Ayako Kamimura, Kohji FukunagaAbstract:Abstract Phospholipids are structural components of cellular membranes that play important roles as precursors for various signaling pathways in modulating neuronal membrane function and maintenance of the intracellular environment. Phosphatidylcholine (PtdCho) is the most abundant cellular phospholipid. Citicoline and docosahexaenoic acid (DHA) are essential intermediates in the synthesis of PtdCho. Both PtdCho intermediates have independently shown neuroprotective effects in cerebral Ischemia, but their combined effect is unknown. This study aimed to investigate the combined effect of oral citicoline and DHA treatment on improvement of cognitive deficits following cerebral Ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. BCCAO ischemic mice were treated for a total of 11 days with a combination of citicoline (40 mg/kg body weight/day) and DHA (300 mg/kg body weight/day) or each alone. Combined citicoline and DHA synergistically and significantly improved learning and memory ability of ischemic mice compared with either alone. Further, citicoline and DHA treatment significantly prevented neuronal cell death, and slightly increased DHA-containing PtdCho in the hippocampus, albeit not significantly. Taken together, these findings suggest that combined citicoline and DHA treatment may have synergistic benefits for partially improving memory deficits following transient Brain Ischemia.
-
Combined citicoline and docosahexaenoic acid treatment improves cognitive dysfunction following transient Brain Ischemia
Elsevier, 2019Co-Authors: Eri Nakazaki, Yasushi Yabuki, Hisanao Izumi, Yasuharu Shinoda, Fumiko Watanabe, Yukihiro Hishida, Ayako Kamimura, Kohji FukunagaAbstract:Phospholipids are structural components of cellular membranes that play important roles as precursors for various signaling pathways in modulating neuronal membrane function and maintenance of the intracellular environment. Phosphatidylcholine (PtdCho) is the most abundant cellular phospholipid. Citicoline and docosahexaenoic acid (DHA) are essential intermediates in the synthesis of PtdCho. Both PtdCho intermediates have independently shown neuroprotective effects in cerebral Ischemia, but their combined effect is unknown. This study aimed to investigate the combined effect of oral citicoline and DHA treatment on improvement of cognitive deficits following cerebral Ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. BCCAO ischemic mice were treated for a total of 11 days with a combination of citicoline (40 mg/kg body weight/day) and DHA (300 mg/kg body weight/day) or each alone. Combined citicoline and DHA synergistically and significantly improved learning and memory ability of ischemic mice compared with either alone. Further, citicoline and DHA treatment significantly prevented neuronal cell death, and slightly increased DHA-containing PtdCho in the hippocampus, albeit not significantly. Taken together, these findings suggest that combined citicoline and DHA treatment may have synergistic benefits for partially improving memory deficits following transient Brain Ischemia. Keywords: Citicoline, DHA, Bilateral common carotid artery occlusion, Neuroprotection, Memor
-
dehydroepiandrosterone administration improves memory deficits following transient Brain Ischemia through sigma 1 receptor stimulation
Brain Research, 2015Co-Authors: Yasushi Yabuki, Hisanao Izumi, Yasuharu Shinoda, Norifumi Shioda, Tatuya Ikuno, Kohji FukunagaAbstract:Abstract Dehydroepiandrosterone (DHEA) is the most abundant neurosteroid synthesized de novo in the central nervous system. Oral DHEA administration elicits neuroprotection and cognitive improvement, but mechanisms underlying these functions in cerebral Ischemia have remained unclear. Since DHEA is the endogenous ligand for the sigma-1 receptor (σ1R), we determined whether oral DHEA administration prevents neuronal cell death and improves cognition via σ1R stimulation in Brain Ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. Twenty-four hours after BCCAO Ischemia, mice were administered DHEA (15 or 30 mg/kg p.o.) daily for 11 consecutive days. Memory deficits following Brain Ischemia were improved by DHEA administration dose-dependently. Accordingly, DHEA administration significantly prevented neuronal cell death in the hippocampal CA1 region in BCCAO mice. Interestingly, DHEA administration rescued decreases in Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) in the CA1 region. Moreover, DHEA administration significantly ameliorated decreases in adenosine 5ʹ-triphosphate (ATP) levels and decreased σ1R expression levels in CA1 following BCCAO Ischemia. Finally, co-treatment of mice with the σ1R antagonist NE-100 (1 mg/kg, p.o.) blocked DHEA effects on memory improvement and neuroprotection in ischemic mice. Taken together, DHEA prevents neuronal cell death and activates CaMKII via σ1R stimulation, thereby improving cognitive deficits following Brain Ischemia.
-
oral l citrulline administration improves memory deficits following transient Brain Ischemia through cerebrovascular protection
Brain Research, 2013Co-Authors: Yasushi Yabuki, Norifumi Shioda, Yui Yamamoto, Miyuki Shigano, Kota Kumagai, Masahiko Morita, Kohji FukunagaAbstract:Abstract l -citrulline ( l -Cit) is known to increase nitric oxide (NO) production via the increase of l -arginine ( l -Arg) concentration in the blood and improve endothelial dysfunction in cardiovascular diseases. However, little is known about the effects of l -Cit on cerebrovascular dysfunction. Here we showed that oral l -Cit administration prevents cerebrovascular injury following cerebral Ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. After BCCAO Ischemia, mice were treated with l -Cit (50, 75, or 100 mg/kg p.o.) for 10 days once a day. l -Cit administration not only prevented neuronal cell death but also prevented capillary loss in the hippocampal region following Brain Ischemia. The cerebrovascular protective effect of l -Cit was associated with the restoration of endothelial nitric oxide synthase (eNOS) expression in the hippocampus. In addition, we devised a novel protocol to analyze NO x − (NO2− and NO3−) productions following l -Arg infusion using in vivo microdialysis and revealed that decreased l -Arg-induced NO x − levels were improved in the hippocampus of BCCAO mice following repeated l -Cit administration. Finally, memory deficits following Brain Ischemia were improved by oral administration of l -Cit. In summary, l -Cit is a potential therapeutic agent that protects cerebrovascular injury and in turn prevents neuronal cell death. Thereby, oral l -Cit administration improves cognitive deficits following Brain Ischemia.
Steven Warach - One of the best experts on this subject based on the ideXlab platform.
-
early blood Brain barrier disruption in human focal Brain Ischemia
Annals of Neurology, 2004Co-Authors: Lawrence L Latour, Julio A. Chalela, Dongwha Kang, Mustapha A. Ezzeddine, Steven WarachAbstract:Loss of integrity of the blood–Brain barrier (BBB) resulting from Ischemia/reperfusion is believed to be a precursor to hemorrhagic transformation (HT) and poor outcome. We used a novel magnetic resonance imaging marker to characterize early BBB disruption in human focal Brain Ischemia and tested for associations with reperfusion, HT, and poor outcome (modified Rankin score >2). BBB disruption was found in 47 of 144 (33%) patients, having a median time from stroke onset to observation of 10.1 hours. Reperfusion was found to be the most powerful independent predictor of early BBB disruption (p = 0.018; odds ratio, 4.09; 95% confidence interval, 1.28–13.1). HT was observed in 22 patients; 16 (72.7%) of those also had early BBB disruption (p 6), early BBB disruption was found to be an independent predictor of HT. Because the timing of the disruption was early enough to make it relevant to acute thrombolytic therapy, early BBB disruption as defined by this imaging biomarker may be a promising target for adjunctive therapy to reduce the complications associated with thrombolytic therapy, broaden the therapeutic window, and improve clinical outcome. Ann Neurol 2004
-
early blood Brain barrier disruption in human focal Brain Ischemia
Annals of Neurology, 2004Co-Authors: Lawrence L Latour, Julio A. Chalela, Dongwha Kang, Mustapha A. Ezzeddine, Steven WarachAbstract:Loss of integrity of the blood-Brain barrier (BBB) resulting from Ischemia/reperfusion is believed to be a precursor to hemorrhagic transformation (HT) and poor outcome. We used a novel magnetic resonance imaging marker to characterize early BBB disruption in human focal Brain Ischemia and tested for associations with reperfusion, HT, and poor outcome (modified Rankin score >2). BBB disruption was found in 47 of 144 (33%) patients, having a median time from stroke onset to observation of 10.1 hours. Reperfusion was found to be the most powerful independent predictor of early BBB disruption (p = 0.018; odds ratio, 4.09; 95% confidence interval, 1.28-13.1). HT was observed in 22 patients; 16 (72.7%) of those also had early BBB disruption (p 6), early BBB disruption was found to be an independent predictor of HT. Because the timing of the disruption was early enough to make it relevant to acute thrombolytic therapy, early BBB disruption as defined by this imaging biomarker may be a promising target for adjunctive therapy to reduce the complications associated with thrombolytic therapy, broaden the therapeutic window, and improve clinical outcome.
Mustapha A. Ezzeddine - One of the best experts on this subject based on the ideXlab platform.
-
early blood Brain barrier disruption in human focal Brain Ischemia
Annals of Neurology, 2004Co-Authors: Lawrence L Latour, Julio A. Chalela, Dongwha Kang, Mustapha A. Ezzeddine, Steven WarachAbstract:Loss of integrity of the blood–Brain barrier (BBB) resulting from Ischemia/reperfusion is believed to be a precursor to hemorrhagic transformation (HT) and poor outcome. We used a novel magnetic resonance imaging marker to characterize early BBB disruption in human focal Brain Ischemia and tested for associations with reperfusion, HT, and poor outcome (modified Rankin score >2). BBB disruption was found in 47 of 144 (33%) patients, having a median time from stroke onset to observation of 10.1 hours. Reperfusion was found to be the most powerful independent predictor of early BBB disruption (p = 0.018; odds ratio, 4.09; 95% confidence interval, 1.28–13.1). HT was observed in 22 patients; 16 (72.7%) of those also had early BBB disruption (p 6), early BBB disruption was found to be an independent predictor of HT. Because the timing of the disruption was early enough to make it relevant to acute thrombolytic therapy, early BBB disruption as defined by this imaging biomarker may be a promising target for adjunctive therapy to reduce the complications associated with thrombolytic therapy, broaden the therapeutic window, and improve clinical outcome. Ann Neurol 2004
-
early blood Brain barrier disruption in human focal Brain Ischemia
Annals of Neurology, 2004Co-Authors: Lawrence L Latour, Julio A. Chalela, Dongwha Kang, Mustapha A. Ezzeddine, Steven WarachAbstract:Loss of integrity of the blood-Brain barrier (BBB) resulting from Ischemia/reperfusion is believed to be a precursor to hemorrhagic transformation (HT) and poor outcome. We used a novel magnetic resonance imaging marker to characterize early BBB disruption in human focal Brain Ischemia and tested for associations with reperfusion, HT, and poor outcome (modified Rankin score >2). BBB disruption was found in 47 of 144 (33%) patients, having a median time from stroke onset to observation of 10.1 hours. Reperfusion was found to be the most powerful independent predictor of early BBB disruption (p = 0.018; odds ratio, 4.09; 95% confidence interval, 1.28-13.1). HT was observed in 22 patients; 16 (72.7%) of those also had early BBB disruption (p 6), early BBB disruption was found to be an independent predictor of HT. Because the timing of the disruption was early enough to make it relevant to acute thrombolytic therapy, early BBB disruption as defined by this imaging biomarker may be a promising target for adjunctive therapy to reduce the complications associated with thrombolytic therapy, broaden the therapeutic window, and improve clinical outcome.
Gary S. Krause - One of the best experts on this subject based on the ideXlab platform.
-
insulin activates the pi3k akt survival pathway in vulnerable neurons following global Brain Ischemia
Neurological Research, 2009Co-Authors: Thomas H Sanderson, Gary S. Krause, Rita Kumar, Alina C Murariudobrin, Andrea B Page, Jonathon M SullivanAbstract:AbstractInsulin is neuroprotective following transient global Brain Ischemia; however, the mechanisms by which insulin exerts its salutary effects remain unclear. Objective: We assessed insulin's effect on the PI3K-Akt survival system and consequent modulation of the pro-apoptotic proteins Bim, Bad and FoxO3a. Methods: We utilized rats subjected to 10 minutes of global Brain Ischemia, with or without insulin administered at the onset of reperfusion. Results: In sham-operated animals, minimal pAkt immunofluorescence was detected in the CA1. Moreover, at 30 minute reperfusion, there was no change in pAkt in CA1 neurons. Single bolus high-dose insulin treatment resulted in an early increase in pAkt after 30 minutes, preservation of CA1 neurons to 14 days of reperfusion and preservation of spatial learning ability. Insulin treatment increased cytoplasmic and nuclear staining for pAkt in both CA1 and cortex. Insulin-induced Akt phosphorylation was suppressed by the PI3K inhibitor wortmannin. Neither reperfusio...
-
perk is responsible for the increased phosphorylation of eif2α and the severe inhibition of protein synthesis after transient global Brain Ischemia
Journal of Neurochemistry, 2005Co-Authors: Cheri R Owen, Rita Kumar, Peichuan Zhang, Barbara C Mcgrath, Douglas R Cavener, Gary S. KrauseAbstract:Reperfusion after global Brain Ischemia results initially in a widespread suppression of protein synthesis in neurons that is due to inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2). To address the role of the eIF2alpha kinase RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) in the reperfused Brain, transgenic mice with a targeted disruption of the Perk gene were subjected to 20 min of foreBrain Ischemia followed by 10 min of reperfusion. In wild-type mice, phosphorylated eIF2alpha was detected in the non-ischemic Brain and its levels were elevated threefold after 10 min of reperfusion. Conversely, there was no phosphorylated eIF2alpha detected in the non-ischemic transgenic mice and there was no sizeable rise in phosphorylated eIF2alpha levels in the foreBrain after Ischemia and reperfusion. Moreover, there was a substantial rescue of protein translation in the reperfused transgenic mice. Neither group showed any change in total eIF2alpha, phosphorylated eukaryotic elongation factor 2 or total eukaryotic elongation factor 2 levels. These data demonstrate that PERK is responsible for the large increase in phosphorylated eIF2alpha and the suppression of translation early in reperfusion after transient global Brain Ischemia.
-
dysfunction of the unfolded protein response during global Brain Ischemia and reperfusion
Journal of Cerebral Blood Flow and Metabolism, 2003Co-Authors: Rita Kumar, Gary S. Krause, Hiderou Yoshida, Kazutoshi Mori, Donald J. DegraciaAbstract:A variety of endoplasmic reticulum (ER) stresses trigger the unfolded protein response (UPR), a compensatory response whose most proximal sensors are the ER membrane-bound proteins ATF6, IRE1alpha, and PERK. The authors simultaneously examined the activation of ATF6, IRE1alpha, and PERK, as well as components of downstream UPR pathways, in the rat Brain after reperfusion after a 10-minute cardiac arrest. Although ATF6 was not activated, PERK was maximally activated at 10-minute reperfusion, which correlated with maximal eIF2alpha phosphorylation and protein synthesis inhibition. By 4-h reperfusion, there was 80% loss of PERK immunostaining in cortex and 50% loss in Brain stem and hippocampus. PERK was degraded in vitro by mu-calpain. Although inactive IRE1alpha was maximally decreased by 90-minute reperfusion, there was no evidence that its substrate xbp-1 messenger RNA had been processed by removal of a 26-nt sequence. Similarly, there was no expression of the UPR effector proteins 55-kd XBP-1, CHOP, or ATF4. These data indicate that there is dysfunction in several key components of the UPR that abrogate the effects of ER stress. In other systems, failure to mount the UPR results in increased cell death. As other studies have shown evidence for ER stress after Brain Ischemia and reperfusion, the failure of the UPR may play a significant role in reperfusion neuronal death.
-
global Brain Ischemia and reperfusion modifications in eukaryotic initiation factors associated with inhibition of translation initiation
Journal of Neurochemistry, 2002Co-Authors: Donald J. Degracia, Robert W. Neumar, Blaine C. White, Gary S. KrauseAbstract:We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors elF-4E, elF-4G, and elF-2 alpha to examine the effects of global Brain Ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from Brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal foreBrain homogenates was approximately 0.4 fmol of leucine/min/microgram of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global Brain Ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of elF-4E. However, we observed in all 90-min-reperfused samples elF-4G fragments that also bound elF-4E. The amount of elF-2 alpha was not altered by Ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated elF-2 alpha in normal samples or in those obtained after Ischemia without reperfusion. However, serine-phosphorylated elF-2 alpha was uniformly present after 90 min of reperfusion and represented 24 +/- 3% of the elF-2 alpha in these samples. The serine phosphorylation of elF-2 alpha and partial fragmentation of elF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.
-
Brain Ischemia and reperfusion molecular mechanisms of neuronal injury
Journal of the Neurological Sciences, 2000Co-Authors: Blaine C. White, Donald J. Degracia, Robert W. Neumar, Jonathon M Sullivan, Brian J Oneil, Lawrence I Grossman, Jose A Rafols, Gary S. KrauseAbstract:Brain Ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of Brain Ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.
Donald J. Degracia - One of the best experts on this subject based on the ideXlab platform.
-
texture analysis of poly adenylated mrna staining following global Brain Ischemia and reperfusion
Computer Methods and Programs in Biomedicine, 2012Co-Authors: Jeffrey J Szymanski, Jill T Jamison, Donald J. DegraciaAbstract:Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1h of reperfusion after 10min of normothermic global cerebral Ischemia. At 1h reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after Brain Ischemia, on mRNA granules. If administered before Ischemia, cycloheximide inhibited mRNA granule formation, but if administered after Ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by Brain Ischemia and reperfusion.
-
hippocampal cellular stress responses after global Brain Ischemia and reperfusion
Antioxidants & Redox Signaling, 2007Co-Authors: George Roberts, Mike J Di Loreto, Monique Marshall, Jie Wang, Donald J. DegraciaAbstract:Brain Ischemia and reperfusion (I/R) induce neuronal intracellular stress responses, including the heat-shock response (HSR) and the unfolded protein response (UPR), but the roles of each in neuronal survival or death are not well understood. We assessed the relative expression of UPR (ATF4, CHOP, GRP78, XBP-1) and HSR-related (HSP70 and HSC70) mRNAs and proteins after Brain I/R. We evaluated these in hippocampal CA1 and CA3 after normothermic, transient global foreBrain Ischemia and up to 42 h of reperfusion. In CA1, chop and xbp-1 mRNA showed maximal 14- and 12-fold increases, and the only protein increase observed was for 30-kDa XBP-1. CA3 showed induction of only xbp-1. GRP78 protein declined in CA1, but increased twofold and then declined in CA3. Transcription of hsp70 was an order of magnitude greater than that of any UPR-induced transcript in either CA1 or CA3. HSP70 translation in CA1 lagged CA3 by ∼24 h. We conclude that (a) in terms of functional end products, the ER stress response after Brain ...
-
dysfunction of the unfolded protein response during global Brain Ischemia and reperfusion
Journal of Cerebral Blood Flow and Metabolism, 2003Co-Authors: Rita Kumar, Gary S. Krause, Hiderou Yoshida, Kazutoshi Mori, Donald J. DegraciaAbstract:A variety of endoplasmic reticulum (ER) stresses trigger the unfolded protein response (UPR), a compensatory response whose most proximal sensors are the ER membrane-bound proteins ATF6, IRE1alpha, and PERK. The authors simultaneously examined the activation of ATF6, IRE1alpha, and PERK, as well as components of downstream UPR pathways, in the rat Brain after reperfusion after a 10-minute cardiac arrest. Although ATF6 was not activated, PERK was maximally activated at 10-minute reperfusion, which correlated with maximal eIF2alpha phosphorylation and protein synthesis inhibition. By 4-h reperfusion, there was 80% loss of PERK immunostaining in cortex and 50% loss in Brain stem and hippocampus. PERK was degraded in vitro by mu-calpain. Although inactive IRE1alpha was maximally decreased by 90-minute reperfusion, there was no evidence that its substrate xbp-1 messenger RNA had been processed by removal of a 26-nt sequence. Similarly, there was no expression of the UPR effector proteins 55-kd XBP-1, CHOP, or ATF4. These data indicate that there is dysfunction in several key components of the UPR that abrogate the effects of ER stress. In other systems, failure to mount the UPR results in increased cell death. As other studies have shown evidence for ER stress after Brain Ischemia and reperfusion, the failure of the UPR may play a significant role in reperfusion neuronal death.
-
global Brain Ischemia and reperfusion modifications in eukaryotic initiation factors associated with inhibition of translation initiation
Journal of Neurochemistry, 2002Co-Authors: Donald J. Degracia, Robert W. Neumar, Blaine C. White, Gary S. KrauseAbstract:We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors elF-4E, elF-4G, and elF-2 alpha to examine the effects of global Brain Ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from Brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal foreBrain homogenates was approximately 0.4 fmol of leucine/min/microgram of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global Brain Ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of elF-4E. However, we observed in all 90-min-reperfused samples elF-4G fragments that also bound elF-4E. The amount of elF-2 alpha was not altered by Ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated elF-2 alpha in normal samples or in those obtained after Ischemia without reperfusion. However, serine-phosphorylated elF-2 alpha was uniformly present after 90 min of reperfusion and represented 24 +/- 3% of the elF-2 alpha in these samples. The serine phosphorylation of elF-2 alpha and partial fragmentation of elF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.
-
Brain Ischemia and reperfusion molecular mechanisms of neuronal injury
Journal of the Neurological Sciences, 2000Co-Authors: Blaine C. White, Donald J. Degracia, Robert W. Neumar, Jonathon M Sullivan, Brian J Oneil, Lawrence I Grossman, Jose A Rafols, Gary S. KrauseAbstract:Brain Ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of Brain Ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.