Hsp70 Overexpression Sequesters AIF and Reduces Neonatal Hypoxic/Ischemic Brain Injury

Apoptosis is implicated in neonatal hypoxic/ischemic (H/I) brain injury among various forms of cell death. Here we investigate whether overexpression of heat shock protein (Hsp) 70, an antiapoptotic protein, protects the neonatal brain from H/I injury and the pathways involved in the protection. Postnatal day 7 (P7) transgenic mice overexpressing rat Hsp70 (Tg) and their wild-type littermates (Wt) underwent unilateral common carotid artery ligation followed by 30 mins exposure to 8% O2. Significant neuroprotection was observed in Tg versus Wt mice on both P12 and P21, correlating with a high level of constitutive but not inducible Hsp70 in the Tg. More prominent injury was observed in Wt and Tg mice on P21, suggesting its continuous evolution after P12. Western blot analysis showed that translocation of cytochrome c, but not the second mitochondria-derived activator of caspase (Smac)/DIABLO and apoptosis-inducing factor (AIF), from mitochondria into cytosol was significantly reduced in Tg 24 h after H/I compared with Wt mice. Coimmunoprecipitation detected more Hsp70 bound to AIF in Tg than Wt mice 24 h after H/I, inversely correlating with the amount of nuclear, but not cytosolic, AIF translocation. Our results suggest that interaction between Hsp70 and AIF might have reduced downstream events leading to cell death, including the reduction of nuclear AIF translocation in the neonatal brains of Hsp70 Tg mice after H/I.

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p53-mediated neuronal cell death in ischemic brain injury

Neuroscience Bulletin ◽

10.1007/s12264-010-1111-0 ◽

2010 ◽

Vol 26 (3) ◽

pp. 232-240 ◽

Cited By ~ 54

Author(s):

Li-Zhi Hong ◽

Xiao-Yuan Zhao ◽

Hui-Ling Zhang

Keyword(s):

Cell Death ◽

Brain Injury ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Ischemic Brain Injury ◽

Ischemic Brain

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Differential Modulation of Estrogen Receptors (ERs) in Ischemic Brain Injury: A Role for ERα in Estradiol-Mediated Protection against Delayed Cell Death

Endocrinology ◽

10.1210/en.2005-1177 ◽

2006 ◽

Vol 147 (6) ◽

pp. 3076-3084 ◽

Cited By ~ 137

Author(s):

Dena B. Dubal ◽

Shane W. Rau ◽

Paul J. Shughrue ◽

Hong Zhu ◽

Jin Yu ◽

...

Keyword(s):

Cell Death ◽

Brain Injury ◽

Estrogen Receptors ◽

Expression Profiles ◽

Artery Occlusion ◽

Aging Brain ◽

Ischemic Brain Injury ◽

Ischemic Brain ◽

Delayed Cell Death ◽

Injured Brain

Abstract Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERα and ERβ, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERα induction occurring early and ERβ modulation occurring later. Finally, we provide evidence for a new and functional role for ERα in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERα.

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Roles of NAD+, PARP-1, and Sirtuins in Cell Death, Ischemic Brain Injury, and Synchrotron Radiation X-Ray-Induced Tissue Injury

Scientifica ◽

10.1155/2013/691251 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

Weihai Ying

Keyword(s):

Cell Death ◽

Brain Injury ◽

Synchrotron Radiation ◽

Brain Damage ◽

Tissue Injury ◽

Ischemic Brain Injury ◽

Biological Processes ◽

Ischemic Brain ◽

X Ray ◽

Parp 1

NAD+plays crucial roles in a variety of biological processes including energy metabolism, aging, and calcium homeostasis. Multiple studies have also shown that NAD+administration can profoundly decrease oxidative cell death and ischemic brain injury. A number of recent studies have further indicated that NAD+administration can decrease ischemic brain damage, traumatic brain damage and synchrotron radiation X-ray-induced tissue injury by such mechanisms as inhibiting inflammation, decreasing autophagy, and reducing DNA damage. Our latest study that applies nano-particles as a NAD+carrier has also provided first direct evidence demonstrating a key role of NAD+depletion in oxidative stress-induced ATP depletion. Poly(ADP-ribose) polymerase-1 (PARP-1) and sirtuins are key NAD+-consuming enzymes that mediate multiple biological processes. Recent studies have provided new information regarding PARP-1 and sirtuins in cell death, ischemic brain damage and synchrotron radiation X-ray-induced tissue damage. These findings have collectively supported the hypothesis that NAD+metabolism, PARP-1 and sirtuins play fundamental roles in oxidative stress-induced cell death, ischemic brain injury, and radiation injury. The findings have also supported “the Central Regulatory Network Hypothesis”, which proposes that a fundamental network that consists of ATP, NAD+and Ca2+as its key components is the essential network regulating various biological processes.

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Ethyl pyruvate protects against hypoxic-ischemic brain injury via anti-cell death and anti-inflammatory mechanisms

Neurobiology of Disease ◽

10.1016/j.nbd.2009.12.010 ◽

2010 ◽

Vol 37 (3) ◽

pp. 711-722 ◽

Cited By ~ 59

Author(s):

Hongxia Shen ◽

Xiaoming Hu ◽

Can Liu ◽

Suping Wang ◽

Wenting Zhang ◽

...

Keyword(s):

Cell Death ◽

Brain Injury ◽

Ethyl Pyruvate ◽

Ischemic Brain Injury ◽

Ischemic Brain ◽

Hypoxic Ischemic Brain Injury ◽

Anti Inflammatory

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Alteration in Downstream Hypoxia Gene Signaling in Neonatal Glutathione Peroxidase Overexpressing Mouse Brain after Hypoxia-Ischemia

Developmental Neuroscience ◽

10.1159/000375369 ◽

2015 ◽

Vol 37 (4-5) ◽

pp. 398-406 ◽

Cited By ~ 2

Author(s):

R. Ann Sheldon ◽

Raha Sadjadi ◽

Matthew Lam ◽

Russell Fitzgerald ◽

Donna M. Ferriero

Keyword(s):

Cell Death ◽

Brain Injury ◽

Glutathione Peroxidase ◽

Apoptotic Cell ◽

Hypoxia Inducible Factor ◽

Artery Ligation ◽

Epo Receptor ◽

Carotid Artery Ligation ◽

Mouse Cortex ◽

Hypoxia Ischemia

We have previously shown that glutathione peroxidase (GPx) overexpressing mice (hGPx-tg) have reduced brain injury after neonatal hypoxia-ischemia (HI) as a consequence of reduced hydrogen peroxide accumulation. However, this protection is reversed with hypoxia preconditioning, raising the question of the roles of the genes regulated by hypoxia-inducible factor-1α (HIF-1α) and their transcription products, such as erythropoietin (EPO), in both the initial protection and subsequent reversal of protection. hGPx-tg and their wild-type (WT) littermates underwent the Vannucci procedure of HI brain injury at postnatal day 9 - left carotid artery ligation followed by exposure to 10% oxygen for 50 min. Brain cortices and hippocampi were subsequently collected 0.5, 4 and 24 h later for the determination of protein expression by Western blot for GPx, HIF-1α, HIF-2α, EPO, EPO receptor, ERK1/2, phospho-ERK1/2, spectrin 145/150 (as a marker of calpain-specific necrotic cell death), and spectrin 120 (as a marker of apoptotic cell death mediated via caspase-3). As expected, the GPx overexpressing mouse cortex had approximately 3 times the GPx expression as WT naïve. Also, GPx expression remained higher in the GPx overexpressing brain than WT at all time points after HI (0.5, 4, 24 h). HIF-1α was not significantly changed in hGPx-tg as a consequence of HI but decreased in the WT cortex 4 h after HI. HIF-2α decreased in the WT hippocampus after HI. EPO was higher in the GPx overexpressing cortex and hippocampus 30 min after HI compared to WT, but the EPO receptor was unchanged by HI. ERK1/2 phosphorylation increased in the hippocampus at 4 h after HI and in the cortex at 24 h after HI in both WT and hGPx-tg. Spectrin 145/150 was increased in the WT cortex at 4 and 24 h after HI, and spectrin 120 increased 24 h after HI, perhaps reflecting greater injury in the WT brain, especially at 24 h when brain injury is more evident. The effect of GPx overexpression does not appear to upregulate the HIF pathway, yet EPO was upregulated, perhaps via ERK. This might explain, in part, why cell death takes a necrotic or apoptotic path. This may also be an explanation for why the GPx overexpressing brain cannot be preconditioned. This information may prove valuable in the development of therapies for neonatal HI brain injury.

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Hsp27 Protects against Ischemic Brain Injury via Attenuation of a Novel Stress-Response Cascade Upstream of Mitochondrial Cell Death Signaling

Journal of Neuroscience ◽

10.1523/jneurosci.4407-08.2008 ◽

2008 ◽

Vol 28 (49) ◽

pp. 13038-13055 ◽

Cited By ~ 88

Author(s):

R. A. Stetler ◽

G. Cao ◽

Y. Gao ◽

F. Zhang ◽

S. Wang ◽

...

Keyword(s):

Cell Death ◽

Brain Injury ◽

Stress Response ◽

Ischemic Brain Injury ◽

Ischemic Brain ◽

Cell Death Signaling

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STAT1 is Activated in Neurons after Ischemia and Contributes to Ischemic Brain Injury

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000034148.72481.f4 ◽

2002 ◽

Vol 22 (11) ◽

pp. 1311-1318 ◽

Cited By ~ 38

Author(s):

Yasushi Takagi ◽

Jun Harada ◽

Alberto Chiarugi ◽

Michael A. Moskowitz

Keyword(s):

Cell Death ◽

Brain Injury ◽

Strain Differences ◽

Cell Types ◽

Interferon Γ ◽

Artery Occlusion ◽

Cytokine Signaling ◽

Ischemic Brain Injury ◽

Ischemic Brain ◽

Phosphorylated Akt

Signal transducers and activators of transcription (STAT) proteins are a family of transcription factors that play a crucial role in growth and differentiation in a variety of cell types. Among them, STAT1, which is expressed in the brain and directly activated by reactive oxygen species, participates in the regulation of cytokine-signaling and cellular responses, particularly to interferon-γ. Very little, however, is known about the importance of STAT1 during brain injury. The authors found that STAT1 was phosphorylated at tyrosine701 and serine727 and translocated into neuronal nuclei within hours after middle cerebral artery occlusion. At later time points, STAT1 immunoreactivity colocalized with TUNEL-positive neurons, thereby suggesting a role in cell death. In mice genetically deficient in STAT1 expression, the volume of ischemic brain injury was reduced, neurologic deficits were less severe, and TUNEL-positive neurons were also less numerous compared with wild-type mice. STAT1-knockout mice showed increased phosphorylated Akt and decreased pro-***caspase-3 cleavage. Major strain differences in phosphorylated STAT3 or cyclooxygenase-2 protein expression were not found after ischemia. These results indicate that STAT1 is activated and translocated within ischemic neurons and may contribute to brain injury by regulating transcription and phosphorylation of proteins related to apoptosis and cell death.

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Neuroprotection against Focal Ischemic Brain Injury by Inhibition of c-Jun N-Terminal Kinase and Attenuation of the Mitochondrial Apoptosis-Signaling Pathway

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600062 ◽

2005 ◽

Vol 25 (6) ◽

pp. 694-712 ◽

Cited By ~ 163

Author(s):

Yanqin Gao ◽

Armando P. Signore ◽

Wei Yin ◽

Guodong Cao ◽

Xiao-Ming Yin ◽

...

Keyword(s):

Cell Death ◽

Brain Injury ◽

Signaling Pathway ◽

Strong Support ◽

Serine Phosphorylation ◽

Ischemic Brain Injury ◽

Mitochondrial Apoptosis ◽

Ischemic Brain ◽

Jnk Activation

c-Jun N-terminal kinase (JNK) is an important stress-responsive kinase that is activated by various forms of brain insults. In this study, we have examined the role of JNK activation in neuronal cell death in a murine model of focal ischemia and reperfusion; furthermore, we investigated the mechanism of JNK in apoptosis signaling, focusing on the mitochondrial-signaling pathway. We show here that JNK activity was induced in the brain 0.5 to 24 h after ischemia. Systemic administration of SP600125, a small molecule JNK-specific inhibitor, diminished JNK activity after ischemia and dose-dependently reduced infarct volume. c-Jun N-terminal kinase inhibition also attenuated ischemia-induced expression of Bim, Hrk/DP5, and Fas, but not the expression of Bcl-2 or FasL. In strong support of a role for JNK in promoting the mitochondrial apoptosis-signaling pathway, JNK inhibition prevented ischemia-induced mitochondrial translocation of Bax and Bim, release of cytochrome c and Smac, and activation of caspase-9 and caspase-3. The potential mechanism by which JNK promoted Bax translocation after ischemia was further studied using coimmunoprecipitation, and the results revealed that JNK activation caused serine phosphorylation of 14-3-3, a cytoplasmic sequestration protein of Bax, leading to Bax disassociation from 14-3-3 and subsequent translocation to mitochondria. These results confirm the role of JNK as a critical cell death mediator in ischemic brain injury, and suggest that one of the mechanisms by which JNK triggers the mitochondrial apoptosis-signaling pathway is via promoting Bax and Bim translocation.

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Ischemic Brain Injury is Mediated by the Activation of Poly(ADP-Ribose)Polymerase

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-199711000-00002 ◽

1997 ◽

Vol 17 (11) ◽

pp. 1143-1151 ◽

Cited By ~ 473

Author(s):

Matthias Endres ◽

Zhao-Qi Wang ◽

Shobu Namura ◽

Christian Waeber ◽

Michael A. Moskowitz

Keyword(s):

Cell Death ◽

Brain Injury ◽

Tissue Injury ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Terminal Deoxynucleotidyl Transferase ◽

Ischemic Brain Injury ◽

Cell Nuclei ◽

Ischemic Brain ◽

Nad Depletion

Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30), an abundant nuclear protein activated by DNA nicks, mediates cell death in vitro by nicotinamide adenine dinucleotide (NAD) depletion after exposure to nitric oxide. The authors examined whether genetic deletion of PARP (PARP null mice) or its pharmacologic inhibition by 3-aminobenzamide (3-AB) attenuates tissue injury after transient cerebral ischemia. Twenty-two hours after reperfusion following 2 hours of filamentous middle cerebral artery occlusion, ischemic injury was decreased in PARP−/− and PARP+/− mice compared with PARP+/+ litter mates, and also was attenuated in 129/SV wild-type mice after 3-AB treatment compared with controls. Infarct sparing was accompanied by functional recovery in PARP−/− and 3-AB–treated mice. Increased poly(ADP-ribose) immunostaining observed in ischemic cell nuclei 5 minutes after reperfusion was reduced by 3-AB treatment. Levels of NAD—the substrate of PARP—were reduced 2 hours after reperfusion and were 35% of contralateral levels at 24 hours. The decreases were attenuated in PARP−/− mice and in 3-AB–treated animals. Poly(ADP-ribose)polymerase cleavage by caspase-3 (CPP-32) has been proposed as an important step in apoptotic cell death. Markers of apoptosis, such as oligonucleosomal DNA damage, total DNA fragmentation, and the density of terminal deoxynucleotidyl transferase dUTP nick-end–labelled (TUNEL +) cells, however, did not differ in ischemic brain tissue of PARP−/− mice or in 3-AB–treated animals versus controls, although there were differences in the number of TUNEL-stained cells reflecting the decrease in infarct size. Thus, ischemic brain injury activates PARP and contributes to cell death most likely by NAD depletion and energy failure, although the authors have not excluded a role for PARP in apoptotic cell death at earlier or later stages in ischemic cell death. Inhibitors of PARP activation could provide a potential therapy in acute stroke.

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