BNIP3 Upregulation and EndoG Translocation in Delayed Neuronal Death in Stroke and in Hypoxia

Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada
Stroke (Impact Factor: 5.72). 06/2007; 38(5):1606-13. DOI: 10.1161/STROKEAHA.106.475129
Source: PubMed

ABSTRACT Delayed neuronal death is a hallmark feature of stroke and the primary target of neuroprotective strategies. Caspase-independent apoptosis pathways are suggested as a mechanism for the delayed neuronal injury. Here we test the hypothesis that one of the caspase-independent apoptosis pathways is activated by BNIP3 and mediated by EndoG.
We performed immunohistochemistry, Western blotting, cell transfection, subcellular fractionation, and RNA interfering to analyze the expression and localization of BNIP3 and EndoG in degenerating neurons in models of stroke and hypoxia.
BNIP3 was upregulated in brain neurons in a rat model of stroke and in cultured primary neurons exposed to hypoxia. The expressed BNIP3 was localized to mitochondria. Both forced expression of BNIP3 by plasmid transfection and induced expression of BNIP3 by hypoxia in neurons resulted in mitochondrial release and nuclear translocation of EndoG and neuronal cell death. Knockdown of BNIP3 by RNAi inhibited EndoG translocation and protected against hypoxia-induced neuronal death.
BNIP3 plays a role in delayed neuronal death in hypoxia and stroke and EndoG is a mediator of the BNIP3-activated neuronal death pathway. The results suggest that BNIP3 may be a new target for neuronal rescue strategies.

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    • "In the present study, co-incubation with ascorbic acid significantly reduced Co(II) and Co-NPs-induced BNIP3 upregulation and Co-NPs-induced GLUT1 upregulation (Figure 5b), while GSH had no effect on cobalt-induced GLUT1 upregulation and only decreased Co(II)-induced BNIP3 mRNA (Figure 5c). BNIP3 belongs to death-inducing mitochondrial proteins, which degrade damaged mitochondrial proteins in response to mitochondrial damage and its mRNA upregulation precedes hypoxia-induced cell death (Zhang et al., 2007). The observed decrease in Co-NPs-induced BNIP3 mRNA in the presence of ascorbic acid but not GSH further confirms a protective role for ascorbic acid and HIF involvement in Co-NP-induced toxicity. "
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    ABSTRACT: Abstract Recent, unexpected high failure rates of metal-on-metal hip implants have reintroduced the issue of cobalt toxicity. An adverse reaction to cobalt ions and cobalt-induced lung injury occurs during environmental exposure and is now strictly controlled. Currently adverse reaction occurs to cobalt nanoparticles during wear and tear of metal-on-metal hip implants of which the underlying mechanism is not fully understood. The putative role of the hypoxia-inducible factor (HIF) pathway in the mechanism of cobalt nanoparticle (Co-NPs) toxicity was examined using the U937 cell line, human alveolar macrophages and monocyte-derived macrophages. Co-NPs (5-20 μg/ml)-induced cytotoxicity (viability ranged from 75% to <20% of control, respectively) and reactive oxygen species (ROS), whereas a comparable concentration of cobalt ions (Co(II); up to 350 μM) did not. Co-NPs induced HIF-1α stabilization. Addition of ascorbic acid (100 µM) and glutathione (1 mM) both prevented the increased ROS. However, only treatment with ascorbic acid reduced HIF-1α levels and prevented cell death, indicating that a ROS-independent pathway is involved in Co-NPs-induced cytotoxicity. Replenishing intracellular ascorbate, which is crucial in preventing HIF pathway activation, modified Co-induced HIF target gene expression and the inflammatory response, by decreasing interleukin-1 beta (IL-1β) mRNA and protein expression. Addition of glutathione had no effect on Co-NPs-induced HIF target gene expression or inflammatory response. Thus, Co-NPs induce the HIF pathway by depleting intracellular ascorbate, leading to HIF stabilization and pathway activation. This suggests a strong, ROS-independent role for HIF activation in Co-NPs-induced cytotoxicity and a possible role for HIF in metal-on-metal hip implant pathology.
    Nanotoxicology 02/2015; DOI:10.3109/17435390.2014.991430 · 6.41 Impact Factor
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    • "Under conditions of oxidative stress and hypoxia, programmed cell death pathways are under control of an atypical BH3-only protein BNIP3 (Bcl2 and adenovirus E1B 19 kDa interacting protein 3) [5], [6], [7], [8], [9]. BNIP3 expression is up-regulated in settings of chronic ischemic injury of the heart, brain, liver and neurons [10], [11], [12], [13], [14]. The activity of BNIP3 is dependent upon cellular pH and redox status [15], [16], [17]. "
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    ABSTRACT: The Goldblatt's 2 kidney 1 clip (2K1C) rat animal model of renovascular hypertension is characterized by ischemic nephropathy of the clipped kidney. 2K1C rats were treated with a specific peroxisome proliferator-activated receptor δ (PPARδ) agonist, HPP593. Clipped kidneys from untreated rats developed tubular and glomerular necrosis and massive interstitial, periglomerular and perivascular fibrosis. HPP593 kidneys did not exhibit any histochemical features of necrosis; fibrotic lesions were present only in perivascular areas. Necrosis in the untreated clipped kidneys was associated with an increased oxidative stress, up regulation and mitochondrial translocation of the pro-death protein BNIP3 specifically in tubules. In the kidneys of HPP593-treated rats oxidative stress was attenuated and BNIP3 protein decreased notably in the mitochondrial fraction when compared to untreated animals. In untreated clipped kidneys, mitochondria were dysfunctional as revealed by perturbations in the levels of MCAD, COXIV, TFAM, and Parkin proteins and AMPK activation, while in HPP593-treated rats these proteins remained at the physiological levels. Nuclear amounts of oxidative stress-responsive proteins, NRF1 and NRF2 were below physiological levels in treated kidneys. Mitochondrial biogenesis and autophagy were inhibited similarly in both treated and untreated 2K1C kidneys as indicated by a decrease in PGC1-α and deficiency of the autophagy-essential proteins LC3-II and ATG5. However, HPP593 treatment resulted in increased accumulation of p62 protein, an autophagic substrate and an enhancer of NRF2 activity. Therefore, inhibition of BNIP3 activation by the preservation of mitochondrial function and control of oxidative stress by PPARδ is the most likely mechanism to account for the prevention of necrotic death in the kidney under conditions of persistent ischemia.
    PLoS ONE 05/2013; 8(5):e64436. DOI:10.1371/journal.pone.0064436 · 3.23 Impact Factor
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    • "In this line, it has been recently described that EndoG is also expressed in the brain (Zhang et al., 2011). Unfortunately, only few studies have addressed the role of EndoG in neuronal function and all of them have focused only in cell death processes (Zhang et al., 2007; Zhao et al., 2009). Thus, if EndoG plays a role in specific neural functions still remains to be explored. "
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    ABSTRACT: Endonuclease G (EndoG) has been largely related with a role in the modulation of a caspase-independent cell death pathway in many cellular systems. However, whether this protein plays a specific role in brain remains to be elucidated. Here we have characterized the behavioral phenotype of EndoG(-/-) null mice and the expression of the nuclease among brain regions. EndoG(-/-) mice showed normal neurological function, learning, motor coordination and spontaneous behaviors. However, these animals displayed lower activity in a running wheel and, strikingly, they were consistently less anxious compared to EndoG(+/+) mice in different tests for anxiety such as plus maze and dark-light test. We next evaluated the expression of EndoG in different brain regions of wild type mice and found that it was expressed in all over but specially enriched in the striatum. Further, subcellular biochemical experiments in neocortical samples from wild type mice revealed that EndoG is localized in pre-synaptic compartments but not in post-synaptic compartments. Altogether these findings suggest that EndoG could play a highly specific role in the regulation of anxiety by modulating synaptic components.
    Experimental Neurology 04/2013; 247. DOI:10.1016/j.expneurol.2013.03.028 · 4.70 Impact Factor
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