Abstract
BackgroundCerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia. However, the brain has the ability to detect and accommodate to hypoxic conditions. This phenomenon, known as preconditioning, is a natural adaptive process highly preserved among species whereby exposure to sub-lethal hypoxia promotes the acquisition of tolerance to a subsequent lethal hypoxic injury. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are found in neurons and their expression is induced by exposure to sub-lethal hypoxia. Accordingly, in this work we tested the hypothesis that the interaction between TWEAK and Fn14 induces tolerance to lethal hypoxic and ischemic conditions.MethodsHere we used in vitro and in vivo models of hypoxic and ischemic preconditioning, an animal model of transient middle cerebral artery occlusion and mice and neurons genetically deficient in TWEAK, Fn14, or tumor necrosis factor alpha (TNF-α) to investigate whether treatment with recombinant TWEAK or an increase in the expression of endogenous TWEAK renders neurons tolerant to lethal hypoxia. We used enzyme-linked immunosorbent assay to study the effect of TWEAK on the expression of neuronal TNF-α, Western blot analysis to investigate whether the effect of TWEAK was mediated by activation of mitogen-activated protein kinases and immunohistochemical techniques and quantitative real-time polymerase chain reaction analysis to study the effect of TWEAK on apoptotic cell death.ResultsWe found that either treatment with recombinant TWEAK or an increase in the expression of TWEAK and Fn14 induce hypoxic and ischemic tolerance in vivo and in vitro. This protective effect is mediated by neuronal TNF-α and activation of the extracellular signal-regulated kinases 1 and 2 pathway via phosphorylation and inactivation of the B-cell lymphoma 2-associated death promoter protein.ConclusionsOur work indicate that the interaction between TWEAK and Fn14 triggers the activation of a cell signaling pathway that results in the induction of tolerance to lethal hypoxia and ischemia. These data indicate that TWEAK may be a potential therapeutic strategy to protect the brain from the devastating effects of an ischemic injury.
Highlights
Cerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia
Other reagents were recombinant tumor necrosis factor-like weak inducer of apoptosis (TWEAK), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (ATCC, Manassas, VA, USA) and the lactate dehydrogenase (LDH) release assay (Roche, Florence, SC, USA), an enzyme-linked immunosorbent assay (ELISA) kit for tumor necrosis factor (TNF)-a (Insight Genomics, Falls Church, VA, USA), antibodies against tumor necrosis factor alpha (TNF-a) and TNF receptor 1 (TNFR1) (R&D Systems), extracellular signal-regulated kinases 1 and 2 (ERK 1/2) phosphorylated at Thr202/Tyr204, total ERK 1/2 and B-cell lymphoma 2 (Bcl-2)-associated death promoter protein (BAD) phosphorylated at Ser112(Cell Signaling, Danvers, MA, USA), b-actin
We found that the concentration of TWEAK in the culture media increased from 6 ± 1.3 pg/mL in cells maintained under normoxic conditions to 10.32 ± 3.64 pg/mL, 14.72 ± 3.47 pg/mL, 13.37 ± 0.7 ng/mL and 6.4 ± 2.5 pg/mL after 30, 60, 180 and 360 minutes of exposure to oxygen-glucose deprivation (OGD) conditions, respectively (n = 8 per experimental group; P < 0.05 when cells exposed to 30, 60 and 180 minutes of OGD were compared to neurons maintained under normoxic conditions; results were non-significant when cells exposed to 6 hours of OGD conditions were compared to control neurons)
Summary
Cerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia. The brain has the ability to detect and accommodate to hypoxic conditions This phenomenon, known as preconditioning, is a natural adaptive process highly preserved among species whereby exposure to sub-lethal hypoxia promotes the acquisition of tolerance to a subsequent lethal hypoxic injury. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are found in neurons and their expression is induced by exposure to sub-lethal hypoxia. In this work we tested the hypothesis that the interaction between TWEAK and Fn14 induces tolerance to lethal hypoxic and ischemic conditions. Exposure to a sub-lethal injury, including a short episode of hypoxia and/or ischemia, renders neurons resistant to a subsequent lethal hypoxic or ischemic insult [2]. Fibroblast growth factor-inducible 14 (Fn14) is the receptor for TWEAK [7] and binding of TWEAK to Fn14 has been reported to stimulate cell proliferation [8,9,10], migration [9,10,11] and differentiation [12], as well as the expression of pro-inflammatory molecules [4,9,13,14,15,16,17]
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