The cytokine tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 have a neuroprotective effect in the central nervous system

Abstract

Background: Cerebral 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.

Methods: Here 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.

Results: We 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.

Conclusions: Our 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.

Figure 1

The interaction between TWEAK and Fn14 induces hypoxic tolerance. (A) Mean cell survival in Wt cerebral cortical neurons incubated 1 or 24 hours with TWEAK 100 ng/mL or 300 ng/mL. *P < 0.05 compared to cells incubated for 60 minutes with TWEAK 100 ng/mL. **P < 0.05 compared to cells incubated for 60 minutes with TWEAK 300 ng/mL. n = 15 per experimental group. Lines denote SD. (B) Mean cell survival in Wt cerebral cortical neurons exposed to 55 minutes of OGD conditions 24 hours after 60 minutes of incubation with TWEAK 0 to 300 ng/mL. n = 20 per experimental group; *, ** and *** P < 0.05 compared to neurons exposed to 55 minutes of OGD conditions without preconditioning. Lines denote SD. Values are given as cell survival compared to cells exposed to OGD conditions without preconditioning with TWEAK. (C) Wt cerebral cortical neurons were preconditioned with TWEAK for 60 minutes 24 hours before exposure to OGD conditions. Mean cell survival was determined with an MTT assay 24, 48 and 72 hours later. n = 12 per experimental group; *P < 0.05 compared to non-preconditioned neurons at each time point. Lines denote SD. (D) Mean cell survival in Wt and Fn14^-/- cerebral cortical neurons exposed to 55 minutes of OGD conditions 24 hours after 60 minutes of incubation with TWEAK 100 ng/mL. n = 10; *P < 0.05 compared to Wt neurons non-preconditioned with TWEAK. Lines denote SD. Fn14: fibroblast growth factor-inducible 14; Ns: non-significant; OGD: oxygen-glucose deprivation; TWEAK: tumor necrosis factor-like weak inducer of apoptosis; Wt: wild-type.

Figure 2

Endogenous TWEAK mediates the neuroprotective effect of hypoxic preconditioning. (A) Mean fold increase in TWEAK and Fn14 mRNA expression in Wt cerebral cortical neurons 1, 3 and 6 hours after exposure to 30 minutes of OGD (sub-lethal hypoxia). n = 8, * and ** P < 0.05 compared to TWEAK mRNA expression in sister cultures maintained under normoxic conditions. ^, § and ¶ P < 0.05 compared to Fn14 expression in sister cultures maintained under normoxic conditions. Lines denote SD. (B-D) Mean cell survival in Wt (B), TWEAK^-/- (C) and Fn14^-/- (D) cerebral cortical neurons exposed to 55 minutes of OGD conditions (lethal hypoxia) 24 hours after 30 minutes of exposure to OGD conditions (hypoxic preconditioning, dark gray bars). A subgroup of neurons was exposed to lethal hypoxia without previous preconditioning (black bar). A sub-set of TWEAK^-/- and Fn14^-/- neurons was incubated with TWEAK 100 ng/mL during the preconditioning phase (hypoxic preconditioning, light gray bars). n = 12 per experimental group; * in B P < 0.05 compared to cells maintained under normoxic conditions; ** in B P <0.05 compared to neuronal cultures exposed to lethal hypoxia without preconditioning. * in C P < 0.05 compared to TWEAK^-/- neurons either exposed to lethal OGD without previous preconditioning or preconditioned in the absence of TWEAK. Lines denote SD. Fn14: fibroblast growth factor-inducible 14; HPC: hypoxic preconditioning; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; Ns: non-significant; OGD: oxygen-glucose deprivation; TWEAK: tumor necrosis factor-like weak inducer of apoptosis.

Figure 3

Treatment with TWEAK induces ischemic tolerance in vivo. Wt and Fn14^-/- mice treated with vehicle (control; black bars) or TWEAK 0.2 mg by intraperitoneal injection (gray bars) 24 hours before tMCAO. The volume of the ischemic lesion was quantified in Wt mice 24 and 48 hours later. *P < 0.05 compared to non-preconditioned Wt mice. n = 10 per experimental group. Lines denote SD. Fn14: fibroblast growth factor-inducible 14; Ns: non-significant; tMCAO: transient middle cerebral artery occlusion; TTC: triphenyltetrazolium chloride; TWEAK: tumor necrosis factor-like weak inducer of apoptosis; Wt: wild-type.

Figure 4

TNF-α mediates the neuroprotective effect of TWEAK. (A) Mean concentration of TNF-α in the culture media of Wt cerebral cortical neurons 0 to 60 minutes after 60 minutes of incubation with TWEAK 100 ng/mL. n = 8, * and ** P < 0.05 compared to untreated sister cultures. Lines denote SD. (B) Mean neuronal survival in Wt cerebral cortical neurons exposed to 55 minutes of OGD conditions 24 hours after 60 minutes of incubation with TWEAK 100 ng/mL alone or in the presence of anti-TNF-α 0.04 μg/mL or anti-TNFR1 100 μg/mL antibodies or an immunoglobulin G isotype control. n = 12, *P < 0.05 compared to neurons exposed to OGD conditions without preconditioning with TWEAK, **P < 0.05 compared to cells preconditioned with TWEAK in the presence of anti-TNF-α or anti-TNFR1 antibodies. Lines denote SD. (C) Mean neuronal survival in TNF-α^-/- neurons incubated for 60 minutes with TWEAK 0 to 300 ng/mL 24 hours before exposure to 55 minutes of OGD conditions. n = 10 per group. (D) Volume of the ischemic lesion in Wt and TNF-α^-/- mice treated with vehicle control (white bars) or TWEAK 0.2 mg by intraperitoneal injection (gray bars) 24 hours before tMCAO. *P < 0.05 compared to non-preconditioned Wt mice. n = 12 per experimental group. Lines denote SD. Ig G: immunoglobulin G; Ns: non-significant; OGD: oxygen-glucose deprivation; TNF: tumor necrosis factor; TNFR1: tumor necrosis factor receptor 1; TWEAK: tumor necrosis factor-like weak inducer of apoptosis; Wt: wild-type.

Figure 5

ERK 1/2 mediates the TWEAK-induced hypoxic and/or ischemic tolerance. (A) Representative western blot analysis for pERK 1/2 in Wt cerebral cortical neurons incubated for 0 to 180 minutes with TWEAK 100 ng/mL alone or in combination with SL327 10 μM. (B) Mean neuronal survival in Wt cerebral cortical neurons exposed to 55 minutes of oxygen-glucose deprivation conditions 24 hours after 60 minutes of incubation with TWEAK 100 ng/mL alone (white bar) or in combination with either wortmannin 100 nM (dark gray bar) or the ERK 1/2 inhibitor SL327 (light gray bar). n = 16 per group, *P < 0.05 compared to neurons maintained under normoxic conditions, **P < 0.05 compared to neurons preconditioned with TWEAK alone. Lines denote SD. (C) Volume of the ischemic lesion in Wt mice treated with vehicle (control, black bar) or TWEAK 0.2 mg alone (white bar) or in combination with the ERK 1/2 inhibitor SL327 10 uM (gray bar) 24 hours before tMCAO. *P < 0.05 compared to non-preconditioned mice and with mice preconditioned with TWEAK alone. n = 8 per experimental group. Lines denote SD. Ns: non-significant; OGD: oxygen-glucose deprivation; pERK: ERK 1/2 phosphorylated at Thr202/Tyr204; tERK: total ERK; tMCAO: transient middle cerebral artery occlusion; TWEAK: tumor necrosis factor-like weak inducer of apoptosis; Wort: wortmannin; Wt: wild-type.

Figure 6

Effect of preconditioning with TWEAK on cerebral ischemia-induced cell death. (A) Representative micrographs of Wt cerebral cortical neurons immunostained for pBAD 3 hours after 60 minutes of treatment with either vehicle (control; panels a-c) or TWEAK 100 ng/mL (panels d-f). Green is pBAD, blue is DAPI. Magnification × 40. (B) Representative western blot analysis for pBAD and total BAD expression 0 to 180 minutes after 60 minutes of incubation with TWEAK alone or in combination with SL327 10 μM. (C) Representative micrographs of TUNEL staining in area of AOI2 in Wt mice intraperitoneally injected with saline solution (panels a and b), TWEAK 0.2 mg (panels b and c), or with a combination of TWEAK and SL327 (panels c and f) 24 hours before tMCAO. Green is TUNEL, blue is DAPI. Magnification × 20. (D) Diagram showing the three different AOIs and percentage of TUNEL-positive cells per field AOI 1, 2 and 3 in the ischemic tissue of Wt mice intraperitoneally injected with saline solution (black bar), TWEAK 0.2 mg (white bar), or TWEAK plus SL327 24 hours before tMCAO. n = 10, *P < 0.05 compared to untreated mice. Lines depict SD. AOI: area of interest; BAD: Bcl-2-associated death promoter protein; DAPI: 4'-6-diamidino-2-phenylindole; pBAD: BAD phosphorylated at Ser112; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; TWEAK: tumor necrosis factor-like weak inducer of apoptosis.