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

Murine strains were TWEAK deficient (TWEAK^-/-) and Fn14 deficient (Fn14^-/-; kindly provided by Dr. Kyungmin Hahm, Biogen Idec Inc., Cambridge, MA, USA) mice, and TNF-α deficient (TNF-α^-/-) mice and their wild-type (Wt) littermate controls on a C57BL/6 J genetic background. Other reagents were recombinant TWEAK (rTWEAK; R&D Systems, Minneapolis, MN, USA), 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 ELISA kit for TNF-α (Insight Genomics, Falls Church, VA, USA), antibodies against TNF-α and TNFR1 (R&D Systems), ERK 1/2 phosphorylated at Thr202/Tyr204 (pERK), total ERK 1/2 and BAD phosphorylated at Ser112 (pBAD)(Cell Signaling, Danvers, MA, USA), β-actin (Sigma Aldrich, St. Louis, MO, USA), the Mitogen Activated Protein Kinase (MAPK) extracellular signal-regulated kinase (MEK) inhibitor SL327 (Tocris Bioscience, Ellisville, MO, USA), wortmannin, the nuclear markers 4'-6-diamidino-2-phenylindole (DAPI) and triphenyltetrazolium chloride (TTC; Sigma-Aldrich), and the ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (Chemicon International, Billerica, MA, USA).

Transient occlusion of the middle cerebral artery (tMCAO) was induced in TWEAK^-/-, Fn14^-/- and TNF-α^-/- mice and their corresponding Wt littermate controls with a 6-0 silk suture advanced from the external carotid artery into the internal carotid artery until the origin of the middle cerebral artery (MCA), as described elsewhere [39]. Briefly, animals were anesthetized with 4% chloral hydrate (400 mg/kg intraperitoneal injection) and a nylon monofilament (6-0, Ethicon, Issy Les Moulineaux, France) coated with silicone was introduced through the external carotid artery and advanced up to the origin of the MCA. The suture was withdrawn after 60 minutes of cerebral ischemia. Cerebral perfusion in the distribution of the MCA was monitored throughout the surgical procedure and after reperfusion with a laser Doppler (Perimed Inc., North Royalton, OH, USA), and only animals with a > 70% decrease in cerebral perfusion after occlusion and complete recovery after suture withdrawal were included in this study. The rectal and masseter muscle temperatures were controlled at 37°C with a homoeothermic blanket. Heart rate, systolic, diastolic and mean arterial blood pressures were controlled throughout the surgical procedure with an IITC 229 System (IITC-Lice Science, Woodland Hills, CA, USA). From the total number of mice used in this study (155), 13 (8.3%) were excluded due to incomplete reperfusion after tMCAO and eight (5.16%) died. To induce ischemic tolerance, a subgroup of mice were intraperitoneally injected 24 hours before tMCAO with 0.1 mL of TWEAK (2 mg/mL) alone or in combination with either the MEK inhibitor SL327 (30 mg/kg) or a comparable volume of saline solution. To measure the volume of the ischemic lesion, animals were deeply anesthetized 24 hours after tMCAO, the brains were harvested, cut onto 2 μm sections and stained with TTC. Each section was photographed and the volume of the ischemic lesion was measured by a blinded investigator with the National Institutes of Health Image Analyzer System as described elsewhere [5]. Each observation was repeated ten times. Results are given as a percentage of the stroke volume in untreated animals. All procedures were approved by the Emory University Institutional Animal Care and Use Committee.

Cerebral cortical neurons were cultured from E16-18 Wt, TWEAK^-/-, Fn14^-/- and TNF-α^-/- mice as described elsewhere [40]. Briefly, the cerebral cortex was dissected, transferred into Hanks' balanced salt solution containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and incubated in trypsin containing 0.02% DNase at 37°C for 15 min. Tissue was then triturated and the supernatant was re-suspended in B27-supplemented neurobasal medium containing 2 mM l-glutamine and plated onto 0.1 mg/mL poly-l-lysine-coated wells.

To study the effect of TWEAK on neuronal survival, Wt cerebral cortical neurons were incubated over 1 or 24 hours with 100 ng/mL or 300 ng/mL of rTWEAK or a comparable volume of vehicle (control), followed 24 hours later by determination of cell survival and/or death with the MTT and LDH release assays following manufacturer's instructions and as described elsewhere [40]. Results are given as a percentage of cell survival or LDH release into the media compared to control cultures. Each experiment was performed in cultures from three different animals and each observation was repeated 15 times.

For TWEAK-induced preconditioning, Fn14^-/-, TWEAK^-/- and Wt cerebral cortical neurons were incubated over 60 minutes with 0 to 300 nM of rTWEAK alone or in combination with antibodies against either TNF-α (0.04 μg/mL) or TNFR1 (100 μg/mL) or an immunoglobulin G isotype control, or with wortmannin 100 nM or SL327 10 μM. Twenty-four hours later, cells were exposed in an anaerobic chamber (Hypoxygen, Frederick, MD, USA) to 55 minutes of oxygen-glucose deprivation (OGD) conditions (< 0.1% oxygen, 94% N₂ and 5% CO₂ at 37°C) in glucose-free media containing CaCl₂ 1.8 mM, MgSO₄ 0.8 mM, KCl 5.3 mM, NaHCO₃ 44.05 mM and NaCl 110.34 mM, followed 24 hours later by determination of cell survival and/or death with the MTT and LDH release assays.

To induce hypoxic preconditioning, neurons were exposed to OGD conditions for 30 minutes. A subset of TWEAK^-/- and Fn14^-/- cells was incubated with rTWEAK 100 ng/mL. The media was then changed to fresh culture media and the cells were returned to the incubator for 24 hours. As controls, sister cultures were kept in OGD media without hypoxia for 30 minutes and then in fresh culture media for 24 hours. After 24 hours, cells were exposed to 55 minutes of OGD conditions and neuronal survival and/or death was studied 24 hours later with the MTT and LDH release assays. Each experiment was performed in cultures from three different animals and each observation was repeated 12 times.

Wt cerebral cortical neurons were either exposed to 30 minutes of OGD conditions or incubated under normoxic conditions for 60 minutes with TWEAK 100 ng/mL. In both experimental groups the media was changed to fresh culture media and cells were harvested 1, 3 or 6 hours later. Total RNA was isolated using the RNeasy mini kit (Qiagen; Valencia, CA) according to the manufacturer's instructions. Equal amounts of RNA were taken for cDNA synthesis using a High-capacity cDNA Kit (Applied Biosystems). Briefly, 2 × reverse transcription master mix was prepared from 10 × Reverse Transcription Buffer, 25 × deoxyribonucleotide triphosphates, 10 × random primers, and MultiScribe Reverse Transcriptase (Applied Biosystems) and mixed with equal parts of total RNA. The PCRs were performed using TaqMan Gene Expression Assays (Applied Biosystems) using forward and reverse primers as well as internal probes Mm00839900_m1, Mm00489103_m1, Bcl-w Mm00432054_m1 and BclxL Mm00437783_m1 for TWEAK, Fn14, Bcl-w and Bcl-xL, respectively. The PCRs were performed using 7500 Fast Real-Time PCR System (Applied Biosystems) under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Each experiment was repeated eight times.

To determine the effect of hypoxia on the release of TWEAK from cerebral cortical neurons, we used an ELISA (Adipobiotech, Santa Clara, CA, USA) to quantify the concentration of TWEAK in the culture media of Wt neurons maintained under normoxic conditions or exposed to 0 to 360 minutes of OGD conditions. Each observation was repeated eight times. To measure the effect of TWEAK on the release of neuronal TNF-α, Wt cerebral cortical neurons were incubated with TWEAK 100 ng/mL or a comparable volume of vehicle (control), followed at 1, 5, 30 or 60 minutes by quantification of TNF-α in the culture media with an ELISA kit (Insight Genomics) following manufacturer's instructions. Each experiment was repeated with neurons cultured from three different animals, and each observation was repeated eight times.

Wt cerebral cortical neurons were incubated for 60 minutes with TWEAK 100 ng/mL alone or in combination with the ERK 1/2 inhibitor 10 μM. After 0 to 180 minutes of incubation, cells were homogenized in radioimmunoprecipitation assay lysis buffer; protein concentration was determined with the bicinchoninic acid protein assay (Thermo Scientific; Canton, GA) and 16 μg of total protein were loaded for SDS-PAGE electrophoresis and immunoblotting with antibodies directed against pERK 1/2, total ERK 1/2, pBAD, total BAD and β-actin. Each observation was repeated four to six times.

Wt mice received an intraperitoneal injection of 0.1 mL of TWEAK (2 mg/mL) or a comparable volume of saline solution followed 24 hours later by tMCAO. Twenty-four hours after tMCAO, brains were harvested and 10 μm frozen sections were stained with the ApopTag Plus Fluorecein In Situ Apoptosis Detection Kit following manufacturer's instructions. Briefly, sections were fixed in 1% paraformaldehyde in PBS, pH 7.4, for 10 minutes at room temperature, permeabilized in a 2:1 ratio of ethanol-acetic acid solution for 5 minutes at -20°C, washed twice for 5 minutes and then incubated in a humidified chamber for 1 hour at 37°C with modified nucleotides coupled with the enzyme terminal deoxynucleotidyl transferase. Samples were then washed, incubated with anti-digoxigenin conjugate for 30 minutes and stained with the nuclear marker DAPI.

To quantify the number of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)-positive cells, each coronal section was divided into 16 square areas (150 mm² each) that involved the necrotic core and the area of ischemic penumbra, and comparable areas in the non-ischemic hemisphere. Two areas of interest (AOI) were chosen in the boundaries between the ischemic penumbra and necrotic core (AOI-1 that includes the frontoparietal zone and AOI-3 that involves the temporoparietal area), and a third zone was located in the necrotic core (AOI-2). To determine the number of TUNEL-positive cells, images were digitized in a Zeiss Axioplan 2 microscope 20 × objective (Munich, Germany) with a Zeiss AxioCam and imported into AxioVision, viewed at 150% of the original with Image MetaMorph Software and the percentage of TUNEL-positive cells in relation to the total number of DAPI-positive cells per AOI recorded. Each observation was repeated eight times.

To study the effect of TWEAK on pBAD expression, Wt cerebral cortical neurons were incubated for 60 minutes with rTWEAK 100 ng/mL or a comparable volume of vehicle (control) and fixed and stained 1, 3 or 6 hours later with an antibody against pBAD. Each observation was repeated four times.

Activation of inflammatory pathways by a preconditioning stimulus is thought to reduce the inflammatory response to a subsequent period of ischemia, leading to neuroprotection [41], and so we decided to investigate whether the cytokine TWEAK induces hypoxic tolerance. However, because it has been reported that 24 hours of incubation with TWEAK induces neuronal death [18, 24], first we investigated whether treatment over a shorter period of time (1 hour) also has an effect on cell survival. Wt cerebral cortical neurons were incubated for 1 or 24 hours with 100 or 300 ng/mL of TWEAK followed by determination of cell survival with the MTT assay as described in the Methods section. We found that, as previously described [24], 24 hours of incubation with 100 or 300 ng/mL of TWEAK is associated with a 25.4% and 37% decrease in neuronal survival, respectively. In contrast, 1 hour of incubation with either 100 or 300 mg/dL of TWEAK did not have an effect on cell survival (Figure 1A; n = 15, P < 0.05).

We then used a previously described in vitro model of preconditioning [40] to investigate whether treatment with sub-lethal concentrations of TWEAK renders neurons resistant to a subsequent lethal hypoxic injury. Wt cerebral cortical neurons were either left untreated or incubated with TWEAK 0 to 300 ng/mL for 60 minutes followed 24 hours later by exposure to 55 minutes of OGD conditions as depicted in Figure 1B (upper panel) and described in the Methods section. Cell survival was quantified 24 hours later with an MTT assay. Our results indicate that exposure to OGD conditions decreases neuronal survival from 100 ± 0.11% to 46.2 ± 2.8% in non-preconditioned cells. In contrast, cell survival in neurons preconditioned with 30, 100 or 300 ng/mL of TWEAK 24 hours before exposure to OGD conditions was 79.4 ± 3.1%, 64.9 ± 2.0% and 58.3 ± 4%, respectively (Figure 1B; n = 20 per experimental condition; P < 0.05 compared to non-preconditioned cells).

To determine whether the protective effect of preconditioning with TWEAK is also observed at later time-points, we quantified cell survival 24, 48 and 72 hours after exposure to 55 minutes of OGD conditions. We found that cell survival decreased from 100 ± 0.8% in control neurons to 54.80 ± 2.4% in neurons exposed to OGD conditions without preconditioning with TWEAK. In contrast, preconditioning with TWEAK 24 hours before exposure to OGD conditions increased neuronal survival to 72.10 ± 1.92%, 77 ± 1.1% and 80 ± 2.0%, when the MTT assay was performed 24, 48 and 72 hours after exposure to OGD, respectively (Figure 1C; n = 12 per experimental condition, P < 0.05).

To investigate whether the protective effect of TWEAK is mediated by its interaction with Fn14, we quantified cell survival in Wt and Fn14^-/- cerebral cortical neurons incubated with TWEAK 100 ng/mL for 1 hour followed 24 hours later by exposure to 55 minutes of OGD conditions. We found that exposure of non-preconditioned neurons to OGD conditions decreased cell survival from 100 ± 0.2% to 50.3 ± 1.2% in Wt neurons and from 100 ± 0.88% to 40.8 ± 2.4% in Fn14^-/- neurons. In contrast, cell survival in Wt and Fn14^-/- neurons preconditioned with TWEAK was 66.13 ± 1.8% and 41.3 ± 1.8%, respectively (Figure 1D; n = 10; P < 0.05), indicating that genetic deficiency of Fn14 abrogates the ability of TWEAK to induce tolerance to the deleterious effects of OGD.

It has been demonstrated that exposure to 30 minutes of OGD conditions (hypoxic preconditioning) not only does not induce cell death [42] but instead renders cerebral cortical neurons tolerant to a lethal hypoxic injury applied at later time points (hypoxic tolerance). Based on these observations we decided to investigate whether endogenous TWEAK plays a role in the protective effect of hypoxic preconditioning. First, we studied the effect of sub-lethal hypoxia (preconditioning) on TWEAK and Fn14 expression. Wt cerebral cortical neurons were exposed to 30 minutes of OGD conditions followed 1, 3 and 6 hours later by quantification of TWEAK and Fn14 mRNA expression by quantitative RT-PCR analysis as described in the Methods section. Our results indicate that sub-lethal hypoxia induces a rapid and transient increase in TWEAK and Fn14 mRNA expression in cerebral cortical neurons that is maximal at 1 hour for TWEAK and 3 hours for Fn14 (Figure 2A).

We then quantified cell survival in Wt, TWEAK^-/- and Fn14^-/- cerebral cortical neurons exposed to sub-lethal hypoxia (30 minutes of OGD conditions) followed 24 hours later by lethal hypoxia (55 minutes of OGD conditions). Sister cultures were exposed to lethal hypoxia without previous preconditioning as controls. A subgroup of TWEAK^-/- and Fn14^-/- neurons was incubated with TWEAK 100 ng/mL during the preconditioning phase. Our results indicate that hypoxic preconditioning induces a 23.44% increase in cell survival in Wt neurons (Figure 2B; n = 12, P < 0.05) and that this effect is abrogated in neurons genetically deficient in either TWEAK or Fn14 (Figure 2C, D; n = 12). Importantly, incubation with TWEAK during the preconditioning phase had a rescue effect in TWEAK^-/- (17.5% increase in neuronal survival) but not in Fn14^-/- neurons.

To investigate whether treatment with TWEAK also has a neuroprotective effect in vivo, we measured the volume of the ischemic lesion in Wt and Fn14^-/- mice intraperitoneally injected with either TWEAK or a comparable volume of saline solution 24 hours before tMCAO, as described in the Methods section. We found that preconditioning with TWEAK decreases the volume of the ischemic lesion from 69.3 ± 7.2 mm³ to 46.41 ± 3.3 mm³ and 54.31 ± 4.8 mm³ 24 and 48 hours after tMCAO, respectively. In contrast, we failed to observe a significant decrease in the volume of the ischemic lesion in Fn14^-/- mice preconditioned with TWEAK (Figure 3; n = 10; P < 0.05 in Wt mice and non-significant in Fn14^-/- animals).

Because it has been reported that TNF-α mediates some of the biological effects of TWEAK [43], we investigated whether TNF-α also mediates TWEAK-induced tolerance. First, we used an ELISA to study the expression of TNF-α in the culture media of Wt cerebral cortical neurons incubated for 1 to 60 minutes with TWEAK 100 ng/mL. Our results indicated that TWEAK induces a rapid increase in the expression of neuronal TNF-α and that this effect is maximum at 30 minutes of incubation (Figure 4A; 12,309 ± 526 pg/mL, n = 8, P < 0.05).

We then used the MTT assay to study cell survival in Wt cerebral cortical neurons incubated for 60 minutes with TWEAK 100 ng/mL alone or in combination with neutralizing antibodies against either TNF-α 0.04 μg/mL or TNFR1 100 μg/mL, or an immunoglobulin G isotype control, followed 24 hours later by exposure to 55 minutes of OGD conditions (lethal hypoxia). We found that, whereas treatment with TWEAK rendered neurons tolerant to a lethal hypoxia insult applied 24 hours later, this preconditioning effect was abrogated by incubation with either anti-TNF-α or anti-TNFR1 antibodies (Figure 4B; n = 12). To further study the role of TNF-α on TWEAK-induced neuroprotection, we quantified cell survival in TNF-α^-/- cerebral cortical neurons incubated for 1 hour with TWEAK 0 to 300 ng/mL followed 24 hours later by exposure to 55 minutes of OGD conditions. Our results indicated that TWEAK fails to induce hypoxic tolerance in TNF-α^-/- neurons (Figure 4C: n = 10, non-significant).

To investigate whether TNF-α also mediates the protective effect of treatment with TWEAK in vivo, we measured the volume of the ischemic lesion in Wt and TNF-α^-/- mice intraperitoneally injected with either TWEAK or a comparable volume of saline solution 24 hours before tMCAO. We found that, whereas preconditioning with TWEAK decreases the volume of the ischemic lesion in Wt mice from 70.5 ± 8.2 mm³ to 49.35 ± 4.4 mm³ in Wt mice, this effect is abrogated by a genetic deficiency of TNF-α (Figure 4D; n = 12).

Because ERK 1/2 mediates the protective effects of several factors that enhance neuronal survival following exposure to hypoxia/ischemia [35], we investigated whether ERK 1/2 also mediates the neuroprotective effect of TWEAK. First, we studied the expression of pERK 1/2 in Wt cerebral cortical neurons incubated for 0 to180 minutes with TWEAK 100 ng/mL. We found that TWEAK induces ERK 1/2 activation, and that this effect is maximal at 5 to 15 minutes of incubation (Figure 5A).

To investigate whether TWEAK-induced hypoxic tolerance is mediated by ERK 1/2 activation, we quantified cell survival in Wt cerebral cortical neurons exposed to 55 minutes of OGD conditions 24 hours after 1 hour of incubation with TWEAK 100 ng/mL either alone or in combination with the ERK 1/2 inhibitor SL327 10 μM. Our results indicate that the preconditioning effect of TWEAK is abrogated by ERK 1/2 inhibition (Figure 5B; n = 12).

Activation of the PI3K/Akt pathway also promotes survival in neurons exposed to hypoxic conditions [40], and so we investigated the effect of PI3K inhibition with wortmannin 20 nM on TWEAK-induced preconditioning. We found that inhibition of the PI3K/Akt pathway does not abrogate the neuroprotective effect of TWEAK (Figure 5B).

To determine whether the protective effect observed following treatment with TWEAK in vivo was also mediated by ERK 1/2 activation we measured the volume of the ischemic lesion in Wt mice intraperitoneally injected with TWEAK, alone or in combination with blood-brain barrier-permeable ERK 1/2 inhibitor SL327, 24 hours before tMCAO. Our results indicate that the beneficial effect of preconditioning with TWEAK in vivo is abrogated by ERK 1/2 inhibition (Figure 5D; n = 8).

Because experimental work with glial cell tumors indicates that TWEAK induces the expression of the anti-apoptotic proteins Bcl-xL and Bcl-w, we used RT-PCR analysis to study Bcl-xL and Bcl-w mRNA expression in Wt cerebral cortical neurons incubated with TWEAK 1, 3, 6 or 24 hours. Our data indicated that TWEAK does not induce the expression of Bcl-xL and Bcl-w in neurons (data not shown).

It has been demonstrated that ERK 1/2 induces the phosphorylation of BAD at Ser¹¹². Because our data indicate that the protective effect of TWEAK is mediated by ERK 1/2 activation, then we investigated the effect of TWEAK on BAD phosphorylation. To test this hypothesis we studied the expression of pBAD 1, 3 and 6 hours after 60 minutes of incubation with TWEAK 100 ng/mL. Our results indicated that TWEAK induces rapid phosphorylation of BAD in cerebral cortical neurons (Figure 6A).

To further characterize these results, we studied the expression of pBAD in Wt cerebral cortical neurons incubated with TWEAK alone or in combination with SL327. We found that TWEAK induced pBAD expression in neurons and that this effect is inhibited by co-treatment with SL327 (Figure 6B).

Because phosphorylation of BAD has an anti-apoptotic effect, we investigated whether preconditioning with TWEAK decreases cerebral ischemia-induced apoptotic cell death. Wt mice were intraperitoneally injected with TWEAK or a comparable volume of saline solution, followed 24 hours later by tMCAO and determination of apoptotic cell death in the ischemic tissue as described in the Methods section. We found that preconditioning with TWEAK decreases the percentage of TUNEL-positive cells per field in the ischemic area from 14.25 ± 3.9% in saline solution-treated animals to 8.1 ± 2.3% in animals pre-treated with TWEAK (Figure 6C, D; n = 10, P < 0.05). Importantly, co-treatment with SL327 not only abrogated the effect of TWEAK on apoptotic cell death but also increased the number of apoptotic cells per field to 23 ± 6% (Figure 6D; n = 10, P < 0.05).