CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons

Abstract

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Fig. 1.

Neuronal cell death in the adult rat brain 3 d after ischemia and reperfusion (n = 5).a, Nissl staining. The ischemic area appears as awhite compartment limited with a red linein the left hemisphere. Regions 1 and2 mark the piriform cortex, ipsilateral and contralateral to the ischemic lesion, respectively. Similar results were obtained in five different animals. b–d, TUNEL-positive cells from region 1 (b, green) exhibit a neuronal phenotype, as detected by antineuronal nuclei (NeuN) antibody (c, red), as assessed by colocalization of NeuN and TUNEL labeling (d, yellow or red andgreen in the same nucleus). Photographed with a 60× objective by confocal microscopy.

Fig. 2.

Enhanced phosphorylation of c-Jun and increased expression of CD95-L and TRAIL in the adult rat brain after ischemia (n = 5) and Jurkat-16 cells after γ radiation and doxorubicin (n = 5).a, Immunohistochemical signals from region 1 (see Fig.1a) 3 d after ischemia and recirculation. Photographed with a 40× objective. 1, Cytoplasmic neuronal CD95-L protein; 2, phosphorylation of serine 73 of c-Jun as detected by a Cy3-labeled secondary antibody (red); 3, TUNEL-positive neurons (green) and double labeling of TUNEL with CD95-L (red; the cell boundary is delineated witharrowheads); 4, colocalization of phosphorylated serine 73 of c-Jun with TUNEL-positive cells (orange–yellow). b, Expression of CD95-L protein after focal ischemia as detected by Western blotting. The specific band of the 39 kDa CD95-L protein was determined in regions1 and 2 (see Fig. 1a) at the indicated time points after ischemia and recirculation and in untreated controls (CO). CD95-L expression was also determined in leukemic Jurkat-16 (J16) T cells either untreated (−) or 3 d after γ irradiation with 10 Gy (γ). Equal protein loading was ensured by Ponceau red staining.c, Induction of CD95-L andTRAIL mRNA after focal ischemia and recirculation at the indicated time points and in untreated controls (CO) as revealed by RT-PCR. n.d., Nondetectable. As a positive control, Jurkat cells (J16) were treated with 500 ng/ml doxorubicin, and RNA was isolated 4 hr later.

Fig. 3.

Expression of CD95-L in neurons 3 d after ischemia and reperfusion in the piriform cortex (corresponding to Fig.1a, region 1) as assessed by double labeling with MAP2 (red) and anti-CD95-L (yellow–green). Photographed with a 100× objective.

Fig. 4.

Induced specific cell death in neuroblastoma cells (SHEP; a), cortical neurons (b), and NT2-N cells (c) measured 24 hr after treatment with recombinant TNF-α, TRAIL, or CD95-L protein (each 100 ng/ml) alone (gray bars) or in the presence of 500 ng/ml cycloheximide (black bars). Cell death was assessed in SHEP cells by FACS analysis using FSC/SSC analysis and in cortical neurons and NT2-N cells by trypan blue exclusion. More than 90% of the NT2-N cells exhibited a neuronal phenotype, as assessed by immunostaining against MAP2 (d).Co, control.

Fig. 5.

FK506 inhibits apoptosis and ischemia- or stress-induced upregulation of death-inducing ligands, c-jun, and c-Jun phosphorylation in vitroand in vivo. a, Neuroblastoma cells (5 × 10⁴ cells per well) were left either untreated (CO) or stimulated with 1 μg/ml doxorubicin alone (D), with doxorubicin in presence of 100 nm FK506 (D/FK), or with 100 nm FK506 alone (CO/FK) for 16 hr.CD95-L, TRAIL, TNF-α, and c-jun mRNA expression was assessed by RT-PCR. Expression of theβ-actin gene served as control for equal conditions.b, Reduction of the ischemic area 3 d after ischemia and reperfusion by FK506 as revealed by Nissl staining. The infarcted area is marked by a red line.c, Expression of CD95-L (1–3), phosphorylation of serine 73 of c-Jun (4–6), and c-Jun (7–9) was examined by immunohistochemistry in region 1 (see Fig.1) of untreated rats (1, 4, 7) 3 d after ischemia and recirculation (2, 5, 8) or 3 d after ischemia and recirculation with intravenous application of FK506 5–15 min after ischemia (3, 6, 9). FK506 antagonized the expression of CD95-L (3) and the phosphorylation of c-Jun (6) but did not affect the c-Jun expression (9). Similar results were obtained in five different experiments.

Fig. 6.

Protection against focal ischemia inlpr mice. a, Infarct volume after transient focal ischemia in wild-type (Control) and lpr mice. Data are presented as the means ± SEM (Control, n = 9;Lpr, n = 9). Significance was determined by t test. **p < 0.004. Data from individual animals are plotted as separate points overlaid on each histogram bar. Infarcted volume was determined by numeric integration of the scanned non-silver-impregnated areas corrected for brain edema. b, Infarct area (white area marked with red line) in the wild-type (Control) and lpr mice as revealed by silver staining. Note that in the lpr mouse despite an infarct volume (∼37 mm³) superior to the mean infarct volume (∼18 mm³), the surrounding cortex remains spared.