Target depletion of distinct tumor necrosis factor receptor subtypes reveals hippocampal neuron death and survival through different signal transduction pathways

Abstract

Tumor necrosis factor receptor-I (TNFRI) and TNFRII are two TNFR subtypes in the immune system, but their roles in the brain remain unclear. Here we present a novel interaction between TNFR subtypes and TNF-alpha in the brain. Our studies on target-depleted TNFR in mice show that TNF-alpha has little effect on hippocampal neurons in which TNFRI, containing an "intracellular death domain," is absent (TNFRI -/-), whereas neurons from TNFRII knock-out mice are vulnerable to TNF-alpha even at low doses. Moreover, little nuclear factor-kappaB (NF-kappaB) translocation is induced by TNF-alpha in neurons of TNFRI -/-, whereas NF-kappaB subunit p65 is still translocated from the cytoplasm into the nucleus in neurons from wild-type and TNFRII -/- mice. Furthermore, p38 mitogen-activated protein (MAP) kinase activity is upregulated in neurons from both wild-type and TNFRI -/-, but no alteration of p38 MAP kinase was found in neurons from TNFRII. Results from overexpression of TNF receptors further support the above findings. NT2 neuronal-like cells transiently transfected with TNFRI are very sensitive to TNF-alpha, whereas TNF-alpha is not toxic and even seems to be trophic to the cells with TNFRII overexpression. Last, our radioligand-binding experiments demonstrate that TNF-alpha binds TNFRI with high affinity (K(d) of 0.6 nm), whereas TNFRII shows lower binding affinity (K(d) of 1.14 nm) to TNF-alpha in NT2 transfected cells. Together, these studies reveal novel neuronal responses of TNF-alpha in mediating consequences of TNF receptor activation differently. Subsequent neuronal death or survival may ultimately depend on a particular subtype of TNF receptor that is predominately expressed in neurons of the brain during neural development or with neurological diseases.

Fig. 1.

Hippocampal neurons from knock-out TNFRI or TNFRII exhibit neuronal death or survival after exposures of TNF-α.A, Morphological changes of hippocampal neurons from TNFRI knock-out (TNFRI −/−) or TNFRII knock-out (TNFRII −/−) mice after exposure to TNF-α for 48 hr. Nikon (Tokyo, Japan) microscope at 200×. Scale bar, 100 μm. TNFRI −/− neurons show resistance to TNF-α insults, but TNFRII −/− demonstrated degeneration after TNF-α treatment. B, The primary cultured neurons from four mice from each type of TNFR knock-out mice; TNFRI −/−, TNFRII −/−, and wild type were incubated for 48 hr with TNF-α. The dead or detached damaged neurons were removed with the supernatant. The remaining neurons were lysed. LDH levels were measured in both supernatant and cell lysates. The data in B are from three independent experiments and represent mean ± SE, which are the percentage of supernatant LDH value relative to total LDH contained in both the supernatant and cell lysates, as described in Materials and Methods.

Fig. 2.

Hippocampal neuronal apoptosis induced by TNF-α treatment is TNFR dependent. TNF-α-induced apoptosis in hippocampal neurons in the absence of TNFRII is demonstrated in TUNEL (A) and DNA fragmentation (B). The hippocampal neurons from TNFRI −/− or TNFRII −/− mice were incubated in TNF-α at 100 pg/ml for 18 hr. The cells were fixed and analyzed for TUNEL reactivity, and neurons were analyzed for apoptosis using TUNEL for fragmented nuclear DNA (A). These images are typical of apoptotic single neurons cultured with TNF-α at 100 pm. For DNA fragmentation, Southern analysis of DNA isolated from neurons from TNFRI −/− or TNFRII −/− mice. Neurons from TNFRI −/−, TNFRII −/−, and wild type were treated with TNF-α (100 pm) for 48 hr, and cychlohexmide was used as positive control (B). The results from TUNEL and Hoechst staining and DNA fragmentation indicate that neurons with TNFRI deletion were resistant to TNF-α-induced apoptosis, whereas neurons with TNFRII deletion were more vulnerable.

Fig. 3.

Overexpressing either TNFRI or TNFRII reveals different vulnerability to TNF-α. Human neurotypic NT2 cells were transiently transfected with either TNFRI or TNFRII cDNA plasmid.A, The protein expression at each TNF receptor subtype after transfection was confirmed by Western blot. B, Overexpression of TNFRI resulted in neuronal vulnerability to TNF-α death even at a low dose (10–100 pm), whereas cells overexpressing TNFRII survive in the presence of TNF-α even at high doses (100–1000 nm). All results were repeated four times from independent experiments. *p < 0.05; **p < 0.01. C, DNA fragmentation was observed in NT2 cells with TNFRI overexpression but not found in the cells with TNFRII overexpression or no transfection.

Fig. 4.

NF-κB p65 translocation and activity in hippocampal neurons are TNFRI dependent. A, Western blot of cytoplasmic or nuclear NF-κB p65 in hippocampal neurons of wild-type mice. Neurons were treated with soluble TNF-α at 0, 1, 10, 100, and 1000 pm for 30 min. At the indicated various doses of TNF-α treatment, cell cytoplasmic or nuclear samples were subjected to SDS-PAGE, and the blots were probed with the antibody to NF-κB p65 or β-actin and lamin as the cytoplasmic and nucleic housekeeping proteins, respectively. Visualization of the proteins was performed with ECL. The result demonstrates that cytoplasmic NF-κB p65 was at high levels and nuclear NF-κB p65 was low without TNF-α treatment. However, TNF-α treatment resulted in low levels of cytoplasmic NF-κB p65 and high levels in the nucleus, suggesting that NF-κB p65 was translocated from the cytoplasm to the nucleus in cortical neurons after TNF-α treatment. B, Western blot of nuclear NF-κB p65 in hippocampal neurons from TNFRI knock-out mice (TNFRI −/−). The neurons from TNFRI knock-out brains were treated with TNF-α at 100 pm for 30 min. The cytoplasmic and nuclear samples were subjected to SDS-PAGE, and the blots were probed with the antibody to NF-κB p65 or β-actin and lamin. We found that little NF-κB p65 was induced by TNF-α at 100 pm compared with that in wild-type or TNFRII −/− hippocampal neurons, whereas NF-κB p65 induced by TNF-α was still translocated from cytoplasm to nucleus in neurons from TNFRII −/− mice. C, EMSA analysis of NF-κB binding activity in hippocampal neurons from TNFRI −/− and TNFRII −/− mice. EMSA assay with nuclear extracts prepared from hippocampal neurons of TNFRI −/− and TNFRII −/− with TNF-α treatment (10 and 100 pm) for 30 min.³²P-labeled oligonucleotide probe used contained the NF-κB p65 subunit and 50-fold molar excess of unlabeled AP2 (nonspecific competitor; NS) probe. The NF-kB activity was abolished in TNFRI-deleted neurons but not in neurons with the TNFRII deletion.

Fig. 5.

p38 MAP kinase expression in hippocampal neurons is TNFRII dependent. Hippocampal neurons from TNFRI −/−or TNFRII −/− mouse brains were treated with either glutamate (0, 10, or 100 μm) or TNF-α (0, 10, or 100 pm) for 24 hr. Extracts were fractionated by SDS-PAGE on 12% Tris-glycine gels and transferred to PVDF membranes; 10 μg of cell lysates were loaded per lane. The p38 MAP kinase was detected with affinity-purified antibody using a horseradish peroxidase-conjugated secondary antibody and processed using ECL detection. We found that glutamate decreased p38 MAP kinase expression, whereas TNF-α increased p38 MAP kinase activity in hippocampal neurons from wild-type or TNFRI −/− mice. These effects are dose dependent. However, no alteration of p38 MAP kinase expression was observed in hippocampal neurons of TNFRII −/− by either glutamate or TNF-α, suggesting that p38 MAP kinase expression induced by either TNF-α or glutamate is TNFRII dependent.

Fig. 6.

Detection of TNFRI and TNFRII in NT2 cells and saturation analysis of specific ¹²⁵I-TNF-α binding to transfected human NT2 neurotypic cells at various TNF-α concentrations. A, Detection of mRNA and protein expression of both TNFRI and TNFRII by using reverse transcription (RT)-PCR and Western blot techniques. For RT-PCR, the negative control reaction in the absence of RT with RNA template yielded no detectable product, whereas the positive control reaction in the TNFRI or TNFRII transfection with RNA template yielded a highly abundant expected band. For Western blot analysis, the same TNFRI- and TNFRII-transfected NT2 cells were used for confirmation of these two-receptor expression. Both results demonstrate little expression of both endogenous TNFRI and TNFRII in NT2 cells (A). Scatchard analyses of the ¹²⁵I-TNFα binding in TNFRI- and TNFRII-transfected NT2 cells are illustrated in Band C, respectively. The assays with transfected and control cells contained 1 × 10⁶ cells per assay. Five independent binding assays were performed, and theK_d averages for TNFRI and TNFRII are 0.6 and 1.14 nm, respectively.