Inhibition of p75 Tumor Necrosis Factor Receptor by Antisense Oligonucleotides Increases Hypoxic Injury and β-Amyloid Toxicity in Human Neuronal Cell Line

Abstract

Recent evidence indicates that tumor necrosis factor-␣ (TNF-␣) is up-regulated following brain injury andin neurodegenerative disorders such as stroke, multiple sclerosis, Parkinson's disease, and Alzheimer's disease. TNF-␣ elicits its biological effects through two distinct TNF receptor (TNFR) subtypes: p55 TNFR (TNFR1) and p75 TNFR (TNFR2). Studies have demonstrated that the p55 TNFR contributes to cell death, whereas the role of the p75 TNFR in neuronal viability is unclear. To better understand the role of p75 TNFR, we treated human neuronal SH-SY5Y cells with phosphorothioate-modified antisense oligonucleotides (ASO) for p75 TNFR and established that ASO inhibited p75 TNFR expression. Treatment of SH-SY5Y cells with ASO alone did not affect cell viability, whereas treatment with both ASO and human TNF-␣ significantly increased cell death relative to treatment with TNF-␣ alone. Moreover, addition of ASO significantly increased the level of cell injury observed following hypoxic conditions or exposure of ␤-amyloid peptide. These results indicate that inhibition of p75 TNFR using ASO increases the vulnerability of neurotypic cells to insults and suggest that the p75 TNFR may not be required for normal neuronal cell viability but rather plays a protective role following injury.Tumor necrosis factor-␣ (TNF-␣), 1 an inflammatory mediator, is recently reported to be up-regulated following brain trauma (1, 2) and ischemic injury (3, 4), and in multiple sclerosis (5, 6), Parkinson's disease (7), and Alzheimer's disease (8). Studies have shown that TNF-␣ can elicit either a trophic or toxic effect, which is dependent on the target cell type. For example, TNF-␣ is toxic to human oligodendrocytes (9) and neuronal cells (10, 11), but it is trophic to rat hippocampal neurons (12). Since TNF-␣ elicits its biological effects through activation of two distinct receptors, p55 TNFR and p75 TNFR, both of which have been cloned (13-15), we hypothesized that the distinct TNF receptor subtypes might contribute to the multiple biological functions of TNF-␣. Reports indicate that the p55 TNFR may contribute to cell death (16), whereas the role of the p75 TNFR in neuronal cell function is not clear. Here we show that inhibition of p75 TNFR by antisense oligonucleotides (ASO) increased the magnitude of neuronal cell death induced by TNF-␣, hypoxic injury or ␤-amyloid peptide (A␤) as measured by lactate dehydrogenase (LDH) release. This novel finding provides evidence that the p75 TNFR might play a protective role following neuronal insult. EXPERIMENTAL PROCEDURESCell Cultures-Human neuronal-like cells, SH-SY5Y, were cultured in a 1:1 ratio of minimum Eagle's medium/F-12 medium, 15% fetal calf serum (Life Technologies, Inc.), and 10 M retinoic acid (RA, Sigma). The cells were seeded at 25,000 cells/well in a 24-well plate for cytotoxicity assays or 3 ϫ 10 6 cells in Petri dishes for radioimmunoprecipitation study. The medium was replaced every 3 days. Treatments were given to the cells in serum-free medium with 1:100 N2-supplement (Li...

Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis

“…Riches, unpublished observations). Interestingly, although our studies with TNFR-deficient lung fibroblasts revealed that ligation of TNF-R1 was required for the increase in Fas expression and susceptibility to Fas ligation-induced apoptosis, they also suggest a role for TNF-R2 in protecting the cells from apoptosis as previously reported (40). Furthermore, because ligation of TNF-R2 has also been shown to activate NF-kB and p38 mapk signaling (41), it is plausible that the incomplete inhibition of caspase-8 activation seen in the presence of Bay11-7082 and SB203580 may be related to loss of antiapoptotic signaling initiated by ligation of TNF-R2.…”

The Low-Density Lipoprotein Receptor-Related Protein Is a Pro-Survival Receptor in Schwann Cells: Possible Implications in Peripheral Nerve Injury

“…The function of TNFRII is less well understood than TNFRI; however, the former receptor demonstrates mitogenic and proinflammatory activity in the immune response (Carpentier et al, 2004). TNFRII also has been implicated in neuronal survival (Shen et al, 1997;Yang et al, 2002). The function of LRP-1 in Schwann cell survival, demonstrated here, and the linkage of TNFRII to LRP-1 expression suggests that TNFRII plays an essential role in glial survival.…”

Integration of cytokine biology and lipid metabolism in stroke

“…The majority of biological effects of TNF-α, including cytotoxicity, ArAc release, and inflammatory responses are mediated through the p55 receptor (84,93) that contains the death domain. The function of p75 remains obscure, although studies have indicated it may participate in protective signaling following injury (94). TNF-α receptors are differentially up-regulated after cerebral ischemia: p55 (contains the death domain) is expressed within 6 hr, whereas p75 increases at 24 hr (93).…”