Characterization of a p75NTR Apoptotic Signaling Pathway Using a Novel Cellular Model

Xin Wang,Johannes H Bauer,Yong Li,Zhihong Shao,Firas S Zetoune,Elena Cattaneo,Claudius Vincenz

doi:10.1074/jbc.m010548200

Xin Wang, Johannes H Bauer + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m010548200

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Abstract

The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.

Highlights

Apoptosis is widespread during development and disease states of the mammalian nervous system [1, 2]
ST14A cells acquire antigenic and electrophysiological properties characteristic of mature neurons and become postmitotic at the non-permissive temperature (39 °C) [34]. Because these neuronal cells are susceptible to a variety of apoptotic stimuli, including the death receptor TNFR1 [35], we investigated their responses to p75 neurotrophin receptor (p75NTR)
A New Cellular Model—The ST14A model presented here is the only model where a strong apoptotic response is elicited by p75NTR alone without any additional stimuli

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Zeocin and ponasterone were obtained from Invitrogen (Carlsbad, CA); hygromycin B was from Roche Molecular Biochemicals; LipofectAMINE was from Life Technologies, Inc.; and NGF was from Harlan Bioproducts (Madison, WI). Measuring p75NTR Expression by FACS Analysis—About 5 ϫ 106 ST14A cells were treated with ponasterone or left untreated. The resulting cell suspensions were pooled with cells in the supernatant, washed with 1% heat-inactivated fetal bovine serum in PBS, blocked with 3% bovine serum albumin in PBS for 30 min, and incubated with ␣-FLAG or ␣-p75NTR antibody (1:100) for 1.5 h and fluorescein isothiocyanate-conjugated secondary antibody (1:2000) for 1 h. Cells were treated with ponasterone for 48 h, washed with PBS, fixed with 4% paraformaldehyde, incubated for 10 min in 0.5 ␮g/ml PI, and washed with PBS. Attached cells and matching supernatant were pooled, fixed with 4% paraformaldehyde, and submitted to terminal deoxytransferase-mediated BrdUTP nickend labeling (TUNEL) using phycoerythrin-conjugated anti-BrdUrd monoclonal antibody flow cytometer following the instructions of the manufacturer.

RESULTS

Findings

DISCUSSION