Death Domain Signaling by Disulfide-Linked Dimers of the p75 Neurotrophin Receptor Mediates Neuronal Death in the CNS

Abstract

The p75 neurotrophin receptor (p75^NTR) mediates neuronal death in response to neural insults by activating a caspase apoptotic pathway. The oligomeric state and activation mechanism that enable p75^NTR to mediate these effects have recently been called into question. Here, we have investigated mutant mice lacking the p75^NTR death domain (DD) or a highly conserved transmembrane (TM) cysteine residue (Cys²⁵⁹) implicated in receptor dimerization and activation. Neuronal death induced by proneurotrophins or epileptic seizures was assessed and compared with responses in p75^NTR knock-out mice and wild-type animals. Proneurotrophins induced apoptosis of cultured hippocampal and cortical neurons from wild-type mice, but mutant neurons lacking p75^NTR, only the p75^NTR DD, or just Cys²⁵⁹ were all equally resistant to proneurotrophin-induced neuronal death. Homo-FRET anisotropy experiments demonstrated that both NGF and proNGF induce conformational changes in p75^NTR that are dependent on the TM cysteine. In vivo, neuronal death induced by pilocarpine-mediated seizures was significantly reduced in the hippocampus and somatosensory, piriform, and entorhinal cortices of all three strains of p75^NTR mutant mice. Interestingly, the levels of protection observed in mice lacking the DD or only Cys²⁵⁹ were identical to those of p75^NTR knock-out mice even though the Cys²⁵⁹ mutant differed from the wild-type receptor in only one amino acid residue. We conclude that, both in vitro and in vivo, neuronal death induced by p75^NTR requires the DD and TM Cys²⁵⁹, supporting the physiological relevance of DD signaling by disulfide-linked dimers of p75^NTR in the CNS.

SIGNIFICANCE STATEMENT A detailed understanding of the physiological significance of distinct structural determinants in the p75 neurotrophin receptor (p75^NTR) is crucial for the identification of suitable drug targets in this receptor. We have tested the relevance of the p75^NTR death domain (DD) and the highly conserved transmembrane residue Cys²⁵⁹ for the ability of p75^NTR to induce apoptosis in neurons of the CNS using gene-targeted mutant mice. The physiological importance of these determinants had been contested in some recent in vitro studies. Our results indicate a requirement for DD signaling by disulfide-linked dimers of p75^NTR for neuronal death induced by proneurotrophins and epileptic seizures. These new mouse models will be useful for clarifying different aspects of p75^NTR physiology.