SUMMARY Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Recent structural studies have shown that neurotrophins interact with dimers of the p75 neurotrophin receptor (p75NTR), but the actual mechanism of receptor activation has remained elusive. Here we show that p75NTR forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys257 in its transmembrane domain. Mutation of Cys257 abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75NTR/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75NTR. FRET experiments revealed a close association of p75NTR intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys257 did not alter the oligomeric state of p75NTR, the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75NTR by a novel mechanism involving rearrangement of disulphide-linked receptor subunits.

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The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEA sulfate (DHEAS), and allopregnanolone (Allo), produced by the CNS and the adrenals, appear to exert a protective effect in hippocampal and cortical neuron ischemia- and excitotoxicity-induced injury. We hypothesized that they may also play a protective role on the adrenal medulla, an important part of the sympathetic nervous system, and the tissue adjacent to their primary site of production. DHEA, DHEAS, and Allo protected rat chromaffin cells and the rat pheochromocytoma PC12 cell line, an established model for the study of adrenomedullary cell apoptosis and survival, against serum deprivation-induced apoptosis. Their effects were time- and dose-dependent, with EC50 1.8, 1.1, and 1.5 nM, respectively. The antiapoptotic effect of DHEA DHEAS and Allo was compared to that of a long list of structurally related compounds and was found to be structure-specific, confined mainly to conformation 3beta-OH-Delta5 for androstenes and 3alpha-OH for pregnanes. Indeed, 3-keto, Delta4, or C7 hydroxylated androstenes and 3beta pregnanes were ineffective. The prosurvival effect of DHEA(S) and Allo was N-methyl-D-aspartate-, GABAA-, sigma1-, or estrogen receptor-independent. It involved the antiapoptotic Bcl-2 proteins, their role being sine qua non for their action because Bcl-2 antisense oligonucleotides reversed their effects. Finally, DHEA(S) and Allo activated cAMP response element-binding protein and NF-kappaB, upstream effectors of antiapoptotic Bcl-2 protein expression. They also activated the antiapoptotic kinase PKCalpha/beta, a posttranslational activator of Bcl-2 protein. Our findings suggest that decline of DHEA(S) and Allo during aging or stress may leave the adrenal medulla unprotected against proapoptotic challenges.

Nongenomic androgen actions imply mechanisms different from the classical intracellular androgen receptor (iAR) activation. We have recently reported the identification of a membrane androgen receptor (mAR) on LNCaP human prostate cancer cells, mediating testosterone signal transduction within minutes. In the present study we provide evidence that activation of mAR by nonpermeable, BSA-coupled testosterone results in 1) inhibition of LNCaP cell growth (with a 50% inhibitory concentration of 5.08 nM, similar to the affinity of testosterone for membrane sites); 2) induction in LNCaP cells of both apoptosis and the proapoptotic Fas protein; and 3) a significant decrease in migration, adhesion, and invasion of iAR-negative DU145 human prostate cancer cells. These actions persisted in the presence of antiandrogen flutamide or after decreasing the content of iAR in LNCaP cells by iAR antisense oligonucleotides. Testosterone-BSA was also effective in inducing apoptosis of DU145 human prostate cancer cells, negative for iAR, but expressing mAR sites. In LNCaP cell-inoculated nude mice, treatment with testosterone-BSA (4.8 mg/kg body weight) for 1 month resulted in a 60% reduction of tumor size compared with that in control animals receiving only BSA, an effect that was not affected by the antiandrogen flutamide. Our findings suggest that activators of mAR may represent a new class of antitumoral agents of prostate cancer.

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The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

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