Abstract
Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.
Highlights
Neurotrophic factors (NGF, brain-derived neurotrophic factor (BDNF) and NT3/4) are large polypeptidic, secreted molecules that exert potent neuroprotective and neurogenic effects (Chao, 2003; Lu et al, 2005)
To evaluate the exact domains of p75 neurotrophin receptor (p75NTR) required for C17-spiroepoxy analog of DHEA (BNN27) binding, we performed competition assays using membranes from human embryonic kidney cell line 293 (HEK293) cells transfected with the p75NTR mutant, lacking the entire extracellular domain (ECD), necessary for nerve growth factor (NGF) binding (Jung et al, 2003)
Bind to membranes isolated from HEK293p75NTR ECD mutants (Figure 1A), suggesting that the presence of the ECD is necessary for its binding to the receptor (Figure 1A)
Summary
Neurotrophic factors (NGF, BDNF and NT3/4) are large polypeptidic, secreted molecules that exert potent neuroprotective and neurogenic effects (Chao, 2003; Lu et al, 2005). Activation of the transcription factor NFkappaB upon the recruitment of RIP2 protein to p75NTR, leads to survival signals on specific neurons (Carter et al, 1996; Vicario et al, 2015), while it simultaneously induces the release of RhoGDI from the receptor and the subsequent RhoA inactivation (Charalampopoulos et al, 2012). These p75NTR-mediated survival-signaling cascades are largely dependent upon mature neurotrophin binding to the receptor. A plethora of other ligands can directly interact with p75NTR receptors, such as Aβ-amyloid, myelin associated glycoprotein (MAG) or Nogo, leading to differential signaling in a cell-context manner (Wang et al, 2002; Wong et al, 2002; Yaar et al, 2002; Knowles et al, 2009)
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