Discussion

The neurotrophin family of growth factors have been well characterized for their effects on the tropomyosin receptor kinase (Trk) family of receptors and their promotion of cell survival. Additionally, these ligands and their precursors are also able to interact with the p75 neurotrophin receptor (p75NTR) to elicit a variety of cellular responses. However the cellular fate caused by downstream signalling in this pathway is less clear. Activation of this receptor by neurotrophins has been implicated in apoptosis, cell proliferation and changes in cell morphology.

In Huntington’s disease (HD) the relative levels of TrkB and p75NTR are disrupted and it is hypothesized that this causes greater signalling of brain derived neurotrophic factor (BDNF) through p75NTR which increases neuronal apoptosis. A small molecule or biologic agent which prevents BDNF binding to p75NTR without inhibiting binding to TrkB may help to reduce the neuronal loss which is characteristic of HD

Our previous attempts to establish a p75NTR assay measuring reported downstream signalling effectors had been unsuccessful and so we evaluated a NanoBRET assay to measure p75NTR receptor conformational changes based on a model described by Ibáñez et al. (2009). Here we describe the successful development of a 96 well microplate assay in HEK293 cells transfected with p75NTR constructs containing HaloTag and Nano-Luciferase (nLuc) c-terminal tags; fluorophores to these tags are used to generate the NanoBRET assay signal if the tags are in adequate proximity for energy transfer. BDNF demonstrated a reproducible time- and concentration-dependent change, with an affinity consistent with the hypothesis that p75NTR is a low-affinity neurotrophin receptor. To support the findings of this assay, we also developed a High Content assay using cells expressing native or transfected p75NTR to detect binding of biotinylated BDNF.

Here we clearly demonstrate the utility of this novel BRET technology for detecting neurotrophin activation of the p75NTR receptor via conformational change of the receptor. We aim to further develop this assay to support 384-well microplate screening and hope to identify compounds which antagonize neurotrophin binding to and activating p75NTR with the aim of exploring their in vivo utility for slowing the progression of HD. Additionally, this technology could be adapted for other difficult to assay intracellular protein-protein interactions, providing a more relevant assay for the intracellular environment than cell-free assays.