HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology.

Perrine Gaub,Philippe Séguéla,Geneviève Dorval,Vincent Soubannier,Julien Gibon,Philip A Barker,Andrès De Léon,Michal Hetman

doi:10.1371/journal.pone.0150601

Perrine Gaub, Philippe Séguéla + Show 6 more

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https://doi.org/10.1371/journal.pone.0150601

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Abstract

Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease.

Highlights

The four mammalian neurotrophins comprise a family of related secreted factors required for differentiation, survival, development and death of specific populations of neurons and nonneuronal cells
From the N-terminal end, HBpF-proBDNF consists of the endogenous gene’s signal peptide, a 6xHis-tag (H), a biotin acceptor peptide (B), a linker, a PreScissionTM Protease cleavage site (p) and a Flag-tag (F); this extension is followed by murine proBDNF containing two site directed mutations, R129A and R130A
ProBDNF has emerged as regulator of cell morphology, neuronal excitability and cell death [2, 30]knowledge of the signaling mechanisms activated by this peptide remains rudimentary

Summary

Introduction

The four mammalian neurotrophins comprise a family of related secreted factors required for differentiation, survival, development and death of specific populations of neurons and nonneuronal cells. The effects of the neurotrophins are mediated by binding to TrkA, TrkB and TrkC receptor tyrosine kinases and to the p75 neurotrophin receptor (p75NTR). The Trk receptors play critical roles mediating neuronal survival and growth and are important modulators of synaptic function [1]. P75NTR is a component of distinct cell surface signaling platforms that function to induce apoptosis and mediate neuronal growth inhibition. P75NTR acts as a Trk co-receptor that increases the binding specificity and affinity of Trk receptors for neurotrophins [2, 3].

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