The effect of nitrative stress on proNGF and BDNF axonal transport in basal forebrain cholinergic neurons

Abstract

Neurotrophins such as pro-nerve growth factor (proNGF) and brain-derived neurotrophic factor (BDNF) are essential for survival and function of basal forebrain cholinergic neurons (BFCNs). ProNGF and BDNF exert their functions through the tropomyosin-related kinase (Trk) receptors, TrkA and TrkB, respectively, and the pan neurotrophin receptor, p75NTR. Neurotrophins binding to their receptors enables retrograde axonal transport, a process necessary for BFCNs to obtain neurotrophins. Neurotrophin transport is impaired in aging, which is associated with BFCN degeneration and cognitive decline. The mechanisms causing loss of neurotrophin transport are unknown. Nitrative stress is elevated in aging and increases JNK activation, but whether this affects neurotrophin transport has not previously been studied. This project investigated how nitrative stress affects proNGF and BDNF transport in BFCNs. BFCNs were cultured in microfluidic chambers and aged for 7-22 days in vitro. Quantum dot labelled proNGF, BDNF, and proNGF mutants that selectively bind to TrkA or p75NTR were added to BFCN axons prior to analysis of their retrograde transport via fluorescence microscopy. Nitrative stress was manipulated using L-NAME, a nitric oxide synthase inhibitor, DEA NONOate, a nitric oxide generator, and SIN-1, a peroxynitrite generator. JNK activity was inhibited using CC401. We determined that nitrative stress decreases retrograde transport of proNGF via TrkA while increasing proNGF transport via p75NTR, which is associated with decreased neurotrophic signalling, increased apoptotic signalling, and neurodegeneration. Additionally, nitrative stress impairs proNGF transport by activating JNK independently of p75NTR. Nitrative stress-induced JNK activation also impairs BDNF transport in young BFCNs but increases BDNF transport in aged BFCNs. Aged BFCNs have decreased levels of TrkB relative to young BFCNs, which may explain the differential effects of nitrative stress on BDNF transport in aged versus young neurons. These results indicate that nitrative stress-induced JNK activation contributes to loss of neurotrophin transport and BFCN degeneration in aging.