Neurotrophins Redirect p75^NTR from a Clathrin‐Independent to a Clathrin‐Dependent Endocytic Pathway Coupled to Axonal Transport

Abstract

The p75 neurotrophin receptor (p75 NTR ) plays multiple roles in neuronal physiology through interactions with many ligands and coreceptors. However, its intracellular neuronal trafficking prior to and after neurotrophin activation is still poorly characterized. We have previously shown that in response to nerve growth factor (NGF), p75 NTR is retrogradely transported along the axons of motor neurons (MNs) in carriers shared with NGF, brainderived neurotrophic factor and the tyrosine kinase receptor TrkB. Here, we report that NGF does not enhance the internalization or degradation of p75 NTR , which undergoes a rapid dynamin-dependent and clathrin-independent recycling process in MNs. Instead, incubation of cells with NGF leads to the redirection of a pool of plasma membrane p75 NTR into clathrin-coated pits. The subsequent internalization of p75 NTR via clathrin-mediated endocytosis, as well as the activity of Rab5, are essential for the sorting of the p75 NTR -containing endosomes to the axonal retrograde transport pathway and for the delivery of p75 NTR to the soma. Our findings suggest that the spatial regulation of p75 NTR signalling is controlled by these ligand-driven routes of endocytosis.

Ligand-independent signaling by disulfide-crosslinked dimers of the p75 neurotrophin receptor

“…Plasmids encoding wild-type and mutant p75 NTR were microinjected into embryonic spinal motoneurons, and axonal trafficking of the expressed receptors was visualized by time-lapse confocal microscopy using antibodies against their N-terminal hemagglutinin (HA) epitope tag. As previously reported (Deinhardt et al, 2007), very few retrograde carriers containing wild-type p75 NTR were detected in the absence of NGF, but these were greatly increased upon NGF addition (Fig. 7A,B).…”

“…First, we looked at axonal retrograde transport of p75 NTR complexes, which is a prerequisite for cell body-mediated responses to p75 NTR signaling in neurons. Although the specific signaling steps leading to retrograde transport of p75 NTR are still unknown, this process has recently been shown to be ligand mediated (Deinhardt et al, 2007). Plasmids encoding wild-type and mutant p75 NTR were microinjected into embryonic spinal motoneurons, and axonal trafficking of the expressed receptors was visualized by time-lapse confocal microscopy using antibodies against their N-terminal hemagglutinin (HA) epitope tag.…”

“…This might be related to the specific complement of downstream effectors expressed by each cell type. Granule neurons were isolated from postnatal day 4 rat cerebellum and motoneurons from E14.5 rat spinal cord (Deinhardt et al, 2007). Cell lines were cultured under standard conditions and primary neurons in serum-free, N2-supplemented DMEM:F12 medium (Gibco).…”

Trafficking Guidance Receptors

“…For other endocytosing receptors, we know that signaling outcomes can differ in important ways depending on the endocytic route taken (clathrin-dependent or -independent) and the subsequent postendocytic trafficking (recycling endosome, late endosome, etc.) (for examples see Deinhardt et al 2007;Le Roy and Wrana 2005). Furthermore, the residence time in these signaling endosomes can also be regulated and greatly influence signaling outcomes (Zoncu et al 2009).…”

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

“…7, D and E). Previous studies have shown that the internalization of TrkA is dependent on dynamin (51,52). We, therefore, used a dominant-negative form of dynamin (dynamin-K44A, DN-dynamin) to test whether we could suppress GFP-TrkA internalization.…”