p75NTR is an obligate signaling receptor required for cues that cause sympathetic neuron growth cone collapse

Abstract

The p75 neurotrophin receptor (p75NTR) is required for the activity of growth cone collapsing factors such as Nogo, MAG, OMgP, and ephrin A. Specifically, p75NTR associates with the Nogo receptor and GPI-linked ephrin A, and unliganded p75NTR mediates the biological effects of those proteins. Here we assess the requirement for p75NTR for the growth cone collapsing responses of semaphorins (Sema) 3A and 3F and ephrin B2 in sympathetic neurons. We show that the ability of Sema 3s or ephrin B2 to collapse growth cones is suppressed in p75NTR−/− sympathetic neurons. Ectopic expression of p75NTR restores the collapsing activity of Sema 3 in p75NTR−/− neurons. Moreover, p75NTR must be bound to its neurotrophin ligands to participate in Sema 3-mediated collapse. Ligand-bound p75NTR participates in Sema 3 and ephrin B2-mediated collapse via the Rho signaling pathway, since inhibition of Rho signaling is sufficient to suppress the effects of Sema 3s and ephrin B2 in p75NTR+/+ but not p75NTR−/− neurons. Our data suggest that in addition to its role as a co-receptor, p75NTR may provide an obligate parallel neurotrophin-activated inhibitory pathway that broadly sensitizes neurons to inhibitory cues.

Introduction

During development, axons are guided by both positive and negative cues that they encounter as they pathfind their way to their targets. These cues bind to receptors on axonal growth cones and newly-formed axons, where they are integrated to culminate in either stabilization or retraction of actin filaments and microtubules, thereby determining the extent and directionality of axonal growth. However, while significant progress has been made in defining the attractive and repulsive cues that are responsible for axon guidance, much less is understood about the way that growth cones and axons integrate the different classes of cues that they encounter.

In the peripheral nervous system, perhaps the best-characterized growth and guidance cues are the neurotrophins. In addition to their well-established roles in peripheral neuron survival and growth (Miller & Kaplan, 2001a, Miller & Kaplan, 2001b, Zhou & Snider, 2006), the neurotrophins regulate peripheral axon pathfinding, target innervation, and pruning (Kohn et al., 1999, Singh et al., 2008). The neurotrophins mediate their positive effects on axonal growth and pathfinding by binding to the Trk neurotrophin receptors, which activate signaling via PI3-kinase and the MEK–ERK pathway to promote axonal growth and growth cone motility (Atwal et al., 2000, Atwal et al., 2003). In contrast, the neurotrophins inhibit axonal growth and cause degeneration of developing axons by binding to the p75 pan-neurotrophin receptor (p75NTR) (Singh et al., 2008), which mediates these effects via Rho-GDI (Yamashita and Tohyama, 2003) and by inhibiting signaling pathways downstream of the Trk receptors (Atwal et al., 2003). In this regard, while neurotrophin binding to p75NTR is sufficient on its own to inhibit axonal growth in some settings (Kohn et al., 1999), p75NTR also acts as an inhibitory co-receptor for other receptors. In particular, the Nogo receptor inhibits growth on myelin substrates by binding to p75NTR and activating the Rho pathway (Yamashita et al., 2005, Yamashita & Tohyama, 2003) and an ephrin A–p75NTR complex reverse signals to mediate retinal axon repulsion during development (Lim et al., 2008). In the latter study, ephrin A mediated its p75NTR-dependent responses via fyn, while both ephrin A and a second ephrin family member, ephrin B2, are thought to primarily signal via RhoA and Cdc42/Rac1, respectively (Cheng et al., 2003, Klein, 2004, Sahin et al., 2005).

These findings raise the intriguing possibility that p75NTR might function as a general and required receptor for growth-inhibitory cues and/or that it might represent an inhibitory signaling system that functions in parallel to sensitize axons to such negative cues. In this regard, two classes of cues well-known for their growth-inhibitory actions in the peripheral and central nervous system are the class 3 semaphorins (Sema 3s), which regulate cell migration, axonal growth and pathfinding, target selection, and axonal pruning (Roth et al., 2009), and B type ephrins which function in axonal guidance and cell migration (Klein, 2004). Sema 3s mediate these effects via a receptor complex comprised of binding subunits, the neuropilins, and signaling components, the Plexin A receptors (He & Tessier-Lavigne, 1997, Takahashi et al., 1999, Tamagnone et al., 1999, Yaron et al., 2005). Interestingly, a number of lines of evidence indicate that the semaphorins interact with the neurotrophins and/or their receptors to regulate peripheral axon growth and guidance. For example, we have previously shown that Sema 3F inhibits Trk signaling to Erk to cause sympathetic neuron growth cone collapse (Atwal et al., 2003) and Ben-Zvi et al. (2007) demonstrated that p75NTR antagonizes Sema 3A to inhibit growth cone collapse in embryonic sensory neurons. Similarly, ephrin B1 can suppress Ras–Erk signaling during neurite retraction mediated by its receptor EphB2 (Elowe et al., 2001).

Here, we have asked whether p75NTR acts in partnership with Sema 3s to inhibit collapse of growth cones, focusing upon peripheral sympathetic neurons. We also asked whether p75NTR is required for the activity of ephrin B2, which we identify as a novel collapsing factor for sympathetic neurons. We show that p75NTR is necessary for Sema 3 and ephrin B2-mediated growth cone collapse. Furthermore, collapse in response to the semaphorins and ephrin B2 requires neurotrophin-mediated activation of a p75NTR–Rho signaling pathway. These findings, together with previous observations that p75NTR signaling is also required for Nogo and ephrin A inhibitory signaling suggest that this receptor is a general co-receptor for ligand–receptor complexes with growth-inhibitory activity.