A novel role for PTEN in the inhibition of neurite outgrowth by myelin-associated glycoprotein in cortical neurons

Abstract

Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG's inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or overexpression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis.

Introduction

The limited regrowth of axons following injury in the mammalian central nervous system (CNS) can result in debilitating and often permanent neurological deficits. Myelin, produced by oliogodendrocytes to facilitate saltatory conduction, expresses a number of proteins that inhibit axonal growth and are thought to contribute to preventing regeneration. These myelin-associated inhibitors include Nogo A, myelin-associated glycoprotein (MAG), Oligodendrocyte myelin glycoprotein (OMgp), Ephrin-B3, and Sema4D (Filbin, 2003, Yiu and He, 2006).

MAG, one of the most extensively studied myelin-associated inhibitors, is a transmembrane glycoprotein expressed on periaxonal myelin membranes in the peripheral and central nervous systems. It plays a physiological role in maintaining myelinated axons as well as contributing to the pathology of demyelinating diseases and the inhibition of CNS regeneration (Filbin, 2003, Quarles, 2007). Several neuronal receptors have been identified for MAG, including the Nogo receptors (NgR1 and NgR2) (Domeniconi et al., 2002, Liu et al., 2002, Venkatesh et al., 2005), gangliosides GD1a and GT1b (Yang et al., 1996, Vinson et al., 2001, Vyas et al., 2002), and the more recently identified paired immunoglobulin-like receptor B (PirB) (Atwal et al., 2008).

Understanding how the various receptors contribute to the inhibition of axon outgrowth by MAG has proven remarkably complex. NgR1 is a glycosylphosphatidylinositol-linked protein that binds MAG and signals through interaction with the p75 neurotrophin receptor (p75NTR) (Wang et al., 2002, Wong et al., 2002) or a homologous protein, Taj/TROY (Park et al., 2005, Shao et al., 2005), and the novel transmembrane protein LINGO-1 (Mi et al., 2004). The NgR complex was recently reported to function in an additive manner with the PirB receptor, such that inhibiting both receptors was necessary to fully reverse the effects of myelin (Atwal et al., 2008). The effects of MAG have also been reported to depend on interaction with gangliosides, at least in some neurons (Vyas et al., 2002, Mehta et al., 2007); however, this remains controversial (Cao et al., 2007, Schnaar and Lopez, 2009). It has been suggested that MAG's inhibitory effects involve different receptors depending on the type of neuron and the nature of the inhibition, such as acute growth cone collapse versus more long-term axon extension (Chivatakarn et al., 2007, Mehta et al., 2007, Venkatesh et al., 2007).

How these various MAG receptors transduce their signals to inhibit axon growth is also not well understood. The NgR1 complex can activate the GTP binding protein RhoA, which regulates the actin-cytoskeleton, leading to growth cone collapse and the prevention of neurite outgrowth (Hall, 1998, Niederost et al., 2002, Wang et al., 2002, Yamashita et al., 2002). However, a role for intracellular calcium (Song et al., 1998, Wong et al., 2002) and protein kinase C (PKC) (Hasegawa et al., 2004, Sivasankaran et al., 2004) has also been suggested downstream of NgR–p75NTR–LINGO. The mechanisms by which gangliosides and PirB signal have yet to be determined, although an association between PirB and the phosphatases Shp-1 and Shp-2 has been reported (Syken et al., 2006).

We set out to investigate the receptors and intracellular signaling pathways involved in MAG-mediated inhibition of neurite outgrowth, specifically in cortical neurons. One of the pathways often damaged in spinal cord injury is the corticospinal tract, resulting in debilitating clinical manifestations including paralysis (Joosten, 1997, McDonald and Sadowsky, 2002). However, very few studies have investigated how cortical neurons respond to myelin-associated inhibitors and the mechanisms underlying their inhibition.

Our results demonstrate that cortical neuron process growth is robustly inhibited by MAG, but the inhibition could not be reversed by deletion of p75NTR or by blocking gangliosides or PirB. Surprisingly, inhibition of Rho signaling only partially reversed the effect of MAG, indicating the presence of additional intracellular signals. We identify PTEN as a downstream effector of MAG-mediated inhibition of cortical neuron process growth. Together, these findings suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis.