Cortical neurons are frequently damaged in spinal cord injury and fail to regenerate, in part, due to the presence of growth inhibitory proteins at the site of injury. Furthermore, recent findings suggest that inhibitors expressed by myelin may normally function in the refinement of cortical circuitry during development (McGee et al., 2005
). However, very little is known about how cortical neurons respond to specific, endogenous inhibitors of axon growth. Here, we investigated the response of cortical neurons to MAG, a myelin protein known to prevent axonal outgrowth in other neurons. Our results indicate that both embryonic and postnatal cortical neurons are particularly sensitive to inhibition of neurite outgrowth by MAG. Interestingly, the effects of MAG were not mediated by any of its known receptors, but involved both the Rho/ROCK pathway and a novel pathway involving the lipid phosphatase PTEN.
MAG is a multifunctional, transmembrane protein expressed on the periaxonal surface of myelinating cells in both the peripheral and central nervous system. It is important for long-term axon-myelin stability and for proper structuring of nodes of Ranvier (Quarles, 2007
; Schnaar and Lopez, 2009
). In addition, MAG regulates axonal growth, being able to promote it or inhibit it depending on the type of neuron as well as the developmental stage. MAG enhances neurite outgrowth from a number of embryonic neurons, including retinal ganglion cells (Cai et al., 2001
), spinal neurons (Turnley and Bartlett, 1998
; Cai et al., 2001
), and dorsal root ganglia (DRG) neurons (Johnson et al., 1989
; Mukhopadhyay et al., 1994
; DeBellard et al., 1996
; Cai et al., 2001
), whereas postnatal axon outgrowth of these neurons is inhibited. In cortical neurons, we observed dramatic inhibition of neurite outgrowth by MAG at both embryonic (E15 to E17) and postnatal (P1-3) ages.
The mechanisms by which MAG mediates these various signals are poorly understood. Due to the relevance for spinal cord injury, much attention has focused on elucidating the signals responsible for inhibiting axonal growth. The best characterized receptor complex mediating the effects of MAG is the one formed by the gpi-linked Nogo receptor, p75NTR or TROY and LINGO. However, we found that p75NTR was not involved in preventing neurite outgrowth in cortical neurons and no expression of TROY was detectable (). These results are in agreement with those of Chivatakarn et al (2007)
who found that cortical neurons from mice with NgR1 deleted were as responsive to MAG-CHO cells as wild-type neurons. Since MAG can also bind NgR2 (Venkatesh et al., 2005
), it is possible their results could be explained by NgR2 substituting for NgR1. However, taken together with our findings, the more likely conclusion is that the NgR-p75NTR-LINGO complex is not required for the inhibition of neurite out growth by membrane-bound MAG in cortical neurons.
The role of the NgR-p75NTR-LINGO complex in mediating the effects of MAG appears to depend on the type of neuron and the form of MAG used. For example, Venkatesh et al reported that wild-type and p75ntr
null retinal ganglion cells (RGCs) and CGNs were equally inhibited by MAG-CHO cells; however, p75ntr
−/− DRG neurons were significantly less inhibited than wild-type neurons, suggesting that membrane-bound MAG signals through p75NTR in DRGs but not RGCs or CGNs (Venkatesh et al., 2007
). In contrast, numerous reports have demonstrated a requirement for p75NTR in mediating the response of CGNs to a soluble fragment of MAG containing the extracellular domain (Wang et al., 2002
; Yamashita et al., 2002
; Yamashita and Tohyama, 2003
). A soluble form of MAG has been detected following incubation of spinal cord extracts in vitro (Tang et al., 2001
), but whether this cleaved product of MAG is generated after neuronal injury is not known. Ultimately, a major objective of studies on MAG’s ability to inhibit neurite outgrowth is to understand how regeneration could be facilitated following axonal damage in vivo. However, p75ntr
−/− mice do not exhibit any significant improvement in regeneration of the CST following spinal cord injury, indicating that this receptor does not have a major role in preventing axonal growth from cortical neurons in vivo (Song et al., 2004
; Zheng et al., 2005
Two other receptors have been reported for MAG, the gangliosides GT1b and GD1a, and PirB, a major histocompatibility complex class 1 receptor. However, we found that neither of these molecules significantly contributed to the inhibitory effects of MAG in cortical neurons. Of course we cannot rule out the possibility that the concentration of PirB antibody used was not high enough to disrupt the interaction of MAG with cortical neurons. Although not statistically significant, there was a trend toward a partial reversal of the inhibition by MAG with anti-PirB. However, taken together, these results suggest that there exists an additional, yet to be identified receptor for membrane-bound MAG that transduces a signal preventing neurite outgrowth in cortical neurons.
Down stream of many signals inhibiting process outgrowth and extension in cells are the small GTPase Rho and its effector kinase ROCK. Increased Rho activity has been detected in neurons following spinal cord injury (Dubreuil et al., 2003
; Madura et al., 2004
) and application of the Rho inhibitor C3 or the ROCK inhibitor Y27632 at the site of injury resulted in significant, although limited, regeneration of corticospinal tract fibers as well as functional improvement in locomotion and coordination (Dergham et al., 2002
; Fournier et al., 2003
). Similarly, genetic deletion of ROCKII revealed that this kinase plays a role in restricting axon outgrowth following spinal cord injury (Duffy et al., 2009
). MAG has been reported to increase Rho activity in neurons (Lehmann et al., 1999
; Niederost et al., 2002
; Yamashita et al., 2002
; Fournier et al., 2003
) and, not unexpectedly, we found that inhibiting Rho or ROCK significantly reversed the effect of membrane-bound MAG on cortical neurons. However, we were surprised to find that blocking Rho or ROCK was only partially effective in the cortical neurons; significant inhibition of neurite outgrowth remained in the neurons plated on MAG-CHO cells when C3 or Y27632 was added. This result suggested that membrane-bound MAG was activating additional growth inhibitory signals.
We identified PTEN as an essential component of MAG’s inhibitory effects on neurite outgrowth in cortical neurons. PTEN is a lipid phosphatase that indirectly inactivates the kinase Akt by reducing the levels of phosphatidylinositol 3,4,5 trisphosphate, which is required for Akt activation. A previous study suggested that the PI3K/AKT/mTOR pathway promotes axon extension and demonstrated that deletion of PTEN or the mTOR repressor, Tuberous sclerosis complex 1 (TSC1), significantly improved regeneration in the optic nerve following a crush injury (Park et al., 2008
). Here, we demonstrated that membrane-bound MAG actively reduced Akt phosphorylation, thereby implicating activation of PTEN by this inhibitor. Suppression of Akt activity would decrease activation of Rac, a GTP binding protein that promotes neurite extension through regulation of actin polymerization, and increase the activation of Glycogen synthase kinase 3 (GSK3), a kinase that regulates microtubule dynamics (Park et al.). Similar to our findings, PTEN was recently identified as a target of Sema3A in triggering growth cone collapse of sensory neurons (Chadborn et al., 2006
). Thus, PTEN appears to be a key component in another signal transduction pathway, like the Rho/ROCK cascade, that is activated by inhibitors of axon growth.
The mechanisms by which PTEN is regulated are not well understood. It can be serine/threonine phosphorylated by casein kinase 2 (CK2) and GSK3 and evidence exists suggesting that this modification inhibits its activity, reduces membrane localization and/or causes destabilization of the protein (Leslie et al., 2008
). Recently, NGF was reported to increase PTEN phosphorylation by CK2 in hippocampal neurons, thereby promoting axonal growth (Arevalo and Rodriguez-Tebar, 2006
). Reactive oxygen species generated in response to various growth factors can also suppress PTEN activity by causing the formation of disulfide bonds (Lee et al., 2002
; Leslie et al., 2003
; Kwon et al., 2004
; Seo et al., 2005
). In contrast, the recruitment to membranes by increasing the local concentration of acidic lipids, such as phosphatidylinositol 3,4 bisphosphate, can activate PTEN (Campbell et al., 2003
; Iijima et al., 2004
; Redfern et al., 2008
). How MAG or Sema3A modulate PTEN activity is not known. Chadborn et al (2006)
suggested that Sema3A induces a local accumulation of PTEN at the growth cone, leading to a depletion of phosphatidylinositol 3,4,5 trisphosphate. Determining the mechanisms by which MAG activates PTEN will be an interesting topic for future studies.
Gaining a more complete understanding of the different mechanisms employed by myelin-associated inhibitors in specific neuron types could help in developing therapeutics that will be effective for specific fiber tracts and brain regions following injury. Our results with cortical neurons suggest that in addition to Rho/ROCK, PTEN is another therapeutic target that may enhance regeneration of these neurons. Furthermore, the identification of PTEN as a downstream effector of MAG in preventing neurite outgrowth may have implications for other processes mediated by MAG such as axon-myelin stability and the refinement of cortical circuitry.