| Unlike the peripheral nervous system (PNS) or embryonic neurons, the adult mammalian central nervous system (CNS) does not spontaneously regenerate after injury. This is due, in part, to the presence of myelin-associated inhibitors, such as myelin-associated glycoprotein (MAG). Our lab has shown that elevation of intracellular cAMP blocks these inhibitors in vitro and in vivo in a transcription-dependent manner. Subsequent microarray analysis revealed that elevation of cAMP results in upregulation of Arginase I (Arg1), a key enzyme in the synthesis of polyamines. Our lab has demonstrated that administration of polyamines is sufficient to block MAG and myelin-induced inhibition of axonal outgrowth in vitro as well as enhance CNS axon regeneration in vivo. Others have shown that binding of polyamines to ionotropic glutamate receptors (iGluRs), which include N-methyl-D-aspartate receptors (NMDAR), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors (KAR), blocks their activity. In addition, it is well established that iGluR activity is disrupted following CNS injury. Thus, since polyamines overcome MAG/myelin-mediated inhibition and given that polyamines block iGluRs activity, we set out to examine the role iGluR-mediated signaling plays in the MAG and myelin inhibitory pathways. We found that blocking NMDAR or AMPA/KA receptor activity with pharmacological antagonists was sufficient to block MAG-induced inhibition of neurite outgrowth in dorsal root ganglia (DRG) and hippocampal neurons (HNs). Likewise, exposure to iGluR agonists increased MAG-mediated inhibition of neurite outgrowth. To determine whether prior exposure of iGluR inhibitors or priming is sufficient to overcome MAG inhibition as in the case of polyamines, HNs were treated with iGluR antagonists 18 hours before exposing neurons to MAG. We found that priming with iGluR antagonists was not sufficient to overcome MAG-mediated inhibition, suggesting that GluR antagonists exert their effect by intercepting MAG inhibition signals, while not changing the ability of neurons to respond to MAG. Because polyamines require priming whereas iGluR antagonists have no effect when used to prime neurons, we therefore conclude that in our model system, polyamines do not overcome inhibition by blocking iGluR activity.;It is well established that upon activation, NMDARs produce influx of Ca2+ inside the cell, which activates Ca2+-dependent kinases such as extracellular signal-regulated kinase (ERK) and conventional protein kinase C (PKC), both of which are important for synaptic plasticity, long term potentiation (LTP), and long term depression (LTD). Importantly, conventional PKC and epidermal growth factor receptor (EGFR)-regulated ERKs are also known to be activated in response to myelin inhibitors in a Ca 2+-dependent manner. Blocking PKCs or EGFRs is sufficient to overcome inhibition by myelin inhibitors in vitro and promote axonal regeneration in vivo. To establish which downstream target activation is affected in response to iGluR activity block as well as myelin, PKC and ERK signaling pathways were examined. We found that myelin induces robust ERK activation, and importantly, inhibiting iGluR activity was sufficient to block ERK activation. Next, to determine whether myelin-induced ERK activation is inhibitory for neurite outgrowth, HNs were treated with the ERK inhibitor U0126 and then subjected neurite outgrowth assay in the presence of MAG. We found that blocking ERK was sufficient to overcome MAG-mediated inhibition in a dose dependent manner. Our findings demonstrate that exposure to myelin induces ERK activation and that ERK activation is regulated by iGluR activity. Since ERK is one of the main downstream targets activated by NMDAR-induced Ca2+ influx, our findings support the idea that exposure to myelin results in iGluR activation.;In synapses, repeated activation of NMDAR leads to long-term potentiation (LTP) through mechanisms involving the activation of PKC and ERK as well as rapid forward trafficking of NMDARs to the membrane. This is consistent with reports that PKC induces phosphorylation in the C-terminal of the NMDA NR1 subunit, leading to increased surface insertion of NMDAR channels. To determine whether myelinassociated inhibitors affect NMDAR trafficking, cortical neurons were treated with myelin and then the amounts of surface NMDARs was assessed. We found that myelin induced a rapid increase in surface NMDAR number. In addition, myelin treatment induced phosphorylation of ser890 and ser896 C-terminal NMDA NR1 subunits, which have been shown to promote forward NMDAR trafficking to the cell surface. Thus, our findings suggest that myelin-induced PKC activation promotes surface incorporation of NMDARs, which can potentially enhance NMDAR-mediated currents. Taken together, our results implicate iGluR activity as well as ERK signaling as new mediators of MAG/myelin-induced inhibition, offering new insights into the molecular mechanism of myelin-induced block of neurite outgrowth. |
