Patrick Doherty

A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration

Following nerve injury, axons in the CNS do not normally regenerate. It has been shown that CNS myelin inhibits neurite outgrowth, though the nature of the molecules responsible for this effect are not known. Here, we demonstrate that the myelin-associated glycoprotein (MAG), a transmembrane protein of both CNS and PNS myelin, strongly inhibits neurite outgrowth from both developing cerebellar and adult dorsal root ganglion (DRG) neurons in vitro. This inhibition is reversed by an anti-MAG antibody. In contrast, MAG promotes neurite outgrowth from newborn DRG neurons. These results suggest that MAG may be responsible, in part, for the lack of CNS nerve regeneration in vivo and may influence, both temporally and spatially, regeneration in the PNS.

Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin.

Cell contact-dependent neurite outgrowth stimulated by CAMs requires activation of a second messenger pathway that requires the function of a tyrosine kinase upstream from calcium influx into neurons. In the present study, we present evidence that implicates activation of the fibroblast growth factor receptor (FGFR) in the pathway underlying neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. We have identified a CAM homology domain in the FGF family of receptors and show that antibodies which bind to this domain specifically inhibit neurite outgrowth stimulated by the above CAMs. We also show that synthetic peptides derived from this domain can differentially and specifically inhibit neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. In addition, a soluble L1-Fc chimera is shown to stimulate an increase in phosphotyrosine on the same set of neuronal proteins that are phosphorylated following activation of the FGFR with basic FGF.

CAM-FGF Receptor Interactions: A Model for Axonal Growth

A number of experimental paradigms have been used to demonstrate that NCAM, N-cadherin, and L1 stimulate axonal growth. The molecular basis of this response has been extensively studied and a range of agents that inhibit neurite outgrowth stimulated by the above CAMs, but not integrins, have now been identified. These studies pointed to the activation of a tyrosine kinase-PLCgamma cascade as being important for the neurite outgrowth responses stimulated by all three CAMs, and this was substantiated by the identification of agents that could activate the cascade and mimic the growth response. In this review we will suggest that the neurite growth response stimulated by these CAMs is mediated by activation of the fibroblast growth factor receptor (FGFR) in neurons and that this results in the recruitment and activation of PLCgamma via interactions of its SH2 domain with the activated receptor. In this context the key events downstream from activation of PLCgamma required for neurite growth appear to be the conversion of diacylglycerol (DAG) to arachidonic acid (AA) via DAG lipase activity, followed by an increased influx of calcium into the neurons. The evolutionary conservation of putative binding motifs between the above CAMs and the FGFR suggests that activation of the FGFR-PLCgamma cascade by the CAMs might involve a direct CAM-FGFR interaction. The identification of the binding motifs also allows for predictions to be made concerning whether other CAMs might directly interact with the FGFR.

Expression of a Dominant Negative FGF Receptor Inhibits Axonal Growth and FGF Receptor Phosphorylation Stimulated by CAMs

The cell adhesion molecules (CAMs) NCAM, N-cadherin, and L1 are homophilic binding molecules that stimulate axonal growth. We have postulated that the above CAMs can stimulate this response by activating the fibroblast growth factor receptor (FGFR) in neurons. In the present study, we demonstrate that activation of NCAM and L1 can lead to phosphorylation of the FGFR. Both this and the neurite outgrowth response stimulated by all three of the above CAMs are lost when a kinase-deleted, dominant negative form of FGFR1 is expressed in PC12 cells. In addition, we have generated transgenic mice that express the dominant negative FGFR under control of the neuron-specific enolase (NSE) promoter. We show that cerebellar neurons isolated from these mice have also lost their ability to respond to NCAM, N-cadherin, and L1. A peptide inhibitor of phospholipase C gamma (PLCgamma) that inhibits neurite outgrowth stimulated by FGF also inhibited neurite outgrowth stimulated by the CAMs. Thus, we conclude that activation of the FGFR is both necessary and sufficient to account for the ability of the above CAMs to stimulate axonal growth, and that PLCgamma is a key downstream effector of this response.

Morphoregulatory activities of NCAM and N-cadherin can be accounted for by G protein-dependent activation of L- and N-type neuronal Ca2+ channels

We present evidence that the morphoregulatory activities of neural cell adhesion molecule (NCAM) and N-cadherin involve activation of intracellular second messenger pathways. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin or NCAM. NCAM and N-cadherin directly induced a transcription-independent change in the morphology of PC12 cells from an adrenal to neuronal phenotype and also specifically increased Thy-1, but not L1/NILE or low affinity NGF receptor, immunoreactivity. The morphological response was more rapid and, in the case of N-cadherin, more substantial than that induced by NGF. It could be fully inhibited by pertussis toxin and a combination of L- and N-type Ca2+ channel antagonists, but not by broad-specificity kinase inhibitors. It was blocked, however, by the kinase inhibitor K-252b. These studies suggest that cell adhesion molecules directly alter cell phenotype and provide direct evidence for transmembrane signaling mediating both the morphological and biochemical responses induced by NCAM and N-cadherin.

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid.

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.

Signal transduction events underlying neurite outgrowth stimulated by cell adhesion molecules

During development of the nervous system, cell adhesion molecules (CAMs) promote cell migration and axonal growth; yet, at other times, CAMs inhibit these events by maintaining stable adhesion between cells. In the present review, we consider recent results that help to explain the paradoxical findings that individual CAMs can both promote and inhibit neuronal plasticity. In particular, we discuss the accumulating evidence that axonal growth stimulated by CAMs depends upon the activation of a second messenger pathway that culminates in calcium entry into neurons rather than on adhesion per se.

A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth

Cerebellar neurons, cultured on monolayers of 3T3 fibroblasts or on a polylysine/extracellular matrix-coated substratum, responded to a soluble recombinant L1-Fc chimera by extending longer neurites than controls. The response was inhibited by pretreating neurons with antibodies to L1 or antibodies to the fibroblast growth factor (FGF) receptor. The response could also be inhibited by a range of pharmacological reagents that inhibit various steps in the signal transduction cascade which underlie a neurite outgrowth response to basic FGF. The response was of a similar magnitude and not additive with that induced by L1 expressed in a cellular substrate. These data show that L1 in neurons is capable of directing a neurite outgrowth response to a soluble L1-Fc chimera, and that neuronal FGF receptor function is required for this response. The data also show that the ability of cell adhesion molecules (CAMs) to stimulate neurite outgrowth can be dissociated from their ability to function as substrate-associated adhesion molecules and point to the potential of using CAM-Fc chimeras to promote nerve regeneration.

Structural mosaicism on the submicron scale in the plasma membrane.

The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using single particle tracking (SPT). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode, or the size of their cytoplasmic domain, were expressed in National Institutes of Health 3T3 cells and C2C12 muscle cells. On a 6.6-s time scale, SPT measurements on both transmembrane and glycosylphosphatidylinositol (GPI) anchored isoforms of NCAM expressed in 3T3 cells could be classified into mobile (Brownian diffusion), slow diffusion, corralled diffusion, and immobile subpopulations. On a 90-s time scale, SPT studies in C2C12 cells revealed that 40-60% of transfected NCAM was mobile, whereas a smaller fraction (approximately 10-30%) experienced much slower diffusion. In addition, a fraction of approximately 30% of both transfected GPI and transmembrane isoforms and endogenous NCAM isoforms in C2C12 cells experienced transient confinement for approximately 8 s within regions of approximately 300-nm diameter. Diffusion within both these and the slow diffusion regions was anomalous, consistent with movements through a dense field of obstacles, whereas diffusion outside these regions was normal. Thus the membrane appears as a mosaic containing regions that permit free diffusion as well as regions in which NCAM is transiently confined to small or more extended domains. These results, including a large, freely diffusing fraction, similar confinement of transmembrane and GPI isoforms, a significant slowly diffusing fraction, and relatively large interdomain distances, are at some variance with the membrane skeleton fence model (Kusumi and Sako, 1996). Possible revisions to the model that incorporate these data are discussed.

The production of arachidonic acid can account for calcium channel activation in the second messenger pathway underlying neurite outgrowth stimulated by NCAM, N-cadherin, and L1.

Abstract: We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)—NCAM, N‐cadherin, and L1—as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N‐and l‐type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 μM) and that this response can be inhibited by N‐and l‐type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A2 or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage‐dependent calcium currents in cardiac myocytes.