Dorte Kornerup Ditlevsen

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons.

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell–cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM‐mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras‐mitogen activated protein kinase (MAPK) and the phosphatidylinositol‐3‐kinase (PI3K)‐Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.

NCAM‐induced intracellular signaling revisited

The neural cell adhesion molecule (NCAM) plays a crucial role in neuronal development, synaptic plasticity, and regeneration. NCAM works as “smart glue” that not only mediates cell–cell adhesion but also induces activation of a complex network of intracellular signaling cascades on homophilic or heterophilic binding. Stimulation of NCAM by homophilic interactions induces neuronal differentiation through activation of a number of signaling molecules, including the fibroblast growth factor receptor, non–receptor kinases Fyn and focal adhesion kinase, growth‐associated protein‐43, the mitogen‐activated protein kinase pathway, intracellular Ca2+, and protein kinases A, C, and G. This review presents and discusses the current knowledge in the area of NCAM signaling with a focus on the events involved in NCAM‐mediated neurite outgrowth.

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival.

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras‐mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM‐ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM‐stimulation. We present here data indicating that phosphatidylinositol 3‐kinase (PI3K) is required for NCAM‐mediated neurite outgrowth from PC12‐E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival‐promoting effect of NCAM‐stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA‐fragmentation and caspase‐3 activation. The survival‐promoting effect of NCAM‐stimulation was also shown to be dependent on PI3K.

Intracellular signaling by the neural cell adhesion molecule

Cell adhesion molecules are known to play far more complex roles than mechanically attaching one cell to an adjacent cell or to components of the extracellular matrix. Thus, important roles for cell adhesion molecules in the regulation of intracellular signaling pathways have been revealed. In this review, we discuss the present knowledge about signaling pathways activated upon homophilic binding of the neural cell adhesion molecule (NCAM). Homophilic NCAM binding leads to activation of a signal transduction pathway involving Ca2+ through activation of the fibroblast growth factor receptor, and to activation of the mitogen-activated protein kinase pathway. In addition, cyclic adenosine monophosphate and protein kinase A are involved in NCAM-mediated signaling. Among these pathways the possibility exists of cross talk or convergence, of which different possible mediators have been suggested. Finally, several downstream effector molecules leading to NCAM-mediated cellular endpoints have been demonstrated, including transcription factors and regulators of the cytoskeleton.

Neuritogenic and survival‐promoting effects of the P2 peptide derived from a homophilic binding site in the neural cell adhesion molecule

The neural cell adhesion molecule (NCAM) plays a pivotal role in neural development, regeneration, and plasticity. NCAM mediates adhesion and subsequent signal transduction through NCAM–NCAM binding. Recently, a peptide ligand termed P2 corresponding to a 12‐amino‐acid sequence in the FG loop of the second Ig domain of NCAM was shown to mimic NCAM homophilic binding as reflected by induction of neurite outgrowth in hippocampal neurons. We demonstrate here that in concentrations between 0.1 and 10 μM, P2 also induced neuritogenesis in primary dopaminergic and cerebellar neurons. Furthermore, it enhanced the survival rate of cerebellar neurons although not of mesencephalic dopaminergic neurons. Moreover, our data indicate that the protective effect of P2 in cerebellar neurons was due to an inhibition of the apoptotic process, in that caspase‐3 activity and the level of DNA fragmentation were lowered by P2. Finally, treatment of neurons with P2 resulted in phosphorylation of the ser/thr kinase Akt. Thus, a small peptide mimicking homophilic NCAM interaction is capable of inducing differentiation as reflected by neurite outgrowth in several neuronal cell types and inhibiting apoptosis in cerebellar granule neurons.

Signalling pathways underlying neural cell adhesion molecule-mediated survival of dopaminergic neurons.

Stimulation of the neural cell adhesion molecule (NCAM) by homophilic interactions is known to lead to neurite outgrowth as well as to neuronal survival. Whereas a complex network of signalling molecules is known to be of importance to NCAM‐mediated neurite extension, only limited information is available regarding signalling underlying NCAM‐mediated neuroprotection. Here, we present data suggesting a difference in the signalling events required for survival of rat dopaminergic neurons as compared with neurite outgrowth from the same cell type. Whereas Fyn, fibroblast growth factor receptor, mitogen‐activated protein and ERK kinase, protein kinase A and protein kinase C are required for both responses to NCAM‐induced signalling, phospholipase C and Ca2+‐calmodulin‐dependent kinase II are only necessary for the neurite outgrowth response, but dispensable for neuroprotection.

Signaling Pathways Involved in NCAM-Induced Neurite Outgrowth

The neural cell adhesion molecule, NCAM, plays important roles in both the developing and adult brains. NCAM, originally, was described as a homophilic cell adhesion molecule abundantly expressed in the central nervous system. This view has been challenged, however, during the last two decades, and NCAM now is considered to be an inducer of complex intracellular signaling cascades in response to extracellular cues. Homo and heterophilic NCAM interactions result in the induction of neurite outgrowth, neuronal survival, and astrocyte proliferation. Here, we review the available data regarding NCAM-induced intracellular signaling and focus on the most intensively studied cellular response, neurite outgrowth.

Cyclic guanosine monophosphate signalling pathway plays a role in neural cell adhesion molecule‐mediated neurite outgrowth and survival

The neural cell adhesion molecule (NCAM) plays a crucial role in neuronal development, regeneration, and synaptic plasticity associated with learning and memory consolidation. Homophilic binding of NCAM leads to neurite extension and neuroprotection in various types of primary neurons through activation of a complex network of signalling cascades, including fibroblast growth factor receptor, Src‐family kinases, the mitogen‐activated protein kinase pathway, protein kinase C, phosphatidylinositol‐3 kinase, and an increase in intracellular Ca2+. Here we present data indicating an involvement of cyclic GMP in NCAM‐mediated neurite outgrowth in both hippocampal and dopaminergic neurons and in NCAM‐mediated neuroprotection of dopaminergic neurons. In addition, evidence is presented suggesting that NCAM mediates activation of cGMP via synthesis of nitric oxide (NO) by NO synthase (NOS) and activation of soluble guanylyl cyclase by NO, leading to an increased synthesis of cGMP and activation by cGMP of protein kinase G.

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen‐activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)‐mediated neurite outgrowth in the PC12‐E2 cell line. We found that both FGFR substrate‐2 and Grb2 play important roles in NCAM as well as in FGF2‐stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.

Relative role of upstream regulators of Akt, ERK and CREB in NCAM- and FGF2-mediated signalling

Although a large number of signalling cascades are known to be activated downstream of NCAM, only little is known regarding the hierarchical relationship between the involved molecules in the individual cascades and the level of cross talk between the cascades. Here, we evaluated the requirement of putative upstream signalling cascades for the phosphorylation of the kinases extracellular signal-regulated kinase (ERK) and Akt and the transcription factor cyclic adenosine monophosphate (cAMP) response-element binding protein (CREB) following stimulation of NCAM in rat cerebellar granule neurons with an NCAM ligand, the C3d peptide. NCAM-mediated ERK phosphorylation depended on activation of the fibroblast growth factor receptor (FGFR), Src-family kinases, MEK (MAP and ERK kinase) and G(0)/G(i)-proteins, whereas NCAM-mediated CREB phosphorylation depended on the activity of Src-family kinases and MEK. NCAM-specific Akt phosphorylation depended on cyclic guanosine monophosphate (cGMP) and phosphatidylinositide 3-kinase (PI3K). All three phosphorylation events were independent of activation of the signalling molecules phospholipase C, protein kinase C, protein kinase A, and CamKII, which all have been demonstrated previously to be involved in NCAM signalling. For comparison, we also evaluated the role of upstream signalling cascades on fibroblast growth factor 2 (FGF2)-mediated phosphorylation of ERK, Akt, and CREB and found that FGF2 required the activity of both FGFR and Src-family kinases for phosphorylation of ERK, Akt, and CREB. MEK was required for phosphorylation of ERK and CREB, but not Akt, whereas G(0)/G(i)-proteins were necessary for phosphorylation of Akt and CREB, and cGMP was necessary for Akt phosphorylation. We thus demonstrate that even though NCAM and FGF2 have many signalling features in common, and even though both are known to activate FGFR, there are a number of differences in the intracellular signalling network activated by the NCAM ligand C3d and the FGFR ligand FGF2.