In spite of advances in understanding the role of the cellular prion protein (PrP) in neural cell interactions, the mechanisms of PrP function remain poorly characterized. We show that PrP interacts directly with the neural cell adhesion molecule (NCAM) and associates with NCAM at the neuronal cell surface. Both cis and trans interactions between NCAM at the neuronal surface and PrP promote recruitment of NCAM to lipid rafts and thereby regulate activation of fyn kinase, an enzyme involved in NCAM-mediated signaling. Cis and trans interactions between NCAM and PrP promote neurite outgrowth. When these interactions are disrupted in NCAM-deficient and PrP-deficient neurons or by PrP antibodies, NCAM/PrP-dependent neurite outgrowth is arrested, indicating that PrP is involved in nervous system development cooperating with NCAM as a signaling receptor.

In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100–insoluble complexes with βI spectrin. Heteromeric spectrin (αIβI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the βI spectrin fragment encompassing second and third spectrin repeats (βI2–3). In NCAM120-transfected cells, βI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-β-cyclodextrin disrupts the NCAM120–spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180–βI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCβ2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180–spectrin–PKCβ2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative βI2–3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCβ2 and NCAM140 or NCAM180. Redistribution of PKCβ2 to NCAM–spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with βI2–3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM–spectrin–PKCβ2 complex is necessary for NCAM-mediated neurite outgrowth.

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The neural cell adhesion molecule (NCAM) forms a complex with p59fyn kinase and activates it via a mechanism that has remained unknown. We show that the NCAM140 isoform directly interacts with the intracellular domain of the receptor-like protein tyrosine phosphatase RPTPα, a known activator of p59fyn. Whereas this direct interaction is Ca2+ independent, formation of the complex is enhanced by Ca2+-dependent spectrin cytoskeleton–mediated cross-linking of NCAM and RPTPα in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTPα-deficient brains and is disrupted by dominant-negative RPTPα mutants, demonstrating that RPTPα is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTPα-deficient neurons, and disruption of the NCAM–p59fyn complex in RPTPα-deficient neurons or with dominant-negative RPTPα mutants blocks NCAM-dependent neurite outgrowth, implicating RPTPα as a major phosphatase involved in NCAM-mediated signaling.

Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural cell adhesion molecule (NCAM) in the plasma membrane. These complexes are translocated along neurites and trapped at sites of initial neurite-to-neurite contacts within several minutes after initial contact formation. The accumulation of TGN organelles at contacts with NCAM-deficient neurons is reduced when compared with wild-type cells, suggesting that NCAM mediates the anchoring of intracellular organelles in nascent synapses.

The Ig superfamily adhesion molecule CHL1, the close homolog of the adhesion molecule L1, promotes neurite outgrowth, neuronal migration, and survival in vitro. We tested whether CHL1, similar to its close homolog L1, has a beneficial impact on recovery from spinal cord injury using adult CHL1-deficient (CHL1 Ϫ/Ϫ ) mice and wild-type (CHL1 ϩ/ϩ ) littermates. In contrast to our hypothesis, we found that functional recovery, assessed by locomotor rating and video-based motion analyses, was improved in CHL1 Ϫ/Ϫ mice compared with wild-type mice at 3-6 weeks after compression of the thoracic spinal cord. Better function was associated with enhanced monoaminergic reinnervation of the lumbar spinal cord and altered pattern of posttraumatic synaptic rearrangements around motoneurons. Restricted recovery of wild-type mice was likely related to early and persistent (3-56 d after lesion) upregulation of CHL1 in GFAP-positive astrocytes at the lesion core. In both the intact spinal cord and cultured astrocytes, enhanced expression of CHL1 and GFAP was induced by application of basic fibroblast growth factor, a cytokine involved in the pathophysiology of spinal cord injury. This upregulation was abolished by inhibitors of FGF receptor-dependent extracellular signal-regulated kinase, calcium/calmodulin-dependent kinase, and phosphoinositide-3 kinase signaling pathways. In homogenotypic and heterogenotypic cocultures of neurons and astrocytes, reduced neurite outgrowth was observed only if CHL1 was simultaneously present on both cell types. These findings and novel in vitro evidence for a homophilic CHL1-CHL1 interaction indicate that CHL1 is a glial scar component that restricts posttraumatic axonal growth and remodeling of spinal circuits by homophilic binding mechanisms.

In searching for binding partners of the intracellular domain of the immunoglobulin superfamily adhesion molecule CHL1, we identified the clathrin-uncoating ATPase Hsc70. CHL1 gene ablation resulted in reduced targeting of Hsc70 to the synaptic plasma membrane and synaptic vesicles, suggesting CHL1 as a synapse-targeting cue for Hsc70. CHL1 accumulates in presynaptic membranes and, in response to synapse activation, is targeted to synaptic vesicles by endocytosis. CHL1 deficiency or disruption of the CHL1/Hsc70 complex results in accumulation of abnormally high levels of clathrin-coated synaptic vesicles with a reduced ability to release clathrin. Generation of new clathrin-coated synaptic vesicles in an activity-dependent manner is inhibited when the CHL1/Hsc70 complex is disrupted, resulting in impaired uptake and release of FM dyes in synaptic boutons. Abnormalities in clathrin-dependent synaptic vesicle recycling may thus underlie brain malfunctions in humans and mice that carry mutations in the CHL1 gene.

The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. We show that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the N-methyl-d-aspartate (NMDA) receptor and Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) in a manner not firmly or directly linked to PSD95 and α-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM–spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM–spectrin–associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIα. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIα to synapses after NMDA receptor activation, which is a critical process in NMDA receptor–dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM.