Prion protein recruits its neuronal receptor NCAM to lipid rafts to activate p59fyn and to enhance neurite outgrowth

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

Neural cell adhesion molecule (NCAM) association with PKCβ2 via βI spectrin is implicated in NCAM-mediated neurite outgrowth

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

The Neural Cell Adhesion Molecule Promotes Maturation of the Presynaptic Endocytotic Machinery by Switching Synaptic Vesicle Recycling from Adaptor Protein 3 (AP-3)- to AP-2-Dependent Mechanisms

Newly formed synapses undergo maturation during ontogenetic development via mechanisms that remain poorly understood. We show that maturation of the presynaptic endocytotic machinery in CNS neurons requires substitution of the adaptor protein 3 (AP-3) with AP-2 at the presynaptic plasma membrane. In mature synapses, AP-2 associates with the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes binding of AP-2 over binding of AP-3 to presynaptic membranes, thus favoring the substitution of AP-3 for AP-2 during formation of mature synapses. The presynaptic endocytotic machinery remains immature in adult NCAM-deficient (NCAM−/−) mice accumulating AP-3 instead of AP-2 and its partner protein AP180 in synaptic membranes and vesicles. NCAM deficiency or disruption of the NCAM/AP-2 complex in wild-type (NCAM+/+) neurons by overexpression of AP-2 binding-defective mutant NCAM interferes with efficient retrieval of the synaptic vesicle v-SNARE synaptobrevin 2. Abnormalities in synaptic vesicle endocytosis and recycling may thus contribute to neurological disorders associated with mutations in NCAM

NCAM induces CaMKIIα-mediated RPTPα phosphorylation to enhance its catalytic activity and neurite outgrowth

Receptor protein tyrosine phosphatase α (RPTPα) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPα activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPα. NCAM associates with T- and L-type voltage-dependent Ca2+ channels, and NCAM clustering at the cell surface results in Ca2+ influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIα (CaMKIIα). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM–RPTPα–CaMKIIα complex, resulting in serine phosphorylation of RPTPα by CaMKIIα. Overexpression of RPTPα with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPα activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity

Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts

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

CHL1 Is a Selective Organizer of the Presynaptic Machinery Chaperoning the SNARE Complex

Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and αSGT. CHL1, Hsc70, CSP and αSGT form predominantly CHL1/Hsc70/αSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and αSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/αSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery

RPTPalpha is essential for NCAM-mediated p59fyn activation and neurite elongation

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 RPTPalpha, 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 RPTPalpha in response to NCAM activation and is accompanied by redistribution of the complex to lipid rafts. Association between NCAM and p59fyn is lost in RPTPalpha-deficient brains and is disrupted by dominant-negative RPTPalpha mutants, demonstrating that RPTPalpha is a link between NCAM and p59fyn. NCAM-mediated p59fyn activation is abolished in RPTPalpha-deficient neurons, and disruption of the NCAM-p59fyn complex in RPTPalpha-deficient neurons or with dominant-negative RPTPalpha mutants blocks NCAM-dependent neurite outgrowth, implicating RPTPalpha as a major phosphatase involved in NCAM-mediated signaling

Age-dependent loss of parvalbumin-expressing hippocampal interneurons in mice deficient in CHL1, a mental retardation and schizophrenia susceptibility gene

In humans, deletions/mutations in the CHL1/CALL gene are associated with mental retardation and schizophrenia. Juvenile CHL1-deficient (CHL1(-/-)) mice have been shown to display abnormally high numbers of parvalbumin-expressing (PV+) hippocampal interneurons and, as adults, display behavioral traits observed in neuropsychiatric disorders. Here, we addressed the question whether inhibitory interneurons and synaptic plasticity in the CHL1(-/-) mouse are affected during brain maturation and in adulthood. We found that hippocampal, but not neocortical, PV+ interneurons were reduced with age in CHL1(-/-) mice, from a surplus of +27% at 1month to a deficit of -20% in adulthood compared with wild-type littermates. This loss occurred during brain maturation, correlating with microgliosis and enhanced interleukin-6 expression. In parallel with the loss of PV+ interneurons, the inhibitory input to adult CA1 pyramidal cells was reduced and a deficit in short- and long-term potentiation developed at CA3-CA1 excitatory synapses between 2 and 9months of age in CHL1(-/-) mice. This deficit could be abrogated by a GABA(A) receptor agonist. We propose that region-specific aberrant GABAergic synaptic connectivity resulting from the mutation and a subsequently enhanced synaptic elimination during brain maturation lead to microgliosis, increase in pro-inflammatory cytokine levels, loss of interneurons, and impaired synaptic plasticity