Characterization of a 21 amino acid peptide sequence of the laminin G2 domain that is involved in HNK-1 carbohydrate binding and cell adhesion

The N-linked HNK-1 carbohydrate expressed by several recognition molecules mediates the adhesion of early postnatal cerebellar neurons to the G2 domain of the terminal globular domain of the laminin α1 chain (H.Hall et al., submitted). To define this binding site more precisely, G2-derived synthetic peptides were used for binding and competition studies. Peptide 5-G2, comprising the amino acid residues 3431-3451 of G2, inhibited the interaction between the HNK-1-carrying glycolipid and laminin in a concentrationdependent and saturable manner. Peptides which overlap only partially with this sequence interfered less. Peptides comprising other amino acid sequences from G2, and peptides derived from G1 and G3 or a scrambled version of peptide 5-G2, did not show significant effects. Direct binding of peptide 5-G2 to the HNK-1 glycolipid was also demonstrated. Furthermore, peptide 5-G2 interfered in a concentration-dependent and saturable manner with the adhesion of early postnatal cerebellar neurons to laminin. These observations indicate that amino acid residues 3431-3451 of the laimnin G2 domain are involved in HNK-1 carbohydratemediated cell adhesio

Electroacupuncture Restores Locomotor Functions After Mouse Spinal Cord Injury in Correlation With Reduction of PTEN and p53 Expression

Background: We previously showed that electroacupuncture (EA) at Jiaji points promotes expression of adhesion molecule L1 in spinal cord tissue after mouse spinal cord injury (SCI) and contributes to recovery of neural functions.Objective: We investigated the effects of EA on downstream signaling molecules of L1 and molecules relevant to apoptosis with the aim to understand the underlying molecular mechanisms.Methods: Female C57BL/6 mice were divided into a sham group, injury group, injury+acupuncture (AP) group and injury+EA group. We investigated the changes in cognate L1-triggered signaling molecules after SCI by immunofluorescence staining and immunoblot analysis.Results: Protein levels of phosphatase and tensin homolog (PTEN) and p53 were decreased by EA at different time points after injury, whereas the levels of phosphorylated mammalian target of rapamycin (pmTOR), p-Akt and phosphorylated extracellular signal-regulatedkinase (p-Erk) were increased. Also, levels of myelin basic protein (MBP) were increased by EA. AP alone showed less pronounced changes in expression of the investigated molecules, when compared to EA.Conclusion: We propose that EA contributes to neuroprotection by inhibiting PTEN and p53 expression and by increasing the levels of pmTOR/Akt/Erk and of MBP after SCI. These observations allow novel insights into the beneficial effects of EA via L1-triggered signaling molecules after injury

Causal Evidence for the Involvement of the Neural Cell Adhesion Molecule, NCAM, in Chronic Stress-Induced Cognitive Impairments

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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

Monoclonal antibodies raised against membrane glycoproteins from mouse brain recognize N-linked oligomannosidic glycans

Monoclonal L3 and L4 antibodies have been shown to recognize carbohydrate epitopes on several neural cell adhesion molecules; these epitopes can be released by treatment with endoglycosidase H. In the present study, we have identified the oligosaccharides released by endoglycosidase H from the cell adhesion molecules AMOG and L1 by fast-atom bombardment mass spectrometry as being solely of the oligomannosidic type. Using neoglycolipids of oligomannosidc glycans, we also report that both antibodies show the highest reactivity with the α-manno-pentaose Manα1-3-[Manα1-6(Manα1-3)Manα1-6]-Man, but decreasing reactivity with the α-manno-hexaose, heptaose, octaose and nonaose glycans. Thus, to our knowledge, we describe here for the first time monoclonal antibodies recognizing N-glycosidically linked oligomannosidic glycan

Structural and Functional Aberrations in the Cerebral Cortex of Tenascin-C Deficient Mice

The extracellular matrix glycoprotein tenascin-C (TNC) has been implicated in neural development and plasticity but many of its functions in vivo remain obscure. Here we addressed the question as to whether the constitutive absence of TNC in mice affects cortical physiology and structure. Defined major cell populations (neurons and inhibitory neuronal subpopulations, astrocytes, oligodendrocytes and microglia) were quantified in the somatosensory and motor cortices of adult TNC deficient (TNC−/−) and wild-type (TNC+/+) mice by immunofluorescence labelling and stereology. In both areas studied we found abnormally high neuronal density, astrogliosis, low density of parvalbumin-positive interneurons and reduced ratios of oligodendrocytes to neurons and of inhibitory to excitatory neurons in the TNC deficient as opposed to the non-deficient animals. Analysis of Golgi-impregnated layer V pyramidal neurons in TNC−/− animals showed aberrant dendrite tortuosity and redistribution of stubby spines within first- to third-order dendritic arbors. Significantly enhanced responses upon whisker stimulation were recorded epicranially over the barrel and the motor cortices of TNC−/− as compared to TNC+/+ animals, and this effect might be associated with the diminished inhibitory circuitry. These results indicate that TNC is essential for normal cortical development and functio

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

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

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