Neural cell adhesion molecule function is regulated by metalloproteinase-mediated ectodomain release

The neural cell adhesion molecule (NCAM) is involved in development of the nervous system, in brain plasticity associated with learning and memory, and in neuronal regeneration. NCAM regulates these processes by influencing cell adhesion, cell migration, and neurite outgrowth. NCAM activates intracellular signaling upon homophilic NCAM binding, and this is a prerequisite for NCAM-stimulated neurite outgrowth. NCAM is synthesized in three main membrane-bound isoforms, NCAM-120, NCAM-140, and NCAM-180. Soluble forms of NCAM in blood and cerebrospinal fluid have also been found, although the functional significance of these forms remains unclear. In this report, we demonstrate that NCAM can be released from primary hippocampal neurons in culture. The release was enhanced by application of ATP and inhibited by the metalloproteinase inhibitor BB-3103. ATP also induced metalloproteinase-dependent release of all three major NCAM isoforms from NCAM-transfected fibroblastoid L-cells. In this model system, the extracellular ATP-binding site of NCAM was shown not to be necessary for ATP-induced NCAM release. Furthermore, inhibition of serine, cysteine, and aspartic proteinases could not prevent ATP-induced down-regulation of NCAM in L-cells, suggesting that NCAM is cleaved directly by a metalloproteinase. Aggregation of hippocampal neurons in culture was increased in the presence of the metalloproteinase inhibitor GM 6001, consistent with a metalloproteinase-dependent shedding of NCAM occurring in these cells. Moreover, NCAM-dependent neurite outgrowth was significantly reduced by application of GM 6001. Taken together, these results suggest that membrane-bound NCAM can be cleaved extracellularly by a metalloproteinase and that metalloproteinase-dependent shedding of NCAM regulates NCAM-mediated neurite outgrowth.     

Cytoplasmic domain of NCAM 180 reduces NCAM-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM) is one of the best-characterized cell adhesion molecules of the immunoglobulin superfamily. In the nervous system, NCAM is involved in cell migration, axon fasciculation and in neurite outgrowth. Neurite outgrowth is mediated by homophilic NCAM-NCAM interactions. Alternative splicing generates three major isoforms of NCAM differing in their intracellular portion. Two of them, NCAM 180 and NCAM 140, are transmembrane proteins with large intracellular domains. The present study is concerned with novel details of the intracellular domains of NCAM 140 and NCAM 180. We expressed these NCAM isoforms consisting only of the transmembrane and intracellular domains (without extracellular domains) in PC12 cells and elaborated their function in neurite outgrowth assays. Our data demonstrate that membrane-associated NCAM 180 interferes with neurite outgrowth, whereas membrane-associated NCAM 140 promotes neurite outgrowth.     

Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by screening a combinatorial peptide library

The neural cell adhesion molecule, NCAM, plays a key role in neural development and plasticity mediating cell adhesion and signal transduction. By screening a combinatorial library of synthetic peptides with NCAM purified from postnatal day 10 rat brains, we identified a nonapeptide, termed NCAM binding peptide 10 (NBP10) and showed by nuclear magnetic resonance analysis that it bound the NCAM IgI module of NCAM. NBP10 modulated cell aggregation as well as neurite outgrowth induced specifically by homophilic NCAM binding. Moreover, both monomeric and multimeric forms of NBP10 stimulated neurite outgrowth from primary hippocampal neurons. The neurite outgrowth response to NBP10 was inhibited by a number of compounds previously shown to inhibit neurite outgrowth induced by homophilic NCAM binding, including voltage-dependent calcium channel antagonists, suggesting that NBP10 induced neurite outgrowth by activating a signal transduction pathway similar to that activated by NCAM itself. Moreover, an inhibitor of intracellular calcium mobilization, TMB-8, prevented NBP10-induced neurite outgrowth suggesting that NCAM-dependent neurite outgrowth also requires mobilization of calcium from intracellular calcium stores in addition to calcium influx from extracellular sources. By single-cell calcium imaging we further demonstrated that NBP10 was capable of inducing an increase in intracellular calcium in PC12E2 cells. Thus, the NBP10 peptide is a new tool for the study of molecular mechanisms underlying NCAM-dependent signal transduction and neurite outgrowth, and could prove to be a useful modulator of regenerative processes in the peripheral and central nervous system.     

Expression of neural cell adhesion molecule in dysmyelinating mutants

The possible role of neural cell adhesion molecule (NCAM) in myelination was studied in the dysmyelinating mouse mutants jimpy and shiverer, by characterizing the expression of the different molecular forms of brain NCAM as a function of age. In jimpy, the expression of NCAM-120 (120,000-Da NCAM) was low and in shiverer both NCAM-120 and NCAM-180 (180,000-Da NCAM) were reduced when compared to controls. In both jimpy and shiverer there was no significant change in the phospholipase C-sensitive NCAM-120. These data further support the possibility that NCAM may be involved in myelination.     

Selective interaction of amyloid precursor protein with different isoforms of neural cell adhesion molecule

Compare to the thoroughly studied beta-amyloid, the physiological function of amyloid precursor protein (APP) is not well understood. We now had identified neural cell adhesion molecule (NCAM)-140 as a potential interaction partner of APP. Our data indicated that NCAM-140, but not NCAM-180, binds to the conserved central extracellular domain of APP. We also found that the phosphorylation levels of ERK1 and ERK2 were increased when cells were co-transfected with NCAM-140 and APP indicate that the interaction between NCAM-140 and APP may involve the MAPK pathway. These findings demonstrated that NCAM-140 interacts with APP, potentially playing a role in neurite outgrowth and neural development.     

Calpain as a therapeutic target in traumatic brain injury

The family of calcium-activated neutral proteases, calpains, appears to play a key role in neuropathologic events following traumatic brain injury (TBI). Neuronal calpain activation has been observed within minutes to hours after either contusive or diffuse brain trauma in animals, suggesting that calpains are an early mediator of neuronal damage. Whereas transient calpain activation triggers numerous cell signaling and remodeling events involved in normal physiological processes, the sustained calpain activation produced by trauma is associated with neuron death and axonal degeneration in multiple models of TBI. Nonetheless, the causal relationship between calpain activation and neuronal death is not fully understood. Much remains to be learned regarding the endogenous regulatory mechanisms for controlling calpain activity, the roles of different calpain isoforms, and the in vivo substrates affected by calpain. Detection of stable proteolytic fragments of the submembrane cytoskeletal protein αII-spectrin specific for cleavage by calpains has been the most widely used marker of calpain activation in models of TBI. More recently, these protein fragments have been detected in the cerebrospinal fluid after TBI, driving interest in their potential utility as TBI-associated biomarkers. Post-traumatic inhibition of calpains, either direct or indirect through targets related to intracellular calcium regulation, is associated with attenuation of functional and behavioral deficits, axonal pathology, and cell death in animal models of TBI. This review focuses on the current state of knowledge of the role of calpains in TBI-induced neuropathology and effectiveness of calpain as a therapeutic target in the acute post-traumatic period.     

Proteolysis of neuronal cytoskeletal proteins by calpain contributes to rat retinal cell death induced by hypoxia

Our previous studies in retina on the mechanism for hypoxia-induced cell death suggested activation of a class of calcium-activated proteases known as calpains. This conclusion was based on data showing proteolysis of a calpain substrate alpha-spectrin, autolysis of activated calpain, and reduction of cell damage by calpain inhibitor SJA6017. Less is known about changes in downstream pathways after calpain activation. Thus, the purpose of the present investigation was to measure proteolysis of neuronal cytoskeletal proteins and apoptotic cell signaling factors during hypoxia-induced retinal cell death. Rat retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. Hypoxia was induced with 95% N2/5% CO2 without glucose. Immunoblotting was used to detect activation of calpain and proteolysis of substrates. Amounts of mRNA for calpain 1 and 2 were determined by quantitative PCR. Twelve times more calpain 2 mRNA than calpain 1 was present in retinas. Activation of calpain 2 and production of a calpain-specific alpha-spectrin breakdown product at 150 kDa were confirmed in hypoxic retinas. Further, pro-caspase-3 at 32 kDa was proteolyzed to a fragment at 30 kDa, tau protein was lost, and p35 was proteolyzed to p25 suggesting prolonged activation of cdk5. SJA6017 partially inhibited the production of these fragments. During hypoxia in rat retinas, calpains may be major proteases causing breakdown of neuronal proteins involved in apoptotic cell death. Calpain inhibitor SJA6017 may have potential for testing as a therapeutic agent against retinal pathologies such those caused by glaucoma, although future studies such as testing in in vivo animal models are required.     

Proteolysis of NR2B by calpain in the hippocampus of epileptic rats

Overactivation of N-methyl-D-aspartate receptors is known to mediate excitotoxicity due to excessive entry of calcium, leading to the activation of several calcium-dependent enzymes. Calpains are calcium-activated proteases that appear to play a role in excitotoxic neuronal death. Several cellular proteins are substrates for these proteases, particularly the N-methyl-D-aspartate receptor. Recently, cleavage of NR2B subunits has been implicated in excitotoxic neurodegeneration in ischemia. In this work, we investigated the proteolysis by calpains of NR2B subunits of the N-methyl-D-aspartate receptor in the hippocampus of epileptic rats. Our results show that cleaved forms of NR2B subunits are formed after status epilepticus, in the same areas of the hippocampus where calpain activation was detected by immunohistochemical staining of calpain-specific spectrin breakdown products.     

Signal functions of NCAM

The neuronal protein of cell adhesion belongs to the immunoglobulin superfamily of cell adhesion proteins. It consists of an extracellular domain providing homo-and heterophilic interactions with surrounding molecules that are located on the cell surface or are components of the extracellular matrix, a transmembrane part, and intracellular domains (NCAM140 and NCAM180). In addition to its role in cell adhesion, NCAMs act as a signal receptor molecule. Adhesion and initiation of signal cascades induced by binding to the NCAM extracellular domains occur interdependently but influence each other. The homo-and heterophilic binding of NCAM can activate a number of intracellular signal cascades resulting in neurite growth, axone guidance, axone myelinization, and formation of nerve fibers. It has been established that the intracellular signal is initiated by the interactions between NCAMs and fibroblast growth factor receptors (FGFR), non-receptor tyrosine kinases (Fyn and FAK), glia-derived neurotrophic factor (GDNF), ATP, prion proteins, and several other molecules. The review discusses possible mechanisms of functioning of these signal cascades.     

Amphibian-specific regulation of polysialic acid and the neural cell adhesion molecule in development and regeneration of the retinotectal system of the salamander Pleurodeles waltl

Antibodies specific to the neural cell adhesion molecule (NCAM-total), the 180 × 10³ My component of NCAM (NCAM-180) and polysialic acid (PSA) were used in immunohistochemistry and Western blots to detect the spatiotemporal dynamics of these molecules in development and regeneration of the retinotectal system of Pleurodeles waltl. NCAM-total and NCAM-180 are continuously expressed in the retina, optic nerve, and tectum of the developing and adult salamander. This is also found for the 140 × 10³ My component of NCAM in Western blots of the retina. In the larval retina, PSA is present in the inner plexiform layer (IPL) and a few cells in all nuclear layers. At metamorphosis, PSA expression in the retina strongly increases in the layer of cone photoreceptor somata. Several cells in the inner nuclear layer and Muller cell processes also begin to express PSA. This pattern persists into adulthood. The optic nerve and the tectum are strongly PSA-immunoreactive throughout development. In the adult optic nerve and optic fiber pathway in the brain, PSA expression is selectively downregulated. In the crush-lesioned adult optic nerve, regenerating fibers are NCAM-180-positive but PSA-negative. This demonstrates a molecular difference between growing nerve fibers of Pleurodeles in development and in regeneration. PSA regulation is closely correlated with metamorphosis, thus suggesting that PSA expression may be under hormonal control. Some aspects of PSA and NCAM isoform expression patterns in the retinotectal system of salamanders differ considerably from that of other vertebrates. The substained expression of NCAM isoforms in adult salamanders might be due to secondary simplification (paedomorphosis). © 1993 Wiley-Liss, Inc.     

Polysialic acid is required for optimal growth of axons on a neuronal substrate.

Formation of axonal pathways involves a variety of molecules that influence cell-cell interactions. The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) is present on neuronal surfaces during process outgrowth. Our studies reveal that the removal of PSA causes a decrease in the rate of elongation of retinal cell processes on a substrate of neuronal membranes derived from chick tectum. This effect was partially reversed by antibodies against the L1 adhesion molecule, but not by antibodies against NCAM, N-cadherin, or beta 1-integrins. This predominant effect of PSA on L1 was also observed in short-term, cell-cell adhesion assays, suggesting that PSA promotes optimal outgrowth on neuronal substrates by limiting the consequences of L1-mediated adhesion.     

Neural cell adhesion molecule and its polysialic acid moiety exhibit opposing and linked effects on neuropathic hyperalgesia

Spinal lamina II, where nociceptive C-fibers terminate, expresses high amounts of the polysialylated form of neural cell adhesion molecule (PSA-NCAM). While enzymatic removal of the PSA moiety from NCAM did not affect normal sensitivity to thermal stimuli, it exacerbated nerve injury-induced neuropathic hyperalgesia. The genetic removal of the NCAM core protein also did not alter thermal sensitivity. However in the presence of a peripheral nerve injury, NCAM-null mutants exhibited a complete suppression of thermal hyperalgesia. This strong NCAM mutant phenotype appears to involve the long form of NCAM's cytoplasmic domain, in that it is duplicated by selective genetic deletion of the NCAM-180 isoform. PSA appears therefore to provide a mechanism for modulation of chronic sensory overload, by means of attenuation of the activity of the NCAM-180 isoform, which reduces nociceptive transmission.     

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 Ca(2+), 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.     

Increased NCAM-180 immunoreactivity and maintenance of L1 immunoreactivity in injured optic fibers of adult mice

The injury related expression of two axon-growth promoting cell adhesion molecules (CAMs), NCAM-180 which is developmentally downregulated and L1 which is regionally restricted, were compared in optic fibers in the adult mouse. The neuron-specific isoform of NCAM (NCAM-180) is present at very low levels in unlesioned adult optic axons. At 7 days after nerve crush, immunoreactivity was strongly and uniformly increased in optic axons within the nerve and throughout retina. Reactivity in surviving axons had returned to control levels at 4 weeks. To induce regrowth of adult retinal ganglion cell axons retinal explants were placed in culture. Strong NCAM-180 staining was observed on these regenerating optic axons. The neuronal cell adhesion molecule L1 is restricted to retina and to the unmyelinated segment of the optic nerve near the optic nerve head in unlesioned adult animals. Following nerve crush, L1 immunoreactivity was retained within retina and proximal nerve and novel staining was detected in the more distal segment of the optic nerve up to the lesion site where it persisted for at least eight months. The capacity of optic fibers to show increased NCAM-180 immunoreactivity and maintain L1 expression after a lesion may explain why these fibers exhibit relatively good potential for regeneration.     

State-dependent changes in the expression levels of NCAM-140 and L1 in the peripheral blood cells of bipolar disorders, but not in the major depressive disorders

Recent postmortem brain and imaging studies provide evidence for disturbances of structural and synaptic plasticity in patients with mood disorders. Several lines of evidence suggest that the cell adhesion molecules (CAMs), neural cell adhesion molecules (NCAM) and L1, play important roles in both structural and synaptic plasticity. Although postmortem brain studies have indicated altered expression levels of NCAM and L1, it is still unclear whether these changes are state- or trait-dependent. In this study, the mRNA levels for various CAMs, including NCAM and L1, were measured using quantitative real-time PCR in peripheral blood cells of major depressive disorder patients, bipolar disorder patients and normal healthy subjects. Reduced expression levels of NCAM-140 mRNA were observed in bipolar disorder patients in a current depressive state. In contrast, L1 mRNA levels were increased in bipolar disorder patients in a current depressive state. NCAM-140 and L1 mRNA levels were not changed in bipolar disorder patients in a remissive state, or in major depressive disorder patients. In addition, there were no significant changes in the expression levels of intercellular adhesion molecule -1, vascular cell adhesion molecule -1, E-cadherin, or integrin alphaD among healthy controls, major depressive or bipolar disorder patients. Our results suggest that the reciprocal alteration in the expression of NCAM-140 and L1 mRNAs could be state-dependent and associated with the pathophysiology of bipolar disorder.     

Acute stress-induced impairment of spatial memory is associated with decreased expression of neural cell adhesion molecule in the hippocampus and prefrontal cortex.

BACKGROUND: There is an extensive literature describing how stress disturbs cognitive processing and can exacerbate psychiatric disorders. There is, however, an insufficient understanding of the molecular mechanisms involved in stress effects on brain and behavior. METHODS: Rats were given spatial memory training in a hippocampus-dependent water maze task. We investigated how a fear-provoking experience (predator exposure) would affect their spatial memory and neural cell adhesion molecule (NCAM) levels in the hippocampus, prefrontal cortex (PFC), amygdala, and cerebellum. RESULTS: Whereas the control (nonstress) group exhibited excellent memory for the hidden platform location in the water maze, the cat-exposed (stress) group exhibited a profound impairment of memory and a marked suppression of levels of the NCAM-180 isoform in the hippocampus. Predator stress produced a more global reduction of NCAM levels in the PFC but had no effect on NCAM levels in the amygdala and cerebellum. CONCLUSIONS: This work provides a novel perspective into dynamic and structure-specific changes in the molecular events involved in learning, memory, and stress. The selective suppression of NCAM-180 in the hippocampus and the more general suppression of NCAM in the PFC provide insight into the mechanisms underlying the great sensitivity of these two structures to be disturbed by stress.     

Assessment of Neural Cell Adhesion Molecule (NCAM) in Autistic Serum and Postmortem Brain

Studies have identified structural abnormalities in areas of the autistic brain, with a pattern suggesting that a neurodevelopmental anomaly took place. Neural cell adhesion molecule (NCAM), which is involved in development of the central nervous system, was previously shown to be decreased in the serum of autistic individuals. In the present study, we measured NCAM protein in the sera from controls, patients with autism, siblings of autistic patients, and individuals with other neurologic disorders, but found no significant differences. We also measured NCAM protein in autistic postmortem brain samples and found the longest isoform, NCAM-180, to be significantly decreased. In addition, we investigated the mRNA expression of NCAM in these brain samples using cDNA microarrays and RT-PCR. Results show that NCAM mRNA levels are not altered in autism.     

NILE/L1 and NCAM-polysialic acid expression on growing axons of isolated neurons

The neuron adhesion molecules NILE/L1 and NCAM may be involved in axonal guidance and cell recognition. To investigate all exposed membrane domains of single neurons, something which has not previously been done for any adhesion molecule, we used digitally processed scanning electron microscopy with a high-energy backscatter electron detector. This allowed a quantitative analysis of immunogold staining densities on all surfaces of isolated rat hippocampal neurons in culture to study NILE/L1 and NCAM expression independent of potentially inductive innervation. During early stages of neuritic extension, all growth cones showed similar NILE/L1 expression, but as soon as a single process extended farther than the others (by 20 hours), this putative axon and its growth cone generally showed a stronger level of NILE/L1 immunogold labeling than the other neurites. This is the earliest evidence of plasma membrane differentiation between axons and dendrites. With further neuritic growth, the relative NILE/L1 expression on axons and their growth cones continued to increase. In contrast to some earlier reports, NILE/L1 was expressed on axonal growth cones growing on both polylysine-coated glass and astrocyte substrates. Strong immunostaining for NCAM-related polysialic acid (PSA) was found on axonal growth cones and filopodia, suggesting that the homophilic adhesive action of NCAM may be reduced during axonal growth. PSA showed greater labeling on distal axons than on other areas of the neuron, indicating a variable NCAM-mediated adhesion on different regions of the same cell. Neither NILE/L1, NCAM, nor PSA appeared to show regional differences in axons fasciculating or defasciculating on themselves. A strong intercellular heterogeneity of NILE/L1, NCAM, and PSA expression levels on neurons in the same culture dish was found, suggesting that subsets of cells from the hippocampus may express biologically relevant differences in adhesion molecules compared to neighboring neurons. In light of the growing body of evidence pointing to the multifaceted array of homophilic and heterophilic binding interactions that NILE/L1 and NCAM may exhibit, and the functional importance of molecular densities, the quantitative data here support the hypothesis that sufficient cellular and subcellular heterogeneity exists for these molecules to be involved in some aspects of axonal guidance. © 1993 Wiley-Liss, Inc.     

Expressions and possible roles of GDNF receptors in the developing dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) has an essential role in the survival and maturation of the dopaminergic (DA) neurons in the substantia nigra (SN) of mammalian embryonic brain. In addition to Ret, cell adhesion molecules (CAMs) were also proposed to function as transmembrane signaling receptors of GDNF. The present study was to investigate whether these transmembrane receptors of GDNF were correlated with the tyrosine hydroxylase (TH) expression of SN DA neurons during early developmental stage. RT-PCR and Western blot were performed to detect TH expression in SN of perinatal rats at mRNA and protein level respectively; meanwhile, Western blot was performed to detect the expressions of the transmembrane proteins including Ret, neural cell adhesion molecule-140 (NCAM-140), integrin β1 and N-cadherin. The results showed that TH mRNA expression was positively correlated with both Ret and N-cadherin protein, while there was no correlation with NCAM-140 and integrin β1; TH protein expression was correlated with all of these transmembrane molecules. These data suggested that the expression of either TH mRNA or TH protein was subject to the mediation of different transmembrane receptor combinations of GDNF.