Martin Grumet, Ph.D.

Juvenile and adult olfactory ensheathing cells bundle and myelinate dorsal root ganglion axons in culture
Babiarz, Joanne; Kane-Goldsmith, Noriko; Basak, Sayantani; Liu, Kai; Young, Wise; Grumet, Martin
2011 May;229(1):72-79, Experimental neurology
Olfactory ensheathing cells (OEC), which normally associate closely with but do not myelinate axons in situ, myelinate axons in the adult mammalian spinal cord. They are of clinical interest as candidate cells for autologous transplantation but the ability of OEC to myelinate axons in vitro has been controversial. To clarify this issue, we isolated OEC from olfactory bulbs (OB) of juvenile and adult rats expressing GFP and analyzed their ability to myelinate axons. Using a well-defined assay for myelination of dorsal root ganglia (DRG) axons in culture, we found that OEC from juvenile pups associated with and then myelinated DRG axons. OEC assembled into bundles with the axons by 1week and required more than a week before myelination on axons was detected. In contrast, rat Schwann cells did not bundle axons and they formed P0(+) and MBP(+) myelin segments after as little as 1week. Most of the OEC in culture exhibited staining for calponin, a marker that was not found on Schwann cells in culture, whereas in both OEC and Schwann cell populations nearly all cells were positive for p75NTR and GFAP. These results confirm previous reports showing only subtle immunological differences between Schwann cells and OEC. Besides differences in the rate of myelination, we detected two additional functional differences in the interactions of OEC and Schwann cells with DRG axons. First, the diameter of OEC generated myelin was greater than for Schwann cell myelin on DRG axons. Second, OEC but not Schwann cells myelinated DRG axons in the absence of vitamin C. OEC isolated from adult OB were also found to bundle and myelinate DRG axons but the latter occurred only after incubation times of at least 3weeks. The results indicate that adult OEC require longer incubation times than juvenile OEC to myelinate axons and suggest that patterns of myelination by OEC and Schwann cells are distinguishable at least on axons in vitro. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair
— id: 131927, year: 2011, vol: 229, page: 72, stat: Journal Article,

Rapid induction of genes associated with tissue protection and neural development in contused adult spinal cord after radial glial cell transplantation
Chang, Yu-Wen; Goff, Loyal A; Li, Hedong; Kane-Goldsmith, Noriko; Tzatzalos, Evangeline; Hart, Ronald P; Young, Wise; Grumet, Martin
2009 Jul;26(7):979-993, Journal of neurotrauma
Cell-based therapy has been widely evaluated in spinal cord injury (SCI) animal models and shown to improve functional recovery. However, host response to cell transplants at gene expression level is rarely discussed. We reported previously that acute transplantation of radial glial cells RG3.6 following SCI promoted early locomotion improvement within one week post injury. To identify rapid molecular changes induced by RG3.6 transplantation in the host tissue, distal spinal cord segments were subjected to microarray analysis. Despite RG3.6 transplantation reduced activity of macrophage as early as 1-2 weeks post injury, the expression levels of inflammatory genes (e.g. IL-6, MIP-2, MCP-1) were not decreased by RG3.6 treatment as compared to medium or other cell type controls at 6-12 hours post injury. However, genes associated with tissue protection (Hsp70 and Hsp32) and neural cell development (Foxg1, Top2a, Sox11, Nkx2.2, Vimentin) were found to be significantly up-regulated by RG3.6 transplants. Foxg1 was the most highly induced gene in the RG3.6 treated spinal cords and its expression by immunocytochemistry was confirmed in the host tissue. Moreover, RG3.6 treatment boosted the number of Nkx2.2 cells in the spinal cord and these cells frequently co-expressed NG2, which marks progenitor cells. Taken together, these results demonstrate that radial glia transplants induced rapid and specific gene expression in the injured host tissue and suggest that these early responses are associated with mechanisms of tissue protection and activation of endogenous neural progenitor cells
— id: 94088, year: 2009, vol: 26, page: 979, stat: Journal Article,

Single, high-dose intraspinal injection of chondroitinase reduces glycosaminoglycans in injured spinal cord and promotes corticospinal axonal regrowth after hemisection but not contusion
Iseda, Tsutomu; Okuda, Tetsuhito; Kane-Goldsmith, Noriko; Mathew, Marlon; Ahmed, Sameer; Chang, Yu-Wen; Young, Wise; Grumet, Martin
2008 Apr;25(4):334-349, Journal of neurotrauma
Chondroitin sulfate proteoglycans (CSPGs) inhibit axonal growth, and treatment with chondroitinase ABC promotes axonal regeneration in some models of central nervous system (CNS) injury. The aims of this study were (1) to compare the spatiotemporal appearance of CSPG expression between spinal cord contusion and hemisection models, and (2) to evaluate chondroitinase treatment effects on axonal regrowth in the two injury models. After hemisection, CSPG-immunoreactivity (IR) in the injury site rose to peak levels at 18 days but then decreased dramatically by 49 days; in contrast, CSPG-IR remained high for at least 49 days after contusion. After hemisection, many anterogradely labeled corticospinal tract (CST) axons remained close to CSPG-rich lesion sites, but after contusion, most CST axons retracted by approximately 1 mm rostral from the rostral-most CSPG-rich cyst. Intraspinal injection of chondroitinase at 0, 1, 2, and 4 weeks following injury dramatically reduced CSPG-IR in both injury models within 4 days, and CSPG-IR remained low for at least 3 weeks. After the chondroitinase treatment, many axons grew around the lesion site in hemisected spinal cords but not in contused spinal cords. We propose that improved axonal growth in hemisected spinal cords is due to decreased inhibition resulting from degradation of CSPGs located adjacent to severed CST axons. However, in spinal cord contusions, retracted CST axons fail to grow across gliotic regions that surround CSPG-rich injury sites despite efficient degradation with chondroitinase, suggesting that other inhibitors of axonal growth persist in the gliotic regions
— id: 94093, year: 2008, vol: 25, page: 334, stat: Journal Article,

Nodes of Ranvier and axon initial segments are ankyrin G-dependent domains that assemble by distinct mechanisms
Dzhashiashvili, Yulia; Zhang, Yanqing; Galinska, Jolanta; Lam, Isabel; Grumet, Martin; Salzer, James L
2007 Jun 4;177(5):857-870, Journal of cell biology
Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and betaIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G
— id: 73381, year: 2007, vol: 177, page: 857, stat: Journal Article,

NrCAM in Addiction Vulnerability: Positional Cloning, Drug-Regulation, Haplotype-Specific Expression, and Altered Drug Reward in Knockout Mice
Ishiguro, Hiroki; Liu, Qing-Rong; Gong, Jian-Ping; Hall, Frank Scott; Ujike, Hiroshi; Morales, Marisela; Sakurai, Takeshi; Grumet, Martin; Uhl, George R
2006 Mar;31(3):572-584, Neuropsychopharmacology
Several lines of evidence support roles for the cell adhesion molecule NrCAM in addictions. Fine mapping within a chromosome 7 region that contains previously linked and associated genomic markers identifies NrCAM haplotypes that are associated with substance abuse vulnerabilities in four samples of abusers and controls. Differential display identifies NrCAM as a drug regulated gene. NrCAM is expressed in neurons linked to reward and memory. NrCAM displays haplotype-specific gene expression in human post-mortem brain samples. Knockout mice display reduced opiate- and stimulant-conditioned place preferences. These observations support NrCAM as a positionally cloned and drug-regulated gene whose variants are likely to change expression and alter substance abuse vulnerabilities in human addictions and animal models of drug reward.Neuropsychopharmacology (2006) 31, 572-584. doi:10.1038/sj.npp.1300855; published online 10 August 2005
— id: 63127, year: 2006, vol: 31, page: 572, stat: Journal Article,

Neurofascin interactions play a critical role in clustering sodium channels, ankyrin(G) and betaIV spectrin at peripheral nodes of Ranvier
Koticha, Darshan; Maurel, Patrice; Zanazzi, George; Kane-Goldsmith, Noriko; Basak, Sayantani; Babiarz, Joanne; Salzer, James; Grumet, Martin
2006 May 1;293(1):1-12, Developmental biology (Orlando)
The Ig cell adhesion molecules (CAM) neurofascin (NF) and Nr-CAM are localized at developing nodes of Ranvier in peripheral myelinated axons prior to clustering of Na(+) channels. Different isoforms of NF are expressed on neurons and glia, and NF binding on both cells has been suggested to play roles in node and paranode formation. To clarify the role of NF further, we analyzed effects of NF-Fc fusion proteins in Schwann cell-DRG neuron myelinating cocultures. NF-Fc significantly inhibited nodal clustering of Na(+) channels, ankyrin(G), and betaIV spectrin, and modestly reduced Caspr clustering at paranodal junctions; it did not significantly affect lengths or numbers of myelin-positive segments, axon initial segments, or accumulations of phosphorylated-ERM proteins in Schwann cell nodal microvilli. NF-Fc binds to Schwann cells but little or no binding to DRG neurons was detected. The results suggest a critical early role for axonal NF in clustering of Na(+) channels at nodes of Ranvier via interactions with receptors on Schwann cells
— id: 63125, year: 2006, vol: 293, page: 1, stat: Journal Article,

A role for Nr-CAM in the patterning of binocular visual pathways
Williams, Scott E; Grumet, Martin; Colman, David R; Henkemeyer, Mark; Mason, Carol A; Sakurai, Takeshi
2006 May 18;50(4):535-547, Neuron
Retinal ganglion cell (RGC) axons diverge within the optic chiasm to project to opposite sides of the brain. In mouse, contralateral RGCs are distributed throughout the retina, whereas ipsilateral RGCs are restricted to the ventrotemporal crescent (VTC). While repulsive guidance mechanisms play a major role in the formation of the ipsilateral projection, little is known about the contribution of growth-promoting interactions to the formation of binocular visual projections. Here, we show that the cell adhesion molecule Nr-CAM is expressed by RGCs that project contralaterally and is critical for the guidance of late-born RGCs within the VTC. Blocking Nr-CAM function causes an increase in the size of the ipsilateral projection and reduces neurite outgrowth on chiasm cells in an age- and region-specific manner. Finally, we demonstrate that EphB1/ephrin-B2-mediated repulsion and Nr-CAM-mediated attraction comprise distinct molecular programs that each contributes to the proper formation of binocular visual pathways
— id: 141829, year: 2006, vol: 50, page: 535, stat: Journal Article,

Dual functional activity of semaphorin 3B is required for positioning the anterior commissure
Falk, Julien; Bechara, Ahmad; Fiore, Roberto; Nawabi, Homaira; Zhou, Heather; Hoyo-Becerra, Carolina; Bozon, Muriel; Rougon, Genevieve; Grumet, Martin; Puschel, Andreas W; Sanes, Joshua R; Castellani, Valerie
2005 Oct 6;48(1):63-75, Neuron
Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue
— id: 63126, year: 2005, vol: 48, page: 63, stat: Journal Article,

Embryonic radial glia bridge spinal cord lesions and promote functional recovery following spinal cord injury
Hasegawa, Koichi; Chang, Yu-Wen; Li, Hedong; Berlin, Yana; Ikeda, Osamu; Kane-Goldsmith, Noriko; Grumet, Martin
2005 Jun;193(2):394-410, Experimental neurology
Radial glial cells are neural stem cells (NSC) that are transiently found in the developing CNS. To study radial glia, we isolated clones following immortalization of E13.5 GFP rat neurospheres with v-myc. Clone RG3.6 exhibits polarized morphology and expresses the radial glial markers nestin and brain lipid binding protein. Both NSC and RG3.6 cells migrated extensively in the adult spinal cord. However, RG3.6 cells differentiated into astroglia slower than NSC, suggesting that immortalization can delay differentiation of radial glia. Following spinal cord contusion, implanted RG3.6 cells migrated widely in the contusion site and into spared white matter where they exhibited a highly polarized morphology. When injected immediately after injury, RG3.6 cells formed cellular bridges surrounding spinal cord lesion sites and extending into spared white matter regions in contrast to GFP fibroblasts that remained in the lesion site. Behavioral analysis indicated higher BBB scores in rats injected with RG3.6 cells than rats injected with fibroblasts or medium as early as 1 week after injury. Spinal cords transplanted with RG3.6 cells or dermal fibroblasts exhibited little accumulation of chondroitin sulfate proteoglycans (CSPG) including NG2 proteoglycans that are known to inhibit axonal growth. Reduced levels of CSPG were accompanied by little accumulation in the injury site of activated macrophages, which are a major source of CSPG. However, increased staining and organization of neurofilaments were found in injured rats transplanted with RG3.6 cells suggesting neuroprotection or regrowth. The combined results indicate that acutely transplanted radial glia can migrate to form bridges across spinal cord lesions in vivo and promote functional recovery following spinal cord injury by protecting against macrophages and secondary damage
— id: 63129, year: 2005, vol: 193, page: 394, stat: Journal Article,

Cell adhesion and neurite outgrowth are promoted by neurofascin NF155 and inhibited by NF186
Koticha, Darshan; Babiarz, Joanne; Kane-Goldsmith, Noriko; Jacob, Jeffrey; Raju, Karthik; Grumet, Martin
2005 Sep;30(1):137-148, Molecular & cellular neurosciences
Neurofascin (NF) is a neural cell adhesion molecule in the L1-family containing six Ig domains and multiple fibronectin type III (FnIII) repeats in its extracellular region. NF has many splicing variants and two of these are exemplars that have different cellular patterns of expression during development. NF186, which is expressed on neurons, contains an unusual mucin-like region and NF155, which is expressed on glia, contains a unique FnIII repeat with an RGD motif. Analysis of Fc fusion proteins representing different extracellular regions of NF indicate that NF186 inhibits cell adhesion and neurite outgrowth, and the inhibition is associated with the region containing the mucin-like domain. NF155 promotes neural cell adhesion and neurite outgrowth, and the RGD motif in its third FnIII repeat is critical for cell spreading and neurite outgrowth. The results suggest that different splicing variants of NF expressed on neurons and glia play distinct roles during neural development
— id: 63128, year: 2005, vol: 30, page: 137, stat: Journal Article,

Signal transduction pathways implicated in neural recognition molecule L1 triggered neuroprotection and neuritogenesis
Loers, Gabriele; Chen, Suzhen; Grumet, Martin; Schachner, Melitta
2005 Mar;92(6):1463-1476, Journal of neurochemistry
The signal transduction pathways involved in adhesion molecule L1-triggered neuritogenesis and neuroprotection were investigated using the extracellular domain of mouse or human L1 in fusion with the Fc portion of human immunoglobulin G or L1 purified from mouse brain by affinity chromatography. Substrate L1-triggered neuritogenesis and neuroprotection depended on distinct but also overlapping signal transduction pathways and on the expression of L1 at the neuronal cell surface. PI3 kinase inhibitors, Src family kinase inhibitors as well as mitogen-activated protein kinase kinase inhibitors reduced both L1-triggered neuritogenesis and neuroprotection. In contrast, fibroblast growth factor receptor inhibitors, a protein kinase A inhibitor, and an inhibitor of cAMP-mediated signal transduction pathways, blocked neuritogenesis, but did not affect L1-triggered neuroprotection. Proteolytic cleavage of L1 or its interaction partners is necessary for both L1-mediated neuritogensis and neuroprotection. Furthermore, L1-triggered neuroprotection was found to be associated with increased phosphorylation of extracellular signal-regulated kinases 1/2, Akt and Bad, and inhibition of caspases. These observations suggest possibilities of differentially targeting signal transduction pathways for L1-dependent neuritogenesis and neuroprotection
— id: 63130, year: 2005, vol: 92, page: 1463, stat: Journal Article,

In vitro analysis of mechanisms underlying age-dependent failure of axon regeneration
Hafidi, Aziz; Grumet, Martin; Sanes, Dan H
2004 Feb 23;470(1):80-92, Journal of comparative neurology
Severed axons of the inferior colliculus (IC) commissure can regenerate across a lesion in organotypic cultures from postnatal day (P) 6 gerbils, but this regenerative capacity is lost by P12 (Hafidi et al. [ 1995] J Neurosci 15:1298-1307, [1999] J Neurobiol 41:267-280). In the present study, we examined the mechanisms underlying this age-dependent failure of axons to regenerate. In P6-P12 heterochronic cultures, the P12 axons failed to cross the lesion site and project to the contralateral P6 IC lobe. In contrast, axons originating from the P6 lobe could regenerate through the lesion and invade the contralateral P12 IC lobe. To determine whether this age-dependent change in regenerative capacity can develop in organotypic cultures, IC slices with an intact commissure were obtained from P6 animals, grown in vitro for 6 days, and then lesioned at the commissure. In these slices, axon regeneration failure was similar to that observed in normal P12 tissue. Several in vitro treatments enhanced axon regeneration: removal of the entire midline region, inhibition of protein synthesis at the lesion site, and exposure to ABC chondroitinase. Furthermore, when the injured commissural axons were provided with a carpet of C6-R cells (a radial glia-like cell line), significantly more axons projected to the contralateral lobe of the IC. Taken together, these results suggest that the maturation of nonneuronal cells within the lesion site lead to failed axon regeneration in mature animals, and show that ameliorative strategies can be evaluated in vitro
— id: 63132, year: 2004, vol: 470, page: 80, stat: Journal Article,

Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors
Li, Hedong; Babiarz, Joanne; Woodbury, Jennifer; Kane-Goldsmith, Noriko; Grumet, Martin
2004 Jul 15;271(2):225-238, Developmental biology (Orlando)
Radial glia are among the first cells that develop in the embryonic central nervous system. They are progenitors of glia and neurons but their relationship with restricted precursors that are also derived from neuroepithelia is unclear. To clarify this issue, we analyzed expression of cell type specific markers (BLBP for radial glia, 5A5/E-NCAM for neuronal precursors and A2B5 for glial precursors) on cortical radial glia in vivo and their progeny in vitro. Clones of cortical cells initially expressing only BLBP gave rise to cells that were A2B5+ and eventually lost BLBP expression in vitro. BLBP is expressed in the rat neuroepithelium as early as E12.5 when there is little or no staining for A2B5 and 5A5. In E13.5-15.5 forebrain, A2B5 is spatially restricted co-localizing with a subset of the BLBP+ radial glia. Analysis of cells isolated acutely from embryonic cortices confirmed that BLBP expression could appear without, or together with, A2B5 or 5A5. The numbers of BLBP+/5A5+ cells decreased during neurogenesis while the numbers of BLBP+/A2B5+ cells remained high through the beginning of gliogenesis. The combined results demonstrate that spatially restricted subpopulations of radial glia along the dorsal-ventral axis acquire different markers for neuronal or glial precursors during CNS development
— id: 63131, year: 2004, vol: 271, page: 225, stat: Journal Article,

The role of the ankyrin-binding protein NrCAM in node of Ranvier formation
Custer, Andrew W; Kazarinova-Noyes, Katia; Sakurai, Takeshi; Xu, Xiaorong; Simon, William; Grumet, Martin; Shrager, Peter
2003 Nov 5;23(31):10032-10039, Journal of neuroscience
Molecular events involved in Na+ channel clustering at the node of Ranvier have been investigated during early development. NrCAM, an ankyrinG-binding protein, precedes Na+ channels at cluster sites adjacent to the tips of Schwann cell processes. Both Na+ channel and ankyrinG sequestration at developing nodes are delayed in NrCAM null mutants. The action of NrCAM is manifest locally at individual nodes, rather than affecting overall neuronal expression, and is linked to glial interactions. During remyelination, Na+ channel clusters at new nodes are initially labile, and anchoring to the cytoskeleton appears to grow progressively with time. The distance between Na+ channel clusters across remyelinating Schwann cells (nascent internodes) increases markedly from 83 to 274 microm during node formation, arguing against schemes in which the loci of nodes are fixed in advance by the axon. A hypothesis for node formation in which axonal Na+ channels move by lateral diffusion from regions of Schwann cell contact, with clustering dependent on linkage to the cytoskeleton by ankyrinG, is proposed and tested in a computational model. To match experimental measurements, this latter reaction needs fast kinetics, and the early arrival of NrCAM is postulated to contribute to this requirement
— id: 63133, year: 2003, vol: 23, page: 10032, stat: Journal Article,

Trauma-induced tumorigenesis of cells implanted into the rat spinal cord
Hasegawa, Koichi; Grumet, Martin
2003 May;98(5):1065-1071, Journal of neurosurgery
OBJECT: Findings in several clinical cases have suggested a correlation between tumor formation and previous injury to the central nervous system (CNS); however, the relationship between trauma and tumorigenesis has not been investigated well experimentally. In this study the authors provide evidence correlating tumorigenesis with trauma in the rat spinal cord. METHODS: A glial cell line, C6R-G/H, which expresses green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT), was implanted into normal and injured rat spinal cords. In all rats in which the cells were implanted into an injured site, locomotor function deteriorated and histological analysis demonstrated glioblastoma multiforme by 6 weeks; tumorigenesis was correlated with a loss of both GFP expression and resistance to hygromycin treatment. In contrast, no evidence of tumor formation was found at 6 weeks in rats in which the cells were implanted into healthy tissue. When C6R-G/H cells were treated with contused spinal cord extract in culture before implantation, they lost GFP expression and hygromycin resistance, and later formed tumors after implantation into normal spinal cord. CONCLUSIONS: The findings of this study indicate that trauma can induce tumorigenesis. Implantation of C6R-G/H cells into traumatized spinal cords resulted in their transformation, which was signaled by loss of GFP expression and hygromycin resistance accompanied by tumor formation. Exposure to extracts derived from injured spinal cord produced similar transformation and gene expression changes, as well as tumor formation after such cells were implanted into normal cords. Care, therefore, should be taken when cells are implanted into an injured CNS because of potential mutagenesis due to trauma-induced factors
— id: 63137, year: 2003, vol: 98, page: 1065, stat: Journal Article,

The L1CAM extracellular region: a multi-domain protein with modular and cooperative binding modes
Haspel, Jeffrey; Grumet, Martin
2003 Sep 1;8:s1210-s1225, Frontiers in biosciences
L1CAM is a neural cell adhesion molecule (CAM) that is critical for proper CNS development in humans. It mediates a myriad of activities important to CNS maturation, including neurite outgrowth, adhesion, fasciculation, migration, myelination and axon guidance. L1CAM promotes these cellular activities by interacting with a diverse group of CAMs, extracellular matrix molecules and signaling receptors through interactions involving its extracellular region. This region is composed of 11 tandem immunoglobulin-like (Ig) domains. This review focuses on the L1CAM extracellular region, and how recent work has clarified important aspects of its structure and function. These studies have provided new insights into how L1CAM binds to several different extracellular molecules, how these binding activities are regulated, and how L1CAM initially folds. Furthermore, these studies suggest that the extracellular region is a dynamic, integrated structure that depends on cooperative interactions among its Ig-like domains for proper functioning
— id: 63135, year: 2003, vol: 8, page: s1210, stat: Journal Article,

Microtubules are critical for radial glial morphology: possible regulation by MAPs and MARKs
Li, Hedong; Berlin, Yana; Hart, Ronald P; Grumet, Martin
2003 Oct;44(1):37-46, Glia
Radial glia are a polarized cell type that in most neural regions appear only transiently during development. They have long been recognized as glia or glial progenitors that support neuronal migration. Recent evidence indicates that radial glia also give rise to neurons and appear to be a major population of dividing precursor cells in the embryonic cortical ventricular zone. Radial glia extend long processes from the ventricular zone to the pial surface that provide guides for neuronal migration. We reasoned that the unique morphology of radial glia is due to the composition and organization of their cytoskeleton. In this present study, we have used C6-R, a radial glial-like cell line and isolated perinatal cerebellar radial glia to ask what are the critical cytoskeletal elements in radial glial cells and how they are regulated. Treatments with nocodazole and cytochalasin D showed that microtubules, but not microfilaments, are critical for the polarized morphology of radial glia. In addition, quantitative real-time PCR indicated that certain mRNAs specific for microtubule-associated proteins (MAPs) are selectively expressed in radial glia. These results together with the known ability of microtubule affinity-regulating kinases to regulate microtubule organization suggest that microtubules and MAPs are critical for the morphology of radial glia
— id: 63136, year: 2003, vol: 44, page: 37, stat: Journal Article,

Soluble cell adhesion molecule L1-Fc promotes locomotor recovery in rats after spinal cord injury
Roonprapunt, Chanland; Huang, Wencheng; Grill, Ray; Friedlander, David; Grumet, Martin; Chen, Suzhen; Schachner, Melitta; Young, Wise
2003 Sep;20(9):871-882, Journal of neurotrauma
Previous studies suggest that the cell adhesion molecule L1 promotes neurite growth by neutralizing white matter associated inhibitors of axonal growth. We made a soluble chimeric dimer by linking mouse L1 to human Fc. This L1-Fc construct (40 microg/mL) markedly facilitated neurite outgrowth, as well as neuronal adhesion to white matter on frozen sections of spinal cord. We applied L1-Fc intrathecally (200 microg/mL at 0.5 microL/h) to rat spinal cords for 2 weeks after a 25-mm weight drop contusion of the T13 spinal cord. Initial experiments indicated that L1-Fc is present in the spinal cord after 2 weeks of intrathecal infusion and significantly improved locomotor recovery by 6-12 weeks after injury. We then randomized 45 rats to intrathecal infusion of L1-Fc (L1), phosphate-buffered saline controls (PBS), and a mouse monoclonal IgM antibody (M1). By 12 weeks after injury, L1-treated rats recovered significantly (p < 0.005) better locomotor function (BBB score 10.57 +/- 0.25, n = 14) than PBS-treated rats (BBB score 9.00 +/- 0.33, n = 14) or M1-treated (BBB score 8.71 +/- 0.16, n = 14). Only two rats of 22 treated with saline recovered weight-supported ambulation. Of 20 L1-Fc-treated rats, however, 18 recovered weight-supported walking by 12 weeks. The L1-Fc-treated rats also showed more consistent hindlimb contact placing than saline controls. We injected biotinylated dextran amine (BDA) into the motor cortices of 14 rats treated with L1-Fc to label corticospinal axons, comparing these with 13 rats treated with saline. In saline-treated rats, BDA-labeled corticospinal axons often grew up to the impact edge and occasionally into the impact site. L1-treated rats showed longer corticospinal tract growth at the injury site. Three rats had BDA-labeled axons that extended beyond the impact center. One L1-Fc-treated rat showed axonal extension and synapse formation in cord distal to the injury. These results indicate that soluble L1-Fc promotes axonal growth and functional recovery after spinal cord injury. However, the limited corticospinal tract growth across the injury site cannot account for the observed locomotor recovery. Thus, L1 may be stimulating growth of other motor tracts or protecting axons and neurons. More studies are required to elucidate the mechanisms of L1-Fc-induced locomotor recovery
— id: 63134, year: 2003, vol: 20, page: 871, stat: Journal Article,

L1 mediated homophilic binding and neurite outgrowth are modulated by alternative splicing of exon 2
Jacob, Jeffrey; Haspel, Jeffrey; Kane-Goldsmith, Noriko; Grumet, Martin
2002 Jun 5;51(3):177-189, Journal of neurobiology
The neural cell adhesion molecule (CAM) L1 is a member of the immunoglobulin superfamily that has been implicated in neuronal adhesion, neurite outgrowth, and axon guidance. The clinical importance of L1 is illustrated by pathological mutations that lead to hydrocephalus, mental retardation, motor defects, and early mortality. The L1 gene is composed of 28 exons, including exons 2 and 27 that are spliced alternatively, and mutations in exon 2 are associated with severe neurological abnormalities in humans. To elucidate the role of L1 exon 2, a recombinant Fc fusion protein called Delta2L1 was constructed lacking the second exon in the extracellular domain of L1. When bound to fluorescent beads, L1 exhibited homophilic binding while Delta2L1 did not. However, L1 beads coaggregated with the Delta2L1 beads. Similarly, in cell binding studies, L1 bound to L1 and Delta2L1 did not bind to Delta2L1 but it bound moderately to L1. Given the reduced binding of Delta2L1, we tested its effect on neurons. By comparison to L1, a lower percentage of dissociated neurons extended neurites on Delta2L1, and there was a modest decrease in the length of the neurites that grew. Neurite outgrowth from reaggregated neurons was much less robust on Delta2L1 than on L1. The combined results indicate that Delta2L1 does not bind homophilically but it can interact with L1 containing exon 2. The reduced binding and neurite promoting activity of Delta2L1 provides an explanation for certain pathological mutations in L1 that lead to clinically apparent disease in the absence of the normal form of L1 in the nervous system
— id: 63138, year: 2002, vol: 51, page: 177, stat: Journal Article,

Differential expression of the cell-adhesion molecule Nr-CAM in hyperplastic and neoplastic human pancreatic tissue
Dhodapkar KM; Friedlander D; Scholes J; Grumet M
2001 Apr;32(4):396-400, Human pathology
Nr-CAM is a member of the immunoglobulin superfamily of neural cell-adhesion molecules initially thought to be expressed mainly in the brain. Here we show the presence of Nr-CAM protein in normal human pancreas and characterize its expression in hyperplastic and neoplastic human pancreatic tissue. Nr-CAM is expressed on the cell surface in normal pancreatic acini with enhanced staining at cell-cell junctions, and weak or no surface staining is seen on normal ductal cells. Nr-CAM expression is markedly up-regulated in intraductal hyperplasia. Expression was well maintained in well or moderately differentiated carcinoma but was reduced or absent from most poorly differentiated tumors. In addition, 4 of 4 human pancreatic adenocarcinoma cell lines tested demonstrated little or no Nr-CAM expression. This differential regulation of Nr-CAM expression suggests that it may be involved in the pathogenesis and invasive/metastatic behavior of pancreatic cancers. HUM PATHOL 32:396-400.
— id: 20692, year: 2001, vol: 32, page: 396, stat: Journal Article,

System for cleavable Fc fusion proteins using tobacco etch virus (TEV) protease
Haspel, J; Blanco, C; Jacob, J; Grumet, M
2001 JAN ;30(1):60-+, Biotechniques
We describe a novel Fc fusion protein system that can be cleaved by tobacco each virus (TEV) protease. This system is desirable because it takes advantage of the high specificity of TEV protease and its activity at 4 degreesC. We produced two TEV-Fe fusion proteins drat contain the first three Ig domains and all six Ig domains of the cell adhesion molecule L1. Both proteins were efficiently cleaved by TEV protease at 4 degreesC. Functional analysis of the cleavage producers in neurite outgrowth assays showed;ed they had similar activities to their parental Fe fusion proteins. Therefore, TEV-Fe fusion proteins may increase the utility and flexibility of the Fc fusion protein system
— id: 55238, year: 2001, vol: 30, page: 60, stat: Journal Article,

Reduced tumorigenicity of rat glioma cells in the brain when mediated by hygromycin phosphotransferase
Hormigo A; Friedlander DR; Brittis PA; Zagzag D; Grumet M
2001 Apr;94(4):596-604, Journal of neurosurgery
OBJECT: A variant of C6 glioma cells, C6R-G/H cells express hygromycin phosphotransferase (HPT) and appear to have reduced tumorigenicity in the embryonic brain. The goal of this study was to investigate their reduced capacity to generate tumors in the adult rat brain. METHODS: Cell lines were implanted into rat brains and tumorigenesis was evaluated. After 3 weeks, all rats with C6 cells showed signs of neurological disease, whereas rats with C6R-G/H cells did not and were either killed then or allowed to survive until later. Histological studies were performed to analyze tumor size, malignancy, angiogenesis, and cell proliferation. Cells isolated from rat brain tumors were analyzed for mutation to HPT by testing their sensitivity to hygromycin. CONCLUSIONS: The results indicate that HPT suppresses tumor formation. Three weeks after implantation, only 44% of animals implanted with C6R-G/H cells developed tumors, whereas all animals that received C6 glioma cells developed high-grade gliomas. The C6R-G/H cells filled a 20-fold smaller maximal cross-sectional area than the C6 cells, and exhibited less malignant characteristics, including reduced angiogenesis, mitosis, and cell proliferation. Similar results were obtained in the brain of nude rats, indicating that the immune system did not play a significant role in suppressing tumor growth. The combination of green fluorescent protein (GFP) and HPT was more effective in suppressing tumorigenesis than either plasmid by itself, indicating that the GFP may protect against inactivation of the HPT. Interestingly. hygromycin resistance was lost in tumor cells that were recovered from a group of animals in which C6R-G/H cells formed tumors, confirming the correlation of HPT with reduced tumorigenicity
— id: 21203, year: 2001, vol: 94, page: 596, stat: Journal Article,

Radial glial cell line C6-R integrates preferentially in adult white matter and facilitates migration of coimplanted neurons in vivo
Hormigo A; McCarthy M; Nothias JM; Hasegawa K; Huang W; Friedlander DR; Fischer I; Fishell G; Grumet M
2001 Apr;168(2):310-322, Experimental neurology
C6-R is a cell line derived from C6 glioma cells that exhibits key properties of radial glia including the ability to support neuronal migration in culture. To explore its potential use in promoting neuronal migration in vivo, we analyzed the behavior of C6-R cells in the intact and injured adult rat CNS. At 6-11 days postimplantation at the splenium of the corpus callosum, green fluorescent protein-labeled C6-R cells were observed primarily in either the corpus callosum or the hippocampus in the brain, and in the spinal cord they migrated more extensively in the white matter than in the grey matter. To determine whether C6-R cells retain their ability to promote neuronal migration in vivo, they were coinjected with labeled neurons into adult brain. When rat embryonic neurons were coimplanted with C6-R cells, the neurons and C6-R cells comigrated through a much larger volume than neurons alone or neurons coimplanted with fibroblasts. In brains preinjured with ibotenic acid, C6-R cells as well as coimplanted neurons distributed widely within the lesion site and migrated into adjacent brain tissue, while transplants with neurons alone were restricted primarily to the lesion site. The results suggest that radial glial cell lines can serve as a scaffold for neuronal migration that may facilitate development of experimental models for neural transplantation and regeneration
— id: 20823, year: 2001, vol: 168, page: 310, stat: Journal Article,

Nr-CAM expression in the developing mouse nervous system: Ventral midline structures, specific fiber tracts, and neuropilar regions
Lustig M; Erskine L; Mason CA; Grumet M; Sakurai T
2001 May 21;434(1):13-28, Journal of comparative neurology
Nr-CAM is a member of the L1 subfamily of cell adhesion molecules (CAMs) that belong to the immunoglobulin superfamily. To explore the role of Nr-CAM in the developing nervous system, we prepared specific antibodies against both chick and mouse Nr-CAM using recombinant Fc fusion proteins of chick Nr-CAM and mouse Nr-CAM, respectively. First, we show the specificity of the new anti-chick Nr-CAM antibody compared with a previously employed antibody using the expression patterns of Nr-CAM in the chick spinal cord and floor plate and on commissural axons, where Nr-CAM has been implicated in axon guidance. Using the anti-mouse Nr-CAM antibody, we then studied the expression patterns of Nr-CAM in the developing mouse nervous system along with the patterns of two related CAMs, L1, which labels most growing axons, and TAG-1, which binds to Nr-CAM and has a more restricted distribution. Major sites that are positive for Nr-CAM are specialized glial formations in the ventral midline, including the floor plate in the spinal cord, the hindbrain and midbrain, the optic chiasm, and the median eminence in the forebrain. Similar to what is seen in the chick spinal cord, Nr-CAM is expressed on crossing fibers as they course through these areas. In addition, Nr-CAM is found in crossing fiber pathways, including the anterior commissure, corpus callosum, and posterior commissure, and in nondecussating pathways, such as the lateral olfactory tract and the habenulointerpeduncular tract. Nr-CAM, for the most part, is colocalized with TAG-1 in all of these systems. Based on in vitro studies indicating that the Nr-CAM-axonin-1/TAG-1 interaction is involved in peripheral axonal growth and guidance in the spinal cord [Lustig et al. (1999) Dev Biol 209:340-351; Fitzli et al. (2000) J Cell Biol 149:951-968], the expression patterns described herein implicate a role for this interaction in central nervous system axon growth and guidance, especially at points of decussation. Nr-CAM also is expressed in cortical regions, such as the olfactory bulb. In the hippocampus, however, TAG-1-positive areas are segregated from Nr-CAM-positive areas, suggesting that, in neuropilar regions, Nr-CAM interacts with molecules other than TAG-1.
— id: 20695, year: 2001, vol: 434, page: 13, stat: Journal Article,

Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier
Lustig M; Zanazzi G; Sakurai T; Blanco C; Levinson SR; Lambert S; Grumet M; Salzer JL
2001 Nov 27;11(23):1864-1869, Current biology. CB
Voltage-dependent sodium (Na(+)) channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na(+) channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, we treated myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc had no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibited Na(+) channel and ankyrin G accumulation at the node. Nr-Fc bound directly to neurons and clustered and coprecipitated neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM
— id: 27406, year: 2001, vol: 11, page: 1864, stat: Journal Article,

Overlapping functions of the cell adhesion molecules Nr-CAM and L1 in cerebellar granule cell development
Sakurai, T; Lustig, M; Babiarz, J; Furley, A J; Tait, S; Brophy, P J; Brown, S A; Brown, L Y; Mason, C A; Grumet, M
2001 Sep 17;154(6):1259-1273, Journal of cell biology
The structurally related cell adhesion molecules L1 and Nr-CAM have overlapping expression patterns in cerebellar granule cells. Here we analyzed their involvement in granule cell development using mutant mice. Nr-CAM-deficient cerebellar granule cells failed to extend neurites in vitro on contactin, a known ligand for Nr-CAM expressed in the cerebellum, confirming that these mice are functionally null for Nr-CAM. In vivo, Nr-CAM-null cerebella did not exhibit obvious histological defects, although a mild size reduction of several lobes was observed, most notably lobes IV and V in the vermis. Mice deficient for both L1 and Nr-CAM exhibited severe cerebellar folial defects and a reduction in the thickness of the inner granule cell layer. Additionally, anti-L1 antibodies specifically disrupted survival and maintenance of Nr-CAM-deficient granule cells in cerebellar cultures treated with antibodies. The combined results indicate that Nr-CAM and L1 play a role in cerebellar granule cell development, and suggest that closely related molecules in the L1 family have overlapping functions
— id: 141830, year: 2001, vol: 154, page: 1259, stat: Journal Article,

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng-CAM
Haspel J; Friedlander DR; Ivgy-May N; Chickramane S; Roonprapunt C; Chen S; Schachner M; Grumet M
2000 Feb 15;42(3):287-302, Journal of neurobiology
Mammalian L1 and avian Ng-CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng-CAM. Our results indicate that Ig domains 1-4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng-CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1-4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1-6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo.
— id: 11857, year: 2000, vol: 42, page: 287, stat: Journal Article,

Contactin-associated protein (Caspr) and contactin form a complex that is targeted to the paranodal junctions during myelination
Rios JC; Melendez-Vasquez CV; Einheber S; Lustig M; Grumet M; Hemperly J; Peles E; Salzer JL
2000 Nov 15;20(22):8354-8364, Journal of neuroscience
Specialized paranodal junctions form between the axon and the closely apposed paranodal loops of myelinating glia. They are interposed between sodium channels at the nodes of Ranvier and potassium channels in the juxtaparanodal regions; their precise function and molecular composition have been elusive. We previously reported that Caspr (contactin-associated protein) is a major axonal constituent of these junctions (Einheber et al., 1997). We now report that contactin colocalizes and forms a cis complex with Caspr in the paranodes and juxtamesaxon. These proteins coextract and coprecipitate from neurons, myelinating cultures, and myelin preparations enriched in junctional markers; they fractionate on sucrose gradients as a high-molecular-weight complex, suggesting that other proteins may also be associated with this complex. Neurons express two contactin isoforms that differ in their extent of glycosylation: a lower-molecular-weight phosphatidylinositol phospholipase C (PI-PLC)-resistant form is associated specifically with Caspr in the paranodes, whereas a higher-molecular-weight form of contactin, not associated with Caspr, is present in central nodes of Ranvier. These results suggest that the targeting of contactin to different axonal domains may be determined, in part, via its association with Caspr. Treatment of myelinating cocultures of Schwann cells and neurons with RPTPbeta-Fc, a soluble construct containing the carbonic anhydrase domain of the receptor protein tyrosine phosphatase beta (RPTPbeta), a potential glial receptor for contactin, blocks the localization of the Caspr/contactin complex to the paranodes. These results strongly suggest that a preformed complex of Caspr and contactin is targeted to the paranodal junctions via extracellular interactions with myelinating glia
— id: 33547, year: 2000, vol: 20, page: 8354, stat: Journal Article,

Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis
Zagzag D; Amirnovin R; Greco MA; Yee H; Holash J; Wiegand SJ; Zabski S; Yancopoulos GD; Grumet M
2000 Jun;80(6):837-849, Laboratory investigation
Vascular changes in gliomas were analyzed by implanting fluorescent-labeled glioma 261 cells in the brains of 28 mice. Seven animals were killed each week for 4 weeks. We investigated the expression of angiopoietin-2 (Ang-2) by in situ hybridization and compared it with the distribution of apoptotic cells identified by DNA strand breaks (using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling [TUNEL] method) and transmission electron microscopy (TEM). As early as 1 week after implantation, tumor cells accumulated around vessels, which expressed Ang-2 and were TUNEL negative. TEM showed tumor cells adjacent to the vascular cells 'lifting up' the normal astrocytic feet processes away from the endothelial cells and disrupting normal pericytic cuffing. After 2 weeks the number of perivascular glioma cells had increased. No increase in the number of blood vessels was detected at this time. Vascular cells remained positive for Ang-2 and rare ones were TUNEL positive. TEM showed closely packed proliferating perivascular tumor cells. After 3 weeks, there was vascular involution with scant zones of tumor necrosis. Ang-2 was still detected in vascular cells, but now numerous vascular cells were TUNEL positive. In addition, TEM showed apoptotic vascular cells. After 4 weeks, there were extensive areas of tumor necrosis with pseudopalisading and adjacent angiogenesis. Ang-2 was detected in vascular cells at the edge of the tumors in the invaded brain and in vessels surrounded by tumor cells. At both 3 and 4 weeks, most of the TUNEL-positive tumor cells lacked morphological features characteristic of apoptosis and displayed features consistent with necrotic cell death as determined by TEM. Only rare tumor cells appeared truly apoptotic. In contrast, the TUNEL-positive endothelial cells and pericytes were round and shrunken, with condensed nuclear chromatin by TEM, suggesting that vascular cells were undergoing an apoptotic cell death. These results suggest that vascular cell apoptosis and involution preceded tumor necrosis and that angiogenesis is a later event in tumor progression in experimental gliomas. Moreover, Ang-2 is detected prior to the onset of apoptosis in vascular cells and could be linked to vascular involution
— id: 9342, year: 2000, vol: 80, page: 837, stat: Journal Article,

Molecular events implicated in brain tumor angiogenesis and invasion
Zagzag D; Friedlander DR; Margolis B; Grumet M; Semenza GL; Zhong H; Simons JW; Holash J; Wiegand SJ; Yancopoulos GD
2000 Jul;33(1):49-55, Pediatric neurosurgery
We have conducted studies designed to help elucidate the molecular mechanisms involved in brain tumor invasion and angiogenesis, which are critical in the growth of malignant tumors of the central nervous system. A variety of molecular factors have been implicated in these processes. Here we focus on three that are of particular importance in the progression of brain tumors. Angiopoietins are involved in the regulation of vascular development. Hypoxia inducible factor-1 is a transcription factor that up-regulates genes, including genes encoding vascular endothelial growth factor under hypoxic conditions. Focal adhesion kinase is associated with infiltration of tumor cells and angiogenesis
— id: 34745, year: 2000, vol: 33, page: 49, stat: Journal Article,

Vascular apoptosis and involution in glioma progression is associated with angiopoietin-2 expression
Zagzag, D; Amirnovin, R; Greco, A; Yee, H; Holash, J; Wiegand, S; Yancopoulos, G; Grumet, M
2000 SEP ;10(4):735-736, Brain pathology
— id: 54520, year: 2000, vol: 10, page: 735, stat: Journal Article,

Endothelial cell apoptosis and vascular regression in gliomas precede neovascularization: A new concept for glioma growth and angiogenesis
Amirnovin, R; Holash, J; Wiegand, S; Yancopoulos, G; Grumet, M; Zagzag, D
1999 MAY ;58(5):537-537, Journal of neuropathology & experimental neurology
— id: 54054, year: 1999, vol: 58, page: 537, stat: Journal Article,

Promotion of neurite outgrowth by regions in human L1
Badrian, C; Haspel, J; Friedlander, D; Grumet, M
1999 MAR 21 ;217(6):U437-U437, Abstracts of papers (American Chemical Society)
— id: 54096, year: 1999, vol: 217, page: U437, stat: Journal Article,

Functional mapping of human L1CAM using recombinant Fc fusion proteins.
Haspel, J; Friedlander, D R; Blanco, C; Badrian, C; Chen, S; Schachner, M; Grumet, M
1999 Oct 23-28;25(1-2):8-8, Abstracts (Society for Neuroscience)
— id: 15873, year: 1999, vol: 25, page: 8, stat: Journal Article,

Integration of embryonic neurons in vivo is enhanced by co-transplantation with a radial glial cell line C6-R
Hormigo, A; McCarthy, M; Nothias, J-M; Fischer, I; Friedlander, D R; Fishell, G; Grumet, M
1999 Oct 23-28;25(1-2):214-214, Abstracts (Society for Neuroscience)
— id: 15869, year: 1999, vol: 25, page: 214, stat: Journal Article,

Nr-CAM promotes neurite outgrowth from peripheral ganglia by a mechanism involving axonin-1 as a neuronal receptor
Lustig M; Sakurai T; Grumet M
1999 May 15;209(2):340-351, Developmental biology (Orlando)
Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development.
— id: 6116, year: 1999, vol: 209, page: 340, stat: Journal Article,

Interactions of Nr-CAM are critical for clustering of ankyrin and sodium channels at the node of Ranvier
Lustig, M; Zanazzi, G; Sakurai, T; Blanco, C; Salzer, J; Grumet, M
1999 Oct 23-28;25(1-2):999-999, Abstracts (Society for Neuroscience)
— id: 15850, year: 1999, vol: 25, page: 999, stat: Journal Article,

Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord
Matise MP; Lustig M; Sakurai T; Grumet M; Joyner AL
1999 Aug;126(16):3649-3659, Development
Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(-/-) embryos. Netrin1 mRNA expression was detected in Gli2(-/-) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(-/-) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(-/-) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(-/-) embryos, although they are less well organized in Gli2(-/-)mutants
— id: 6162, year: 1999, vol: 126, page: 3649, stat: Journal Article,

Caspr and contactin co-localize in the paranodal and internodal membranes of myelinated axons
Rios, J C; Lustig, M; Grumet, M; Gollan, L; Peles, E; Hemperly, J J; Salzer, J L
1999 Oct 23-28;25(1-2):999-999, Abstracts (Society for Neuroscience)
— id: 15848, year: 1999, vol: 25, page: 999, stat: Journal Article,

Generation and initial characterization of Nr-CAM null mice
Sakurai, Takeshi; Lustig, Marc; Grumet, Martin
1999 Oct 23-28;25(1-2):8-8, Abstracts (Society for Neuroscience)
— id: 15874, year: 1999, vol: 25, page: 8, stat: Journal Article,

In situ expression of angiopoietins in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis
Zagzag D; Hooper A; Friedlander DR; Chan W; Holash J; Wiegand SJ; Yancopoulos GD; Grumet M
1999 Oct;159(2):391-400, Experimental neurology
Angiopoietin-1 (Ang-1) and its naturally occurring antagonist angiopoietin-2 (Ang-2) are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on endothelial cells. Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and endothelial cell interactions with supporting pericytes/smooth muscle cells. We investigated the expression of Ang-1 and Ang-2 in human astrocytomas by in situ hybridization and compared them to the distribution of pericytes/smooth muscle cells by immunohistochemistry for alpha-smooth muscle actin (SMA). Ang-1 mRNA was localized in tumor cells and Ang-2 mRNA was detected in endothelial cells of hyperplastic and nonhyperplastic tumor vessels. Ang-2 was also expressed in partially sclerotic vessels and in vascular channels surrounded by tumor cells in brain adjacent to the tumor. Neither Ang-1 nor Ang-2 was detected in normal brain. Dynamic changes in SMA expression during glioma tumorigenesis appear to progress from fragmentation in early vascular hyperplasia to subsequent reassociation and enhanced expression in later stages of vascular proliferation in hyperplastic complexes in high-grade gliomas. All these vessels displaying dynamic changes in SMA immunoreactivity also expressed Ang-2 mRNA. Moreover, SMA immunoreactive intratumoral vascular channels lacking morphological evidence of hyperplasia also showed upregulation of Ang-2. These results suggest that angiopoietins are involved in the early stage of vascular activation and in advanced angiogenesis, and they identify Ang-2 as an early marker of glioma-induced neovascularization. The association between Ang-2 expression and alterations in SMA immunoreactivity suggests a role for Ang-2 in tumor-associated activation of pericytes/smooth muscle cells.
— id: 6215, year: 1999, vol: 159, page: 391, stat: Journal Article,

Generation of a radial-like glial cell line
Friedlander DR; Brittis PA; Sakurai T; Shif B; Wirchansky W; Fishell G; Grumet M
1998 Nov 5;37(2):291-304, Journal of neurobiology
Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase beta, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (approximately 60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (approximately 30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration
— id: 7568, year: 1998, vol: 37, page: 291, stat: Journal Article,

Multi-ligand interactions with receptor-like protein tyrosine phosphatase beta: implications for intercellular signaling
Peles E; Schlessinger J; Grumet M
1998 Apr;23(4):121-124, Trends in biochemical sciences
Receptor-like protein tyrosine phosphatase beta (RPTP beta) shows structural and functional similarity to cell adhesion molecules (CAMs). It binds to several neuronal CAMs and extracellular matrix (ECM) proteins that combine to form cell-recognition complexes. Here, the authors discuss the implications of such complexes for intercellular signaling, and the regulation of RPTP activity by cell-cell and cell-ECM contact
— id: 7739, year: 1998, vol: 23, page: 121, stat: Journal Article,

Purification of Ig-fusion proteins from medium containing Ig
Sakurai T; Roonprapunt C; Grumet M
1998 Sep;25(3):382-385, Biotechniques
— id: 7775, year: 1998, vol: 25, page: 382, stat: Journal Article,

Angiopoietin-1 expression in sporadic and Von-Hippel-Lindau disease-associated hemangioblastomas
Zagzag, D; Hooper, A; Chan, W; Friedlander, D; Yancopoulos, G; Grumet, M
1998 MAY ;57(5):526-526, Journal of neuropathology & experimental neurology
— id: 53448, year: 1998, vol: 57, page: 526, stat: Journal Article,

Nr-CAM: a cell adhesion molecule with ligand and receptor functions
Grumet M
1997 Nov;290(2):423-428, Cell & tissue research
Nr-CAM Ng-CAM-related cell adhesion molecule) is expressed only in the nervous system on a subset of neurons and non-neuronal cells, including floor-plate cells in the spinal cord and Schwann cells. It is a member of the immunoglobulin (Ig) superfamily and can bind by a homophilic mechanism but its heterophilic interactions may be of greater biological significance. Nr-CAM functions as a neuronal receptor for neurite-growth-promoting stimuli provided by contactin/F11, neurofascin, and RPTPbeta. In addition, by binding to neuronal receptors such as axonin-1, it can modulate axonal guidance. Nr-CAM can also interact laterally with contactin/F11 within the plasma membrane in the form of a complex that may transmit signals to regulate axonal growth. This review summarizes the structure and expression of Nr-CAM and discusses its potential functions as a ligand and as a receptor during neural development
— id: 12278, year: 1997, vol: 290, page: 423, stat: Journal Article,

Tenascin-C expression in the cyst wall and fluid of human brain tumors correlates with angiogenesis
Jallo GI; Friedlander DR; Kelly PJ; Wisoff JH; Grumet M; Zagzag D
1997 Nov;41(5):1052-1059, Neurosurgery
OBJECTIVE: Tenascin-C (TN) is an extracellular matrix glycoprotein with a characteristic six-armed structure. The aim of this study was to determine whether the concentration of TN in the cyst fluid of brain tumors can be used as a marker for angiogenesis and glioma grade. METHODS: We investigated the expression of TN in the cyst wall and cyst fluid of human brain tumors by immunohistochemistry, immunoprecipitation, and immunoblotting. The tumors included 12 astrocytomas (5 glioblastoma multiforme tumors, 1 anaplastic astrocytoma, 1 low-grade astrocytoma, 4 juvenile pilocytic astrocytomas, and 1 mixed glioma), 2 dysembryoplastic neuroepithelial tumors, 3 craniopharyngiomas, 2 ependymomas, 2 metastatic carcinomas, 3 arachnoid cysts, 1 glial ependymal cyst, and 1 inflammatory cyst. RESULTS: We detected no expression of TN in the cyst fluids of the ependymomas, craniopharyngiomas, and nonpilocytic low-grade astrocytoma. By contrast, TN was detected in the cyst fluids of all the other tumors. Results of quantitative immunoblotting using a PhosphorImager unit (Molecular Dynamics, Sunnyvale, CA) revealed that, on average, a 5-fold higher signal was observed in the glioblastoma multiforme tumors as compared with the anaplastic astrocytoma, and a 10-fold higher signal as compared with the mixed glioma, juvenile pilocytic astrocytomas, and dysembryoplastic neuroepithelial tumors. Results of TN immunohistochemistry in the astrocytomas correlated with glioma grade, with stronger staining of the hyperplastic vessels and tumor cells being observed in higher grade gliomas. No TN immunoreactivity was detected in the walls of the ependymomas, arachnoid cysts, and glial ependymal cyst that lack hyperplastic vessels, and minimal TN immunoreactivity was observed in the perivascular gliotic rim of the craniopharyngiomas. No TN was detected in the cyst fluid of these cystic processes. CONCLUSION: The presence of TN in and around the hyperplastic vessels and tumor cells present in the cyst walls of astrocytomas and its deposition in the intratumoral cyst fluid in which angiogenic factors have been detected further suggests a role for TN as an angiogenic modulator. These preliminary results suggest that immunodetection of TN in the tumor cyst fluid may indicate tumor type and grade
— id: 9349, year: 1997, vol: 41, page: 1052, stat: Journal Article,

Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions
Peles E; Nativ M; Lustig M; Grumet M; Schilling J; Martinez R; Plowman GD; Schlessinger J
1997 Mar 3;16(5):978-988, EMBO journal
Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin-associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development
— id: 62341, year: 1997, vol: 16, page: 978, stat: Journal Article,

Induction of neurite outgrowth through contactin and Nr-CAM by extracellular regions of glial receptor tyrosine phosphatase beta
Sakurai T; Lustig M; Nativ M; Hemperly JJ; Schlessinger J; Peles E; Grumet M
1997 Feb 24;136(4):907-918, Journal of cell biology
Receptor protein tyrosine phosphatase beta (RPTPbeta) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (beta C) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, betaCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than betaCF. betaS had no effect by itself, but it potentiated neurite growth when mixed with betaCF. Neurite outgrowth induced by betaCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound betaCFS. NIH-3T3 cells transfected to express betaCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPbeta to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation
— id: 7439, year: 1997, vol: 136, page: 907, stat: Journal Article,

Cell adhesion molecules regulate guidance of dorsal root ganglion axons in the marginal zone and their invasion into the mantle layer of embryonic spinal cord
Shiga T; Lustig M; Grumet M; Shirai T
1997 Dec 1;192(1):136-148, Developmental biology (Orlando)
In order to elucidate the mechanisms regulating the projections of dorsal root ganglion (DRG) axons in the dorsal funiculus and invasion into target regions in the mantle layer (prospective gray matter) of the spinal cord, we examined the interactions between DRG axons and spinal cord. DRG neurons were dissociated from chick embryos and cultured for 1-2 days on cryostat sections of the spinal cord at embryonic day 5 (E5) or at E9. E5 and E9 DRG neurons extended neurites onto both marginal zone (prospective white matter) and mantle layer (prospective gray matter) of the spinal cord, suggesting that both of these regions are permissive for neurite growth. When E5 DRG neurites approached cryosections of E5 spinal cord from outside, most of them ran in the marginal zone without invading the mantle layer. In contrast, about half of E9 DRG neurites entered the mantle layer after crossing the marginal zone of E9 spinal cord. These growth patterns of DRG neurites on spinal marginal zone and mantle layer are similar to the pathway formation of DRG axons at comparable stages in vivo; DRG axons run exclusively in the prospective dorsal funiculus before E6, and enter the mantle layer (prospective dorsal horn) to reach the target regions by E9. Perturbation of functions of Ng-CAM, Nr-CAM, and axonin-1/SC2 by adding the specific antibodies in the culture medium increased the ratio of DRG neurites entering the mantle layer of E5 spinal cord, suggesting that these cell adhesion molecules are involved in keeping DRG neurites in the marginal zone. Taken together with the expression of Ng-CAM, Nr-CAM, and axonin-1/SC2, these CAMs on DRG axons may regulate the guidance of these axons in the marginal zone before E6, and the subsequent decrease in the relative levels of these CAMs might allow DRG axons to invade the target mantle layer.
— id: 7792, year: 1997, vol: 192, page: 136, stat: Journal Article,

Migration of brain tumor cells on extracellular matrix proteins in vitro correlates with tumor type and grade and involves alphaV and beta1 integrins
Friedlander DR; Zagzag D; Shiff B; Cohen H; Allen JC; Kelly PJ; Grumet M
1996 Apr 15;56(8):1939-1947, Cancer research
An important contributor to the malignancy of brain tumors is their ability to infiltrate the brain. Extracellular matrix molecules and cell adhesion molecules on cell surfaces play key roles in cell migration. In the present study, we used reaggregates of dissociated cells from freshly excised human brain tumors to analyze the migration of cells from human brain tumors of different types and grades on many different adhesion proteins adsorbed to glass substrates. Proteins were chosen based on their presence in normal or neoplastic nervous tissue, and included the extra-cellular matrix molecules fibronectin, collagens, fibrinogen, laminin, tenascin-C, thrombospondin, and the neuron-glia cell adhesion molecule, Ng-CAM. Cells from astrocytomas (n = 24) migrated on a variety of substrates, in contrast to cells from primitive neuroectodermal tumors cells (n=6), which only migrated well on laminin, fibronectin, or type IV collagen but not on the other substrates. Typically, migrating cells from astrocytomas of all grades had long, slender processes, were usually bipolar, and their cell bodies did not spread well on any substrate. Although there was variability in the migration of cells from astrocytomas of the same grade, cells from high-grade astrocytomas tended to migrate more extensively (42.3 +/- 4.7 micrometers/16 h: n = 16) than cells from lower grade astrocytomas (28.9 +/- 3.9 micrometers/16 h; P = 0.07; n = 8); the most striking differences were observed for collagen substrates, on which cells from lower grade astrocytomas migrated at very low levels (7.6 +/- 2 .6 micrometers/16 h) and cells from high-grade astrocytomas at higher levels (24.4 +/- 5.2 micrometers;P = 0.01). In contrast to primary cells from glioblastomas (n = 13), glioblastoma cell lines (n = 10) consistently spread on various substrates and migrated at high levels (69.5 +/- 7.6 versus 46.4 +/-5.7 micrometers/16 h; P = 0.03), in particular, on collagens (108.4 +/- 20.2 versus 28.0 +/- 6.1 micrometers/16 h; P= 0.001). Specific monoclonal antibodies to alphaV and beta1 integrin monomers completely inhibited the migration of astrocytoma cells on most substrates, suggesting that alphaV and beta1 integrins play a crucial role in brain tumor infiltration. These studies also suggest that although a large number of extracellular matrix molecules may promote tumor cell migration, disrupting the function of only a few tumor cell receptors may be critical for tumor infiltration in the brain
— id: 8091, year: 1996, vol: 56, page: 1939, stat: Journal Article,

Functions of brain chondroitin sulfate proteoglycans during developments: interactions with adhesion molecules
Grumet M; Friedlander DR; Sakurai T
1996 ;3(4):319-330, Perspectives on developmental neurobiology
Chondroitin sulfate proteoglycans (CSPGs), including neurocan and phosphocan, are believed to be major components of brain extracellular matrix that interact with other matrix proteins and cell surface receptors. In addition, several brain CSPGs such as receptor protein tyrosine phosphatase beta are expressed as cell surface receptors that interact with proteins in the extracellular matrix and with receptors on neural cells. Recent in vitro studies demonstrate that, although the brain CSPGs neurocan and phosphocan can promote transient adhesion of neuronal cells, they inhibit stable cell adhesion and neurite growth promoted by the cell adhesion molecule Ng-CAM/L1. Neurocan and phosphocan bind with high affinity to Ng-CAM/L1 and N-CAM which may be their major receptors on neurons. These CSPGs also bind to other adhesion molecules, such as tenascin-C, and can differentially modulate adhesion of glia of tenascin-C. Both the glycosaminoglycan and the core glycoproteins contribute to the function of the brain CSPGs. When expressed in regions containing low levels of adhesion molecules, various CSPGs including phosphocan, neurocan, versican, aggrecan, and NG2 proteoglycan may act as barriers to cell migration and axonal growth. In regions containing high levels of adhesion proteins, brain CSPGs may still act to maintain certain boundaries while allowing selective axonal extension to proceed. There are numerous regions of overlap in the expression patterns of CSPGs and adhesion molecules in vivo, and the relative levels of these molecules as well as the organization of the extracellular matrix may be important factors that regulate the rate of axonal growth locally. Differential expression of CSPGs may be important for modulating cell adhesion as well as axonal growth and guidance during neural development, and continued expression may prevent these processes in the normal nature nervous system as well as following brain injury
— id: 57527, year: 1996, vol: 3, page: 319, stat: Journal Article,

Heterophilic interactions of the neural cell adhesion molecules Ng-CAM and Nr-CAM with neural receptors and extracellular matrix proteins
Grumet, M; Sakurai, T
1996 DEC ;8(6):379-389, Seminars in the neurosciences
Ng-CAM/L1 and Nr-CAM are closely related neural cell adhesion molecules expressed on neurons and Schwann cells. Ng-CAM/L1 is prevalent on axons and is a potent promoter of neurite growth and axonal fasciculation. Although Nr-CAM does not appear to play a major role in these functions, its transient expression in the developing floor plate of the spinal cord is important for guidance of commissural axons. Ng-CAM and Nr-CAM can bind homophilicly and heterophilicly to several adhesion molecules. Different complexes of adhesion molecules are likely to control axonal growth and guidance as a consequence of their differential localizations in vivo
— id: 52694, year: 1996, vol: 8, page: 379, stat: Journal Article,

Proteoglycans in neural development - Preface
Margolis, RU; Grumet, M
1996 DEC ;3(4):243-243, Perspectives on developmental neurobiology
— id: 52691, year: 1996, vol: 3, page: 243, stat: Journal Article,

Expression of polypeptide variants of receptor-type protein tyrosine phosphatase beta: the secreted form, phosphacan, increases dramatically during embryonic development and modulates glial cell behavior in vitro
Sakurai T; Friedlander DR; Grumet M
1996 Mar 15;43(6):694-706, Journal of neuroscience research
Glial cells express three splicing variants of a receptor-type protein tyrosine phosphatase called RPTP beta. Two are receptor forms that differ in a large extracellular domain. The third is a secreted proteoglycan called phosphacan that lacks the cytoplasmic phosphatase domains. We have now identified, by immunoblotting, proteins corresponding to these three forms of RPTP beta in rat C6 glioma cells and brain. The short receptor form is much more prevalent than the full-length receptor in C6 glioma cells. Phosphacan is much more abundant than either of the receptor forms in rat brain, and its expression increases progressively during embryonic development, while the receptor forms show only moderate changes. In contrast to the long form and phosphacan that were detected as proteoglycans, the short receptor form, lacking the large alternatively spliced domain, was not detected as a chondroitin sulfate proteoglycan. We recently showed that phosphacan binds to the neuron-glia cell adhesion molecule, Ng-CAM, and we now report that glia expressing RPTP beta adhere and extend processes on substrates coated with Ng-CAM. After one day in culture, however, the glia retract their processes and often lift off the substrate. Conditioned medium from glial cells, which contains large amounts of phosphacan, inhibits glial adhesion to Ng-CAM, and depletion of phosphacan from the conditioned medium by immunoadsorption reduces the inhibitory activity. The results show that phosphacan increases dramatically during development, and indicate that secreted forms of RPTP beta can modulate glial cell adhesion and behavior
— id: 56843, year: 1996, vol: 43, page: 694, stat: Journal Article,

Tenascin-C expression by angiogenic vessels in human astrocytomas and by human brain endothelial cells in vitro
Zagzag D; Friedlander DR; Dosik J; Chikramane S; Chan W; Greco MA; Allen JC; Dorovini-Zis K; Grumet M
1996 Jan 1;56(1):182-189, Cancer research
The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis
— id: 56858, year: 1996, vol: 56, page: 182, stat: Journal Article,

THE CARBONIC-ANHYDRASE DOMAIN OF RECEPTOR TYROSINE PHOSPHATASE-BETA IS A FUNCTIONAL LIGAND FOR THE AXONAL CELL RECOGNITION MOLECULE CONTACTIN
PELES, E; NATIV, M; CAMPBELL, PL; SAKURAI, T; MARTINEZ, R; LEV, S; CLARY, DO; SCHILLING, J; BARNEA, G; PLOWMAN, GD; GRUMET, M; SCHLESSINGER, J
1995 JUL 28 ;82(2):251-260, Cell
Receptor-type protein tyrosine phosphatase beta (RPTP beta) is expressed in the developing nervous system and contains a carbonic anhydrase (CAH) domain as well as a fibronectin type III repeat in its extracellular domain. Fusion proteins containing these domains were used to search for ligands of RPTP beta. The CAH domain bound specifically to a 140 kDa protein expressed on the surface of neuronal cells. Expression cloning in COS7 cells revealed that this protein is contactin, a GPI membrane-anchored neuronal cell recognition molecule. The CAH domain of RPTP beta induced cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. These responses were blocked by antibodies against contactin, demonstrating that contactin is a neuronal receptor for RPTP beta. These experiments show that an individual domain of RPTP beta acts as a functional ligand for the neuronal receptor contactin. The Interaction between contactin and RPTP beta may generate unidirectional or bidirectional signals during neural development
— id: 87254, year: 1995, vol: 82, page: 251, stat: Journal Article,

Tenascin expression in astrocytomas correlates with angiogenesis
Zagzag D; Friedlander DR; Miller DC; Dosik J; Cangiarella J; Kostianovsky M; Cohen H; Grumet M; Greco MA
1995 Feb 15;55(4):907-914, Cancer research
We investigated the expression and distribution of the extracellular matrix protein tenascin (TN) in 59 astrocytomas and 11 samples of normal brain by Western blot analysis and immunohistochemistry using antibodies against human TN. The tumors included 14 juvenile pilocytic astrocytomas (grade 1), 13 low grade fibrillary astrocytomas (grade II), 8 anaplastic astrocytomas (grade III), and 24 glioblastomas multiforme (grade IV). Proliferation indices were calculated by computer-based image analysis after immunostaining with the MIB-1 antibody against the Ki-67 proliferation-associated antigen. Western blot analysis for TN on fresh frozen tumor tissue from 23 of the 59 astrocytomas indicated up to 4-fold higher TN expression in glioblastomas multiforme than in nontumorous control tissues. Enhanced intercellular expression of TN was observed by immunohistochemistry in glioblastomas multiforme. More-over, TN immunostaining was consistently greater within and around the walls of hyperplastic blood vessels than nonhyperplastic vessels of both high grade tumors and juvenile pilocytic astrocytomas. Juvenile pilocytic astrocytomas with increased TN expression by Western blot analysis had vascular hyperplasia by light microscopy. Proliferation indices moderately correlated with tumor grade. Enhanced immunohistochemical expression of TN was associated with higher tumor grade with higher proliferation indices. The strong association of TN and vascular hyperplasia, regardless of tumor grade, suggests that TN may play a crucial role in angiogenesis
— id: 6786, year: 1995, vol: 55, page: 907, stat: Journal Article,

Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin
Barnea G; Grumet M; Milev P; Silvennoinen O; Levy JB; Sap J; Schlessinger J
1994 May 20;269(20):14349-14352, Journal of biological chemistry
The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous immunohistochemical studies indicated that both RPTP beta and the extracellular matrix protein tenascin are localized in similar regions of the central nervous system. We have performed co-aggregation assays with red and green Co-vaspheres coated with tenascin and 3F8 PG, respectively, showing that the extracellular domain of RPTP beta (3F8 PG) binds specifically to tenascin. The interaction between a receptor tyrosine phosphatase and an extracellular matrix protein may have a role in development of the mammalian central nervous system
— id: 6318, year: 1994, vol: 269, page: 14349, stat: Journal Article,

Close similarity between receptor-linked tyrosine phosphatase and rat brain proteoglycan
Barnea G; Grumet M; Sap J; Margolis RU; Schlessinger J
1994 Jan 28;76(2):205-205, Cell
— id: 17822, year: 1994, vol: 76, page: 205, stat: Journal Article,

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth
Friedlander DR; Milev P; Karthikeyan L; Margolis RK; Margolis RU; Grumet M
1994 May;125(3):669-680, Journal of cell biology
We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules
— id: 8072, year: 1994, vol: 125, page: 669, stat: Journal Article,

Interactions with tenascin and differential effects on cell adhesion of neurocan and phosphacan, two major chondroitin sulfate proteoglycans of nervous tissue
Grumet M; Milev P; Sakurai T; Karthikeyan L; Bourdon M; Margolis RK; Margolis RU
1994 Apr 22;269(16):12142-12146, Journal of biological chemistry
We have studied interactions of tenascin with two chondroitin sulfate proteoglycans, neurocan and phosphacan. Neurocan is a multi-domain proteoglycan with a 136-kDa core protein that is synthesized by neurons and binds to hyaluronic acid, whereas the 173-kDa core protein of phosphacan, which is synthesized by glia, represents an extracellular variant of the receptor-type protein tyrosine phosphatase RPTP zeta/beta. Keratan sulfate-containing glycoforms of phosphacan (designated phosphacan-KS) are also present in brain. Immunocytochemical studies of early postnatal rat cerebellum demonstrated that the localization of neurocan, phosphacan, and phosphacan-KS all overlap extensively with that of tenascin, an extracellular matrix protein that modulates cell adhesion and migration. Binding studies using purified proteins covalently attached to fluorescent microbeads demonstrated that proteoglycan-coated beads co-aggregated with differently fluorescing beads coated with tenascin. The co-aggregation was specifically inhibited by Fab' fragments of antibodies against tenascin or the proteoglycans and by soluble neurocan, phosphacan, and tenascin. A solid phase radioligand binding assay confirmed that neurocan, phosphacan, and phosphacan-KS bind to tenascin but not to laminin and fibronectin. Chondroitinase treatment of the proteoglycans or addition of free chondroitin sulfate had no significant effect, indicating that the binding activity is mediated largely via the core glycoproteins. Scatchard analysis demonstrated high affinity binding of 125I-phosphacan, phosphacan-KS, and neurocan to a single site in tenascin, and neurocan and various glycoforms of phosphacan all inhibited binding of 125I-phosphacan to tenascin. In studies of cell adhesion to proteins adsorbed to Petri dishes, phosphacan inhibited adhesion of C6 glioma cells to tenascin whereas neurocan had no effect. Our results suggest that tenascin binds phosphacan and neurocan in vivo and that interactions between chondroitin sulfate proteoglycans and tenascin may play important roles in nervous tissue histogenesis, possibly by modulating signal transduction across the plasma membrane
— id: 6399, year: 1994, vol: 269, page: 12142, stat: Journal Article,

Regions of the human neurokinin A receptor involved in the generation of second messengers and in receptor desensitization
Josiah SM; Cyr CR; Chu V; Grumet M; Gardner JP; Kris RM
1994 Mar 15;199(2):626-632, Biochemical & biophysical research communications
Deletion analysis was used to study sites of human Neurokinin A receptor (HNKAR) necessary for signal transduction in CHO cells. Deletion of 62 and 81 amino acids from the c-terminus of HNKAR forms mutant receptors HNKAR delta 62 and HNKAR delta 81, which bind neurokinin A with high affinity but are functionally different. Wild type HNKAR and HNKAR delta 62 are functionally active whereas HNKAR delta 81 is functionally inactive. In addition, HNKAR and HNKAR delta 62 were both desensitized to the neurokinin A signal within 5 minutes. The data indicates: 1) an intact cytoplasmic tail of the HNKAR is not critical for signal transduction, but the n-terminal amino acids of the cytoplasmic tail are necessary for signaling and 2) the c-terminal 62 amino acids are not necessary for desensitization
— id: 6416, year: 1994, vol: 199, page: 626, stat: Journal Article,

STRUCTURE AND FUNCTIONS OF NERVOUS-TISSUE PROTEOGLYCANS
MARGOLIS, RU; MAUREL, P; MILEV, P; KARTHIKEYAN, L; FRIEDLANDER, DR; GRUMET, M; MARGOLIS, RK
1994 JUL 5 ;62(14):S30-S30, Journal of neurochemistry
— id: 52418, year: 1994, vol: 62, page: S30, stat: Journal Article,

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules
Milev P; Friedlander DR; Sakurai T; Karthikeyan L; Flad M; Margolis RK; Grumet M; Margolis RU
1994 Dec;127(6 Pt 1):1703-1715, Journal of cell biology
Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system
— id: 6692, year: 1994, vol: 127, page: 1703, stat: Journal Article,

Biochemical characterization and immunolocalization of SC2 protein: SC2 protein is indistinguishable from the cell adhesion molecule axonin-1
Sakurai T; Shiga T; Shirai T; Tanaka H; Grumet M
1994 Nov 18;83(1):99-108, Brain research. Developmental brain research
SC2 is a monoclonal antibody that was previously shown to recognize a subset of neurons in the developing nervous system of the chick. We have now used the SC2 monoclonal antibody to purify from chick embryo brain membranes a glycoprotein that migrates at approximately 125 kDa on SDS/PAGE. The size of this protein and its distribution pattern in the spinal cord are similar to that observed for axonin-1. A polyclonal anti-axonin-1 antibody R26 specifically reacted with the SC2 protein from brain. This antibody, as well as polyclonal antibody (369) against purified SC2 protein, reacted with 115-130 kDa proteins in vitreous humor, a rich source of axonin-1, and with similar sized proteins precipitated from vitreous humor by the 369, and SC2 antibodies. Treatment of SC2 protein isolated from chick brain membranes with PI-PLC indicated that it contains a glycophosphatidylinositol (GPI) moiety. Co-aggregation experiments using Covaspheres with covalently bound proteins indicated that SC2 protein binds heterophilically to Ng-CAM. Immunohistochemical analysis of chick embryos showed that SC2 protein is abundant in the sensory nerve bundles of both the central and peripheral nervous systems during development. Its expression was restricted and it was specifically localized in the dorsal funiculus of the spinal cord, as well as in olfactory, retinal, trigeminal, vestibulocochlear, glossopharyngeal and vagal nerve fibers. The biochemical and immunohistochemical data show that SC2 protein is axonin-1, and the immunolocalization studies support the hypothesis that SC2 protein may play a role during development of particular fiber systems by interacting with other cell adhesion molecules such as Ng-CAM
— id: 57403, year: 1994, vol: 83, page: 99, stat: Journal Article,

Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding
Sap J; Jiang YP; Friedlander D; Grumet M; Schlessinger J
1994 Jan;14(1):1-9, Molecular & cellular biology
Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation
— id: 6511, year: 1994, vol: 14, page: 1, stat: Journal Article,

Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules
Grumet M; Flaccus A; Margolis RU
1993 Feb;120(3):815-824, Journal of cell biology
Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system
— id: 13273, year: 1993, vol: 120, page: 815, stat: Journal Article,

Evidence for the binding of Ng-CAM to laminin
Grumet M; Friedlander DR; Edelman GM
1993 Sep;1(2):177-190, Cell adhesion & communications
Ng-CAM is a cell adhesion molecule mediating neuron-glia and neuron-neuron adhesion via different binding mechanisms. While its binding can be homophilic as demonstrated by the self-aggregation of Ng-CAM coated beads (Covaspheres), Ng-CAM has also been shown to bind to glia by a heterophilic mechanism. In the present study, we found that the extent of Ng-CAM Covasphere aggregation was strongly diminished in the presence of the extracellular matrix glycoprotein laminin. When proteolytic fragments of laminin were tested, the P1' fragment (obtained from the short arms by pepsin treatment) was found to inhibit aggregation of Ng-CAM-Covaspheres while the elastase fragments E3 and E8 (from the long arm) were ineffective. To provide other means of analyzing interactions between laminin and Ng-CAM, the two proteins were covalently linked to differently fluorescing Covaspheres and tested for coaggregation. Laminin-Covaspheres coaggregated with Ng-CAM-Covaspheres, and this binding was inhibited both by anti-Ng-CAM and by anti-laminin antibodies. Covaspheres coated with other proteins including BSA and fibronectin did not coaggregate with Ng-CAM-Covaspheres. Moreover, using a solid phase binding assay, we found that 125I-labeled Ng-CAM bound to laminin and to Ng-CAM but not to fibronectin. The results suggest that regions in the short arms of laminin can bind to Ng-CAM. To test whether Ng-CAM present on neurons could be involved in binding to laminin, adhesion of neurons to substrates coated with various proteins was tested in the presence of specific antibodies. Anti-Ng-CAM Fab' fragments inhibited neuronal binding to laminin but not binding to fibronectin. The combined results open the possibility that Ng-CAM on the surface of neurons may mediate binding to laminin in vivo, and that interactions with laminin can modulate homophilic Ng-CAM binding
— id: 13089, year: 1993, vol: 1, page: 177, stat: Journal Article,

INTERACTIONS AND CELLULAR SITES OF SYNTHESIS OF CHONDROITIN SULFATE PROTEOGLYCANS OF BRAIN
MILEV, P; FRIEDLANDER, D; GRUMET, M; MARGOLIS, RK; MARGOLIS, RU
1993 JUL 16 ;61(1):S110-S110, Journal of neurochemistry
— id: 52268, year: 1993, vol: 61, page: S110, stat: Journal Article,

DIFFERENTIAL EXPRESSION OF NEURON-GLIA CELL-ADHESION MOLECULE (NG-CAM) ON DEVELOPING AXONS AND GROWTH CONES OF INTERNEURONS IN THE CHICK-EMBRYO SPINAL-CORD - AN IMMUNOELECTRON MICROSCOPIC STUDY
SHIGA, T; SHIRAI, T; GRUMET, M; EDELMAN, GM; OPPENHEIM, RW
1993 MAR 22 ;329(4):512-518, Journal of comparative neurology
To elucidate the role of neuron-glia cell adhesion molecule (Ng-CAM) in axonal pathway formation of avian spinal interneurons, we have examined the ultrastructural expression of Ng-CAM in the developing spinal cord, by using a preembedding immunocytochemical method. Ng-CAM immunoreactivity was punctate and was restricted to cell surfaces. In accordance with our previous light microscopic observations (Shiga et al., '90), the earliest developing spinal interneurons were Ng-CAM-positive on their cell bodies, axons, and growth cones. Axons and growth cones that were either fasciculated or in contact with each other strongly expressed Ng-CAM, thus indicating the possible involvement of Ng-CAM in fasciculation of axons and in the contact guidance of growth cones along preexisting axons. By using higher resolution immunoelectron microscopy, the present study has also revealed new information on the subcellular localization of Ng-CAM on developing spinal interneurons, neuroepithelial cells, and floor plate cells. Although Ng-CAM immunoreactivity was prominent on both axons and growth cones, these structures were Ng-CAM-negative when they contacted the basal lamina around the spinal cord. By contrast, Ng-CAM was detectable on the surface of both neuroepithelial cells and floor plate cells only when they made contact with the Ng-CAM-positive axons and growth cones of interneurons. These results suggest that the subcellular distribution of Ng-CAM is regulated differentially, depending on the apposing cell surfaces, and that such differential and developmentally regulated expression may contribute to the elongation, fasciculation, and guidance of spinal axons
— id: 54359, year: 1993, vol: 329, page: 512, stat: Journal Article,

Structure, expression, and function of Ng-CAM, a member of the immunoglobulin superfamily involved in neuron-neuron and neuron-glia adhesion
Grumet, M
1992 Jan;31(1):1-13, Journal of neuroscience research
The neuron-glia cell adhesion molecule (Ng-CAM) mediates neuron-neuron adhesion by a homophilic mechanism and neuron-astrocyte adhesion by a heterophilic mechanism. The protein is expressed on neurons and Schwann cells but not on astrocytes. It is most prevalent during development on cell bodies of migrating neurons and on axons during formation of nerves. Ng-CAM expression is greatly increased following nerve injury. Anti-Ng-CAM antibodies inhibited migration of granule cells along Bergmann glia in cerebellar explants and fasciculation of neurites in outgrowths from explants of dorsal root ganglia. The combined results indicate that Ng-CAM on neurons binds to Ng-CAM on adjacent neurons and to as yet unidentified ligands on astrocytes. Ng-CAM is synthesized in chicken neurons from a 6 kb mRNA as Mr approximately 200,000 forms which are cleaved to yield two components of Mr 135,000 and 80,000. It is glycosylated and can be phosphorylated. Amino acid sequence analysis indicates that it contains six immunoglobulin domains, five fibronectin type III repeats, a transmembrane domain and a cytoplasmic region. Structural analyses indicate that Ng-CAM is most closely related to the mammalian glycoprotein L1 but significant differences between them strongly suggest that they are not equivalent molecules. The recent identification of another structurally related molecule in the chicken called Nr-CAM underscores the notion that these molecules are members of a subfamily of neural cell adhesion molecules within the immunoglobulin superfamily that have related or complementary functions in the nervous system
— id: 133251, year: 1992, vol: 31, page: 1, stat: Journal Article,

Cell adhesion molecules and their subgroups in the nervous system
Grumet M
1991 Oct;1(3):370-376, Current opinion in neurobiology
Structural relationships among cell adhesion molecules have been used to classify two large families of these molecules into subgroups. The cell adhesion molecules within each subgroup share several structural features that indicate that they may function in similar or complementary ways either simultaneously or at different times and locations
— id: 8301, year: 1991, vol: 1, page: 370, stat: Journal Article,

Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules
Grumet, M; Mauro, V; Burgoon, M P; Edelman, G M; Cunningham, B A
1991 Jun;113(6):1399-1412, Journal of cell biology
We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 133253, year: 1991, vol: 113, page: 1399, stat: Journal Article,