Homophilic NCAM interactions interfere with L1 stimulated neurite outgrowth

The cell adhesion molecules NCAM and L1 are considered to play key roles in neuronal development and plasticity. L1 has been shown to interact with NCAM, possibly through NCAM binding to oligomannosidic glycans present in L1. We investigated the effect of recombinant immunoglobulin (Ig) modules of NCAM involved in homophilic NCAM binding, on L1 induced neurite outgrowth from PC12-E2 cells and found a complete inhibition of L1 induced neurite outgrowth after addition of Ig-modules 1, 2 and 3 of NCAM, suggesting that the ligation state of NCAM is crucial for normal L1 signaling.     

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

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.     

Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1

The effects of L1-Fc and CHL1-Fc fusion proteins on neuronal survival were investigated. Cerebellar granule neurons of mouse and hippocampal neurons of rat embryo undergo apoptosis when cultured in serum-free medium. Treatment with chimeric proteins containing the extracellular domains of the neural adhesion molecules L1 or CHL1 fused to the Fc region of human immunoglobulin significantly enhanced the survival of neurons. Compared to the control, the percentage of surviving neurons increased about 60% and 45% with L1 and CHL1 fusion proteins, respectively. A fusion protein containing the extracellular domain of NCAM had no effect on survival. The L1 and CHL1 fusion proteins were effective both in soluble form or when offered as a substrate, with the maximal effect at about 1 microg/mL. To explore the intracellular events related to the neuronal survival effects of L1-Fc fusion protein, Bcl-2 and c-Jun expression were analyzed by Western blotting. The level of Bcl-2 in cerebellar granule neurons was increased by treatment with L1-Fc at both 1 and 5 days of culture. The level of c-Jun was not significantly affected at the early time point and was reduced by L1-Fc fusion protein after long-term culture. The results demonstrate that the neural adhesion molecule L1 and its relative CHL1 are potential neuronal survival factors for neurons of the central nervous system. Bcl-2 may serve as one of the intracellular mediators of the neuronal survival effects of L1.     

Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV- 1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin- 1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.     

Elevated phenylalanine levels interfere with neurite outgrowth stimulated by the neuronal cell adhesion molecule L1 in vitro

Elevated levels of phenylalanine (Phe) as observed in patients with phenylketonuria interfere with proper neuronal development, leading to severe psychomotor deficits and mental retardation. We have analyzed the effects of Phe on neurite outgrowth in vitro. When expressed in fibroblasts, the neuronal cell adhesion molecules L1 and plexin B3 strongly increase the length of neurites emanating from cerebellar neurons in co-culture experiments. Elevated Phe blocks L1-mediated, but not plexin B3-mediated outgrowth, whereas tyrosine is ineffective. Elevated Phe also interferes with aggregation of fibroblasts overexpressing L1, suggesting that the pathological effect of elevated Phe occurs by interfering with L1-mediated cell adhesion.     

L1 cell adhesion molecules as regulators of tumor cell invasiveness

Fast growing malignant cancers represent a major therapeutic challenge. Basic cancer research has concentrated efforts to determine the mechanisms underlying cancer initiation and progression and reveal candidate targets for future therapeutic treatment of cancer patients. With known roles in fundamental processes required for proper development and function of the nervous system, L1-CAMs have been recently identified as key players in cancer biology. In particular L1 has been implicated in cancer invasiveness and metastasis, and has been pursued as a powerful prognostic factor, indicating poor outcome for patients. Interestingly, L1 has been shown to be important for the survival of cancer stem cells, which are thought to be the source of cancer recurrence. The newly recognized roles for L1CAMs in cancer prompt a search for alternative therapeutic approaches. Despite the promising advances in cancer basic research, a better understanding of the molecular mechanisms dictating L1-mediated signaling is needed for the development of effective therapeutic treatment for cancer patients.Key words: L1CAMs, cancer, metastasis, axon guidance, cancer stem cell, migration, invasionA major obstacle in oncology is the early diagnosis and curative therapeutic intervention of locally invasive cancers that rapidly disseminate from the primary tumor to form metastases. The standard treatment for malignant tumors consists of surgical removal of the tumor mass followed by chemo- and radiotherapy in order to eradicate the remaining cancer cells. Despite such aggressive intervention, a population of resistant cancer cells often remains intact and is thought to be the source of cancer recurrence.During the past decades, cancer basic research has focused on determining the molecular mechanisms underlying cancer initiation and progression that can provide a basis for the development of new and effective therapeutic treatments for cancer patients. An important finding was the discovery that cancer onset and development are often associated with alterations in the expression of cell adhesion molecules, which are likely to stimulate tumor cell invasiveness by signaling mechanisms that enhance cell migration.¹ The L1 family of neural cell adhesion molecules (L1-CAMs), which is comprised of four structurally related transmembrane proteins L1, CHL1, NrCAM and neurofascin (Fig. 1), is now in the spotlight of cancer research due to their upregulation in certain human tumors. L1-CAMs are transmembrane molecules of the immunoglobulin superfamily, characterized by an extracellular region of six immunoglobulin-like domains and four to five fibronectin type III repeats, followed by a highly conserved cytoplasmic domain, which is reversibly linked to the cell cytoskeleton through binding to ankyrin and ERM proteins (ezrin-radixin-moesin).² Its multi-domain structure allows complex heterophilic interactions with diverse cell receptors, although homophilic interactions also have a crucial role in L1-CAMs mediated signaling.Open in a separate windowFigure 1L1-CAMs: All have 6 Ig domains and 4–5 FN domains. The 186 kD Neurofascin isoform has a mucin-like Pro/Ala/Thr-rich (PAT) domain, while the 155 kD has only the 4 FN domains. RGD and DGEA motifs interact with integrins, while the FigQ/AY motif binds to ankyrin. ERM binding sites are indicated. The RSLE motif in L1 recruits AP2/clathrin adaptor for endocytosis.A wealth of studies has revealed L1-CAMs as pivotal components for proper development of the nervous system through regulation of cell-cell interactions. L1-CAMs have critical roles in neuronal migration and survival, axon outgrowth and fasciculation, synaptic plasticity and regeneration after trauma.² Neither CHL1 nor L1 is present on mature astrocytes, oligodendroglia or endothelial blood vessel cells in the brain, but CHL1 is upregulated in astrocytes upon injury³ and is present on oligodendroglial precursors.^4,5 During neural development, L1 plays an important role in the migration of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.⁶ In the cerebellum, L1 is required for the inward migration of granule neurons from the external granular layer and cooperates with NrCAM in regulating neuronal positioning.² Similarly, CHL1 controls area-specific migration and positioning of deep layer cortical neurons in the neocortex.⁷ In addition to its role in neuronal precursor positioning, L1 plays a crucial role in axon guidance, which is governed by repellent and attractive response mechanisms directed by Ephrins and Semaphorins and their receptors (Ephs, Neuropilins, Plexins).² The importance of L1-CAMs in the development and function of the nervous system is exemplified by developmental neuropsychiatric disorders that are associated with mutation or genetic polymorphisms in genes encoding L1 (X-linked mental retardation) and CHL1 (low IQ, speech and motor delay). Polymorphisms in L1 and CHL1 genes are also associated with schizophrenia, and NrCAM gene polymorphisms are linked to autism in some populations.²Recent studies have described upregulation of L1 in a variety of tumor types. Overexpression of L1 correlates with tumor progression and metastasis in certain human gliomas,⁸ melanoma,⁹ ovarian¹⁰ and colon carcinomas.^11–13 Interestingly, L1 was found to be present only in cells at the invasive front of colon cancers but not in the tumor mass.¹² L1 is also associated with micrometastasis to both lymph nodes and bone marrow in patients bearing other cancers, suggesting a potential role in early metastatic spread.¹¹ L1 has now been pursued as both a biomarker and a powerful prognostic factor, indicative of poor outcome for patients as observed for epithelial ovarian carcinoma¹⁰ and colorectal cancer.¹¹ More recently, L1 has been shown to be overexpressed in a small fraction of glioma cells, termed glioma stem cells, which are capable of self-renewal and generate the diverse cells that comprise the tumor.¹⁴ First characterized in acute myeloid leukemia,¹⁵ cancer stem cells have been recently described in a variety of solid tumors, including breast cancer, lung cancer and gastrointestinal tumors.¹⁶ In gliomas, L1 expression was shown to be required for maintaining the growth and survival of glioma stem cells.¹⁴ These findings suggest that L1 may be implicated not only in cancer invasiveness but also in cancer survival. It will be important to determine if L1 is also upregulated in other cancer stem cells as well as to define the role of L1-mediated signaling in other cancers. Although not extensively investigated, NrCAM has also been shown to be overexpressed in glioblastoma cell lines and several cases of high grade astrocytoma¹⁷ and ependymomas.¹⁸ Studies are needed to address whether CHL1 and neurofascin play analogous roles in cancer onset and progression.The molecular mechanisms of L1-mediated signaling that govern the migration of neuronal precursors and guidance of axons during the development of the nervous system may also be used by cancer cells to facilitate invasion and cancer progression. Integrins are well-characterized cooperative partners for L1-CAMs, and signal transduction pathways activated by this complex are known to promote cell adhesion and directional motility. L1/integrin-mediated signaling may converge with growth factor signaling networks to promote motility. Like L1, CHL1 cooperates with integrins to stimulate migration. All L1-CAMs reversibly engage the actin cytoskeleton through a conserved motif FigQ/AY in the cytoplasmic domain that contains a crucial tyrosine residue required for binding the spectrin adaptor ankyrin. Phosphorylation of the FigQY tyrosine decreases ankyrin binding, whereas dephosphorylation promotes L1-ankyrin interaction. Dynamic adhesive interactions controlled by phosphorylation/dephosphorylation of the ankyrin motif in L1 family members may enable a cell to cyclically attach and detach from the ECM substrate or from neighboring cells, thus facilitating migration.¹ Another way L1 promotes cell migration is by stimulating endocytosis of integrins, reducing cell adhesion to the extracellular matrix.¹⁹ Thus, it is reasonable to speculate that upregulation of L1 in cancer may result in increased L1-mediated signaling and, consequently, increased cell migration.L1-CAMs are cleaved by metalloproteases, releasing functionally active ectodomain fragments that are laid down as “tracks” on the extracellular matrix (ECM). These fragments can cause autocrine activation of signal transduction pathways, promoting cell migration through heterophilic binding to integrins.²⁰ Specifically, L1 is cleaved constitutively or inducibly by the ADAM family metalloproteases (a disintegrin and metalloprotease) ADAM10 and ADAM17, which stimulates cell migration and neurite outgrowth during brain development.^20,21 In colon cancer, L1 colocalizes with ADAM 10 at the invasive front of the tumor tissue, suggesting that L1 shedding may play a role in cancer invasiveness.¹² Similarly, CHL1 is shed by ADAM8, which was reported to promote cell migration and invasive activity of glioma cells in vitro and is highly expressed in human brain tumors including glioblastoma multiforme, correlating with invasiveness in vivo.²² Furthermore, NrCAM, found in pancreatic, renal and colon cancers, is subject to ectodomain shedding,²³ but its function in regulating cell migration or invasion has not yet been studied.Given the newly recognized roles of L1 in tumor progression, a growing body of experimental studies has explored novel therapeutic approaches targeting L1-CAMs. Antibody-based therapeutic strategies are being pursued to functionally inhibit homophilic and heterophilic interactions of cell adhesion molecules to suppress tumor invasive motility. L1 monoclonal antibodies reduce in vivo growth of human ovarian and colon carcinoma cells in mouse xenograft models.^13,24,25 L1 targeting using lentiviral-mediated short hairpin RNA (shRNA) interference decreases growth and survival of glioma stem cells in vitro, suppresses tumor growth, and increases survival of tumor-bearing animals.¹⁴ These findings raise the possibility that L1 represents a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. Cancer stem cells represent a potential target for future treatment of different cancer as these cells are believed to be responsible for cancer recurrence.²⁶ Promoting cancer stem cell differentiation by drug treatment could potentially reduce stem cells properties of self-renewal and proliferation, leading to inhibition of tumor growth.Inhibitors of metalloproteases that block L1-CAM shedding represent a potentially novel approach to curtailing tumor invasiveness. Chemical inhibitors of ADAMS are appealing for glioma therapy due to their diffusability, which circumvents blood-brain barrier limitations. Another novel approach involves the secreted axon repellent protein, Semaphorin 3A (Sema3A). L1-CAMs serve as co-receptors for Sema3A by cis binding in the plasma membrane to Neuropilin-1, important for repellent axon guidance.² Interestingly, Sema3A inhibits invasiveness of prostate cancer cells²⁷ and migration and spreading of breast cancer cells in in vitro assays,²⁸ and thus may also be mediated by L1-CAMs. Such an approach could be potentially useful in mitigating invasion of cancer cells in gliomas and other tumors that are known to express L1 and Neuropilins. However, effective strategies for some types of cancer can promote cancer progression in other types. For example, Sema3A has been shown to contribute to the progression of pancreatic cancer²⁹ and colon cancer.³⁰ Thus, it is imperative that the molecular mechanisms underlying L1-mediated signaling are understood in a tissue specific manner. Despite the promising advances in cancer basic research, much more research is needed to better design strategies for cancer therapy.     

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.     

Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination

The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function‐triggering L1 antibody leads to cathepsin E‐mediated generation of a sumoylated 30 kDa L1 fragment (L1‐30) and to import of L1‐30 into the nucleus. Mutation of the sumoylation site at K₁₁₇₂ or the cathepsin E cleavage site at E₁₁₆₇ abolishes generation of L1‐30, while mutation of the nuclear localization signal at K₁₁₄₇ prevents nuclear import of L1‐30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1‐30 and inhibits L1‐induced migration of cerebellar neurons and Schwann cells as well as L1‐dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1‐stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non‐mutated L1. In addition, L1‐stimulated migration of HEK293 cells expressing non‐mutated L1 is also abolished upon knock‐down of cathepsin E expression and enhanced by over‐expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1‐30 regulate neuronal and Schwann cell migration as well as myelination.

     

Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth

Although mutations in the human doublecortin gene (DCX) cause profound defects in cortical neuronal migration, a genetic deletion of Dcx in mice produces a milder defect. A second locus, doublecortin-like kinase (Dclk), encodes a protein with similar "doublecortin domains" and microtubule stabilization properties that may compensate for Dcx. Here, we generate a mouse with a Dclk mutation that causes no obvious migrational abnormalities but show that mice mutant for both Dcx and Dclk demonstrate perinatal lethality, disorganized neocortical layering, and profound hippocampal cytoarchitectural disorganization. Surprisingly, Dcx(-/y);Dclk(-/-) mutants have widespread axonal defects, affecting the corpus callosum, anterior commissure, subcortical fiber tracts, and internal capsule. Dcx/Dclk-deficient dissociated neurons show abnormal axon outgrowth and dendritic structure, with defects in axonal transport of synaptic vesicle proteins. Dcx and Dclk may directly or indirectly regulate microtubule-based vesicle transport, a process critical to both neuronal migration and axon outgrowth.     

Substrate-bound beta-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures

Accumulation of the beta-amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism of Abeta toxicity remains unclear. Abeta can bind to the extracellular matrix, a structure that regulates adhesive events such as neurite outgrowth and synaptogenesis. The binding of Abeta to the extracellular matrix suggests that Abeta may disrupt cell-substrate interactions. Therefore, the effect of substrate-bound Abeta on the growth of isolated chick sympathetic and mouse cortical neurons was examined. Abeta1-40 and Abeta1-42 had dose-dependent effects on cell morphology. When tissue culture plates were coated with 0.1-10 ng/well Abeta, neurite outgrowth increased. Higher amounts of Abeta peptides (> or =3 microg/well) inhibited outgrowth. The inhibitory effect was related to aggregation of the peptide, as preincubation of Abeta1-40 for 24 h at 37 degrees C (a process known to increase amyloid fibril formation) was necessary for inhibition of neurite outgrowth. Abeta29-42, but not Abeta1-28, also inhibited neurite outgrowth at high concentrations, demonstrating that the inhibitory domain is located within the hydrophobic C-terminal region. Abeta1-40, Abeta1-42, and Abeta29-42 also inhibited cell-substrate adhesion, indicating that the effect on neurite outgrowth may have been due to inhibition of cell adhesion. The results suggest that accumulation of Abeta may disrupt cell-adhesion mechanisms in vivo.     

MAP1B is required for Netrin 1 signaling in neuronal migration and axonal guidance

BACKGROUND: The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. RESULTS: Here we show that Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. We also show that map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. CONCLUSIONS: These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Cell adhesion molecules in the early developing mouse retina: Retinal neurons show preferential outgrowth in vitro on L1 but not N-CAM

Both L1 and N-CAM are present on optic axons early in the developing mouse retina and optic nerve. In in vitro assays on substrates of purified cell adhesion molecules cells derived from E13 mouse retinae showed vigorous neurite extension on L1 but not on N-CAM. Although retinal neurons on N-CAM showed only limited attachment to the substrate, they were able to form lamellipodia immediately around the cell perimeter. In contrast, similarly derived cortical cells showed extensive neurite outgrowth on both substrates. Under these culture conditions, nearly all of the L1 and N-CAM present in the cell membrane appeared to be sequestered on the lower surface of the growth cones and neurites, indicating that most of these cell adhesion molecules were involved in homophilic interactions. Our results suggest differential roles for L1 and N-CAM in intitiation and establishment of the optic pathway. © 1994 John Wiley & Sons, Inc.     

MEKK4 signaling regulates filamin expression and neuronal migration

Periventricular heterotopia (PVH) is a congenital malformation of human cerebral cortex frequently associated with Filamin-A (FLN-A) mutations but the pathogenetic mechanisms remain unclear. Here, we show that the MEKK4 (MAP3K4) pathway is involved in Fln-A regulation and PVH formation. MEKK4(-/-) mice developed PVH associated with breaches in the neuroependymal lining which were largely comprised of neurons that failed to reach the cortical plate. RNA interference (RNAi) targeting MEKK4 also impaired neuronal migration. Expression of Fln was elevated in MEKK4(-/-) forebrain, most notably near sites of failed neuronal migration. Importantly, recombinant MKK4 protein precipitated a complex containing MEKK4 and Fln-A, and MKK4 mediated signaling between MEKK4 and Fln-A, suggesting that MKK4 may bridge these molecules during development. Finally, we showed that wild-type FLN-A overexpression inhibited neuronal migration. Collectively, our results demonstrate a link between MEKK4 and Fln-A that impacts neuronal migration initiation and provides insight into the pathogenesis of human PVH.     

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein- free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD- dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell- matrix interactions during pattern formation or tumor progression.      

Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti- adhesive

Regulation by the extracellular matrix (ECM) of migration, motility, and adhesion of olfactory neurons and their precursors was studied in vitro. Neuronal cells of the embryonic olfactory epithelium (OE), which undergo extensive migration in the central nervous system during normal development, were shown to be highly migratory in culture as well. Migration of OE neuronal cells was strongly dependent on substratum- bound ECM molecules, being specifically stimulated and guided by laminin (or the laminin-related molecule merosin) in preference to fibronectin, type I collagen, or type IV collagen. Motility of OE neuronal cells, examined by time-lapse video microscopy, was high on laminin-containing substrata, but negligible on fibronectin substrata. Quantitative assays of adhesion of OE neuronal cells to substrata treated with different ECM molecules demonstrated no correlation, either positive or negative, between the migratory preferences of cells and the strength of cell-substratum adhesion. Moreover, measurements of cell adhesion to substrata containing combinations of ECM proteins revealed that laminin and merosin are anti-adhesive for OE neuronal cells, i.e., cause these cells to adhere poorly to substrata that would otherwise be strongly adhesive. The evidence suggests that the anti- adhesive effect of laminin is not the result of interactions between laminin and other ECM molecules, but rather an effect of laminin on cells, which alters the way in which cells adhere. Consistent with this view, laminin was found to interfere strongly with the formation of focal contacts by OE neuronal cells.