Extracellular vesicles from UTX-knockout endothelial cells boost neural stem cell differentiation in spinal cord injury.

BACKGROUND: Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI. METHODS: Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model. RESULTS: In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation. CONCLUSIONS: In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI. Video Abstract.

Viral delivery of L1CAM promotes axonal extensions by embryonic cerebral grafts in mouse brain.

Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy.

Effects of neuronal cell adhesion molecule L1 and nanoparticle surface modification on microglia.

Microelectrode arrays for neural recording suffer from low yield and stability partly due to the inflammatory host responses. A neuronal cell adhesion molecule L1 coating has been shown to promote electrode-neuron integration, reduce microglia activation and improve recording. Coupling L1 to surface via a nanoparticle (NP) base layer further increased the protein surface density and stability. However, the exact L1-microglia interaction in these coatings has not been studied. Here we cultured primary microglia on L1 modified surfaces (with and without NP) and characterized microglia activation upon phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) stimulation. Results showed L1 coatings reduced microglia's superoxide production in response to PMA and presented intrinsic antioxidant properties. Meanwhile, L1 decreased iNOS, NO, and pro-inflammatory cytokines (TNF alpha, IL-6, IL-1 beta), while increased anti-inflammatory cytokines (TGF beta 1, IL-10) in LPS stimulated microglia. Furthermore, L1 increased Arg-1 expression and phagocytosis upon LPS stimulation. Rougher NP surface showed lower number of microglia attached per area than their smooth counterpart, lower IL-6 release and superoxide production, and higher intrinsic reducing potential. Finally, we examined the effect of L1 and nanoparticle modifications on microglia response in vivo over 8 weeks with 2-photon imaging. Microglial coverage on the implant surface was found to be lower on the L1 modified substrates relative to unmodified, consistent with the in vitro observation. Our results indicate L1 significantly reduces superoxide production and inflammatory response of microglia and promotes wound healing, while L1 immobilization via a nanoparticle base layer brings added benefit without adverse effects. STATEMENT OF SIGNIFICANCE: Surface modification of microelectrode arrays with L1 has been shown to reduce microglia coverage on neural probe surface in vivo and improves neural recording, but the specific mechanism of action is not fully understood. The results in this study show that surface bound L1 reduces superoxide production from cultured microglia via direct reduction reaction and signaling pathways, increases anti-inflammatory cytokine release and phagocytosis in response to PMA or LPS stimulation. Additionally, roughening the surface with nanoparticles prior to L1 immobilization further increased the benefit of L1 in reducing microglia activation and oxidative stress. Together, our findings shed light on the mechanisms of action of nanotextured and neuroadhesive neural implant coatings and guide future development of seamless tissue interface.

PSA mimetic 5-nonyloxytryptamine protects cerebellar neurons against glutamate induced excitotoxicity: An in vitro perspective.

PSA-NCAM is a molecule of therapeutic interest for its key role in promoting neuritogenesis and synaptic plasticity. The current study was aimed to investigate the neuroregenerative potential of 5-nonyloxytryptamine (5-NOT) as PSA mimetic compound against glutamate induced excitotoxicity. 2D and 3D cultures of cerebellar neurons challenged with glutamate were used to ascertain the effect of 5-NOT on neurite outgrowth, migration and expression of neuronal plasticity markers. Glutamate excitotoxicity is one of the major underlying pathological factor responsible for neurodegeneration in various neurological disorders such as trauma, stroke, ischemia, epilepsy and neurodegenerative diseases.5-NOT treatment was observed to promote axonal growth and defasiculation in glutamate challenged neurons as well as promoted the migration of cerebellar neurons in both wound scratched area and cerebellar explant cultures. Further, 5-NOT treatment upregulated the expression of synaptic plasticity and cell survival pathway proteins which showed reduced expression after glutamate induced excitotoxicity. Thus, this preliminary data reveals thatPSA-mimetic,5-NOT may prove to be a potential neuroprotective candidate for neurodegenerative diseases.

NCAM2 Fibronectin type-III domains form a rigid structure that binds and activates the Fibroblast Growth Factor Receptor.

NCAM1 and NCAM2 have ectodomains consisting of 5 Ig domains followed by 2 membrane-proximal FnIII domains. In this study we investigate and compare the structures and functions of these FnIII domains. The NCAM1 and -2 FnIII2 domains both contain a Walker A motif. In NCAM1 binding of ATP to this motif interferes with NCAM1 binding to FGFR. We obtained a structural model of the NCAM2 FnIII2 domain by NMR spectroscopy, and by titration with an ATP analogue we show that the NCAM2 Walker A motif does not bind ATP. Small angle X-ray scattering (SAXS) data revealed that the NCAM2 FnIII1-2 double domain exhibits a very low degree of flexibility. Moreover, recombinant NCAM2 FnIII domains bind FGFR in vitro, and the FnIII1-2 double domain induces neurite outgrowth in a concentration-dependent manner through activation of FGFR. Several synthetic NCAM1-derived peptides induce neurite outgrowth via FGFR. Only 2 of 5 peptides derived from similar regions in NCAM2 induce neurite outgrowth, but the most potent of these peptides stimulates neurite outgrowth through FGFR-dependent activation of the Ras-MAPK pathway. These results reveal that the NCAM2 FnIII domains form a rigid structure that binds and activates FGFR in a manner related to, but different from NCAM1.

L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways.

L1 cell adhesion molecule (L1CAM) is highly expressed in various types of cancer cells and has been implicated in the control of cell proliferation and motility. Recently, L1CAM was reported to induce the motility of melanoma cells, but the mechanism of this induction remains poorly understood. In this study, we investigated the molecular mechanisms by which L1CAM induces the motility of melanoma cells. Unlike other types of cancer cells, B16F10 melanoma cells highly expressed L1CAM at both the RNA and protein levels, and the expression of L1CAM induced AP-1 activity. In accordance to AP-1 activation, MAPK signaling pathways were activated by L1CAM. Inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38. However, no significant change was observed in JNK activation. As expected, upstream MAP2K, MKK3/6, MAP3K, and TAK1 were also deactivated by the inhibition of L1CAM expression. L1CAM induced the motility of B16F10 cells. Inhibition of L1CAM expression suppressed migration and invasion of B16F10 cells, but no suppressive effect was observed on their proliferation and anti-apoptotic resistance. Treatment of B16F10 cells with U0126, an ERK inhibitor, or SB203580, a p38 inhibitor, suppressed the migration and invasion abilities of B16F10 cells. Taken together, our results suggest that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways. This finding provides a more detailed molecular mechanism of L1CAM-mediated induction of melanoma cell motility.

Ethanol disrupts axon outgrowth stimulated by netrin-1, GDNF, and L1 by blocking their convergent activation of Src family kinase signaling.

Pre-natal alcohol exposure causes fetal alcohol spectrum disorders (FASD), the most common, preventable cause of developmental disability. The developing cerebellum is particularly vulnerable to the effects of ethanol. We reported that ethanol inhibits the stimulation of axon outgrowth in cerebellar granule neurons (CGN) by NAP, an active motif of activity-dependent neuroprotective protein (ADNP), by blocking NAP activation of Fyn kinase and its downstream signaling molecule, the scaffolding protein Cas. Here, we asked whether ethanol inhibits the stimulation of axon outgrowth by diverse axon guidance molecules through a common action on the Src family kinases (SFK). We first demonstrated that netrin-1, glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule L1 stimulate axon outgrowth in CGNs by activating SFK, Cas, and extracellular signal-regulated kinase 1 and 2 (ERK1/2). The specific SFK inhibitor, PP2, blocked the stimulation of axon outgrowth and the activation of the SFK-Cas-ERK1/2 signaling pathway by each of these axon-guidance molecules. In contrast, brain-derived neurotrophic factor (BDNF) stimulated axon outgrowth and activated ERK1/2 without first activating SFK or Cas. Clinically relevant concentrations of ethanol inhibited axon outgrowth and the activation of the SFK-Cas-ERK1/2 pathway by netrin-1, GDNF, and L1, but did not disrupt BDNF-induced axon outgrowth or ERK1/2 activation. These results indicate that SFK, but not ERK1/2, is a primary target for ethanol inhibition of axon outgrowth. The ability of ethanol to block the convergent activation of the SFK-Cas-ERK1/2 pathway by netrin-1, GDNF, L1, and ADNP could contribute significantly to the pathogenesis of FASD.

PSA-NCAM-dependent GDNF signaling limits neurodegeneration and epileptogenesis in temporal lobe epilepsy.

Polysialylated neuronal cell adhesion molecule (PSA-NCAM), a polysialylated protein constitutively expressed in the hippocampus, is involved in neuronal growth, synaptic plasticity and neurotrophin signaling. In particular, PSA-NCAM mediates Ret-independent glial-derived neurotrophic factor (GDNF) signaling, leading to downstream FAK activation. GDNF has potent seizure-suppressant action, whereas PSA-NCAM is upregulated by seizure activity. However, the involvement of Ret-independent GDNF signaling in temporal lobe epilepsy (TLE) is not established. We tested the effects of PSA-NCAM inactivation on neurodegeneration and epileptogenesis in a mouse model of TLE. In this model, unilateral intrahippocampal kainic acid (KA) injection induced degeneration of CA1, CA3c and hilar neurons, followed by spontaneous recurrent focal seizures. In the contralateral, morphologically preserved hippocampus, a long-lasting increase of PSA-NCAM immunoreactivity was observed. Inactivation of PSA-NCAM by endoneuraminidase (EndoN) administration into the contralateral ventricle of KA-treated mice caused severe degeneration of CA3a,b neurons and dentate gyrus granule cells in the epileptic focus, and led to early onset of focal seizures. This striking trans-hemispheric alteration suggested that PSA-NCAM mediates GDNF signaling, leading to transport of neuroprotective signals into the lesioned hippocampus. This hypothesis was confirmed by injecting GDNF antibodies into the contralateral hippocampus of KA-treated mice, thereby reproducing the enhanced neurodegeneration seen after PSA-NCAM inactivation. Furthermore, contralateral EndoN and anti-GDNF treatment decreased GDNF family receptor alpha1 immunoreactivity and FAK phosphorylation in the epileptic focus. Thus, Ret-independent GDNF signaling across the commissural projection might protect CA3a,b neurons and delay seizure onset. These findings implicate GDNF in the control of epileptogenesis and offer a possible mechanism explaining lesion asymmetry in mesial TLE.

Balanced Vav2 GEF activity regulates neurite outgrowth and branching in vitro and in vivo.

We have investigated the role of Vav2, a reported Rac1/Cdc42 GEF, on the development of Xenopus spinal neurons in vitro and in vivo. Both gain and loss of Vav2 function inhibited the rate neurite extension on laminin (LN), while only GFP-Vav2 over-expression enhanced process formation and branching. Vav2 over-expression protected neurons from RhoA-mediated growth cone collapse, similar to constitutively active Rac1, suggesting that Vav2 activates Rac1 in spinal neurons. Enhanced branching on LN required both Vav2 GEF activity and N-terminal tyrosine residues, but protection from RhoA-mediated collapse only required GEF activity. Interestingly, wild-type spinal neurons exhibited increased branching on the cell adhesion molecule L1, which required Vav2 GEF function, but not N-terminal tyrosine residues. Finally, we find that Vav2 differentially affects the Rohon-Beard peripheral and central process extension but promotes neurite branching of commissural interneurons near the ventral midline. Together, we suggest that balanced Vav2 activity is necessary for optimal neurite outgrowth and promotes branching by targeting GEF activity to branch points.

The cell adhesion molecule L1 controls growth cone navigation via ankyrin(B)-dependent modulation of cyclic AMP.

During development, asymmetric Ca(2+) signals across the growth cone mediate bidirectional axon guidance depending on intracellular levels of cyclic AMP: Ca(2+) signals trigger attractive or repulsive turning when cyclic AMP levels are high or low, respectively. Here, we report that the cell adhesion molecule L1 elevates cyclic AMP levels in neurons via ankyrin(B), a protein that links the L1 cytoplasmic tail with the spectrin network. We also show that the loss of ankyrin(B) expression converts Ca(2+)-triggered attraction to repulsion when the growth cone migrates via an L1-dependent mechanism. These results indicate that ankyrin(B) regulates axon guidance via cyclic AMP.

Adeno-associated virus-mediated L1 expression promotes functional recovery after spinal cord injury.

Paucity of permissive molecules and abundance of inhibitory molecules in the injured spinal cord of adult mammals prevent axons from successful regeneration and, thus, contribute to the failure of functional recovery. Using an adeno-associated viral (AAV) vector, we expressed the regeneration-promoting cell adhesion molecule L1 in both neurons and glia in the lesioned spinal cord of adult mice. Exogenous L1, detectable already 1 week after thoracic spinal cord compression and immediate vector injection, was expressed at high levels up to 5 weeks, the longest time-period studied. Dissemination of L1-transduced cells throughout the spinal cord was wide, spanning over more than 10 mm rostral and 10 mm caudal to the lesion scar. L1 was not detectable in the fibronectin-positive lesion core. L1 overexpression led to improved stepping abilities and muscle coordination during ground locomotion over a 5-week observation period. Superior functional improvement was associated with enhanced reinnervation of the lumbar spinal cord by 5-HT axons. Corticospinal tract axons did not regrow beyond the lesion scar but extended distally into closer proximity to the injury site in AAV-L1-treated compared with control mice. The expression of the neurite outgrowth-inhibitory chondroitin sulphate proteoglycan NG2 was decreased in AAV-L1-treated spinal cords, along with reduction of the reactive astroglial marker GFAP. In vitro experiments confirmed that L1 inhibits astrocyte proliferation, migration, process extension and GFAP expression. Analyses of intracellular signalling indicated that exogenous L1 activates diverse cascades in neurons and glia. Thus, AAV-mediated L1 overexpression appears to be a potent means to favourably modify the local environment in the injured spinal cord and promote regeneration. Our study demonstrates a clinically feasible approach of promising potential.

Polysialylated neural cell adhesion molecule is involved in induction of long-term potentiation and memory acquisition and consolidation in a fear-conditioning paradigm.

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM-NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

Cell adhesion molecule L1 modulates nerve-growth-factor-induced CGRP-IR fiber sprouting.

Overexpression of nerve growth factor (NGF) using adenoviruses (Adts) after spinal cord injury induces extensive regeneration and sprouting of calcitonin-gene-related peptide immunoreactive (CGRP-IR) fibers, whereas overexpression of cell adhesion molecules (CAMs) has no effect on the normal distribution of these fibers. Interestingly, co-expression of cell adhesion molecule L1 and NGF significantly decreases (p<0.0001) CGRP-IR fiber sprouting within the spinal cord, when compared to NGF alone. Co-expression of cell adhesion molecules NCAM or N-cadherin had no effect on NGF-induced CGRP-IR fiber sprouting. These data demonstrate that reduced sprouting is specific to L1 co-expression and not other cell adhesion molecules. In vitro studies carried out to address potential mechanisms show that neurite outgrowth over astrocytes overexpressing L1 in the presence of NGF is comparable to controls, indicating that other factors present in vivo might be involved in the L1-mediated reduction in sprouting. One potential factor is semaphorin 3A (sema3A), which mediates growth cone collapse of CGRP-positive axons. Recent studies have shown that L1 is important in sema3A receptor signaling for cortical neurons. In our study, co-expression of sema3A indeed reduces neurite outgrowth from DRG neurons by about 40% on L1-expressing astrocytes. Based on these results, we hypothesize that overexpression of L1 potentiates sema3A signaling resulting in reduced sprouting.

Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

Neurite extension and in vitro myelination within three-dimensional modified fibrin matrices.

The deposition of fibrin clots in vivo occurs after injury in the peripheral nervous system and their removal correlates with nerve regeneration. Fibrin clots provide a provisional matrix for invading cells, induce wound healing, and become proteolytically removed by regenerating tissue. Here, neurite extension and in vitro myelination were studied within three-dimensional fibrin matrices that were covalently modified with the sixth Ig-like domain of cell adhesion molecules L1 containing N-terminal transglutaminase substrate sequences (TG-L1Ig6) for covalent incorporation into fibrin matrices. TG-L1Ig6 is a specific receptor for alphavbeta3-integrin involved in neurite extension of PC12 cells and dorsal root ganglion neurons (DRGs). Neurite extension of PC12 cells depended on interactions between cell surface alphavbeta3 and RGD-sites provided by TG-L1Ig6. In addition, matrix properties such as fibrin crosslink density and matrix degradation by serine proteases were crucial. No involvement of matrix metalloproteinases was found. DRG neurite extension in native fibrin matrices was retarded as compared to neurite extension within L1Ig6-modified and laminin-1-containing matrices. Moreover, myelinated structures were almost exclusively found in TG-L1Ig6-modified and laminin-1-containing matrices. These results indicate that potential use of three-dimensional matrices in a biomaterials-based healing device to induce and/or help in vivo nerve regeneration requires specific structural and biological signals.

Heterophilic interactions between cell adhesion molecule L1 and alphavbeta3-integrin induce HUVEC process extension in vitro and angiogenesis in vivo.

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with alpha(v)beta3 to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab alpha(v)beta3 or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface alpha(v)beta3 and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab alpha(v)beta3 or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with alpha(v)beta3 suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of alpha(v)beta3 and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of alpha(v) and alpha(v)beta3 followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50-60% increased alpha(v) and alpha(v)beta3 protein expression and in vivo angiogenesis indicated by approximately 50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of alpha(v)beta3.

Adhesion molecule L1 stimulates neuronal migration through Vav2-Pak1 signaling.

Organized neuronal migration and guided axon outgrowth are key determinants of the development of the functional nervous system. L1, a member of the Ig superfamily of cell surface receptors, stimulates cell migration and neurite outgrowth through the MAP kinases ERK1, 2. The signaling molecules participating in this signaling cascade have only partly been identified. Here it is shown that L1 clustering activates the guanine nucleotide exchange factor (GEF) Vav2 and the Rac1 effector p21 associated kinase 1 (Pak1). Also, we found that Pak1 kinase activity contributes to ERK activation by L1, and is necessary for L1-potentiated haptotactic cell migration. A signaling pathway is proposed from L1 through Vav2, Rac1, Pak1 and ERK that may be important for L1 mediated neuronal cell migration.

Promotion of neurite and filopodium formation by CD47: roles of integrins, Rac, and Cdc42.

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1-Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1-Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin beta3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the beta3 subunit participate in the effect of CD47 on neurite formation.

L1/Laminin modulation of growth cone response to EphB triggers growth pauses and regulates the microtubule destabilizing protein SCG10.

During development, EphB proteins serve as axon guidance molecules for retinal ganglion cell axon pathfinding toward the optic nerve head and in midbrain targets. To better understand the mechanisms by which EphB proteins influence retinal growth cone behavior, we investigated how axon responses to EphB were modulated by laminin and L1, two guidance molecules that retinal axons encounter during in vivo pathfinding. Unlike EphB stimulation in the presence of laminin, which triggers typical growth cone collapse, growth cones co-stimulated by L1 did not respond to EphB. Moreover, EphB exposure in the presence of both laminin and L1 resulted in a novel growth cone inhibition manifested as a pause in axon elongation with maintenance of normal growth cone morphology and filopodial activity. Pauses were not associated with loss of growth cone actin but were accompanied by a redistribution of the microtubule cytoskeleton with increased numbers of microtubules extending into filopodia and to the peripheral edge of the growth cone. This phenomenon was accompanied by reduced levels of the growth cone microtubule destabilizing protein SCG10. Antibody blockade of SCG10 function in growth cones resulted in both changes in microtubule distribution and pause responses mirroring those elicited by EphB in the presence of laminin and L1. These results demonstrate that retinal growth cone responsiveness to EphB is regulated by co-impinging signals from other axon guidance molecules. Furthermore, the results are consistent with EphB-mediated axon guidance mechanisms that involve the SCG10-mediated regulation of the growth cone microtubule cytoskeleton.

ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles.

Cells can release membrane components in a soluble form and as membrane vesicles. L1, an important molecule for cell migration of neural and tumor cells, is released by membrane-proximal cleavage, and soluble L1 promotes cell migration. Release of L1 is enhanced by shedding inducers such as phorbol ester and pervanadate, but it is also enhanced by depletion of cellular cholesterol with methyl-beta-cyclodextrin (MCD). How such different compounds can induce shedding is presently unknown. We show here that ADAM10 is involved in L1 cleavage, which occurs at the cell surface and in the Golgi apparatus. MCD and pervanadate treatment induced the release of microvesicles containing full-length L1 and the active form of ADAM10. L1 cleavage occurred in isolated vesicles. L1-containing microvesicles could trigger haptotactic cell migration. Only the neural L1 form carrying the RSLE signal for clathrin-dependent endocytosis was recruited and cleaved in vesicles. Phorbol ester treatment activated L1 cleavage predominantly at the cell surface. Our results provide evidence for two pathways of L1 cleavage, based on ADAM10 localization, that can be activated differentially: 1) direct cleavage at the cell surface, and 2) release and cleavage in secretory vesicles most likely derived from the Golgi apparatus. The findings establish a novel role for ADAM10 as a vesicle-based protease.