L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

L1CAM induces perineural invasion of pancreas cancer cells by upregulation of metalloproteinase expression.

Pancreas cancer cells have a tendency to invade along nerves. Such cancerous nerve invasion (CNI) is associated with poor outcome; however, the exact mechanism that drives cancer cells to disseminate along nerves is unknown. Immunohistochemical analysis of human pancreatic ductal adenocarcinoma (PDAC) specimens showed overexpression of the L1 cell adhesion molecule (L1CAM) in cancer cells and in adjacent Schwann cells (SC) in invaded nerves. By modeling the neural microenvironment, we found that L1CAM secreted from SCs acts as a strong chemoattractant to cancer cells, through activation of MAP kinase signaling. L1CAM also upregulated expression of metalloproteinase-2 (MMP-2) and MMP-9 by PDAC cells, through STAT3 activation. Using a transgenic Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, we show that treatment with anti-L1CAM Ab significantly reduces CNI in vivo. We provide evidence of a paracrine response between SCs and cancer cells in the neural niche, which promotes cancer invasion via L1CAM secretion.

Cell Recognition Molecule L1 Regulates Cell Surface Glycosylation to Modulate Cell Survival and Migration.

Background: Cell recognition molecule L1 (L1) plays an important role in cancer cell differentiation, proliferation, migration and survival, but its mechanism remains unclear. Methodology/Principal: Our previous study has demonstrated that L1 enhanced cell survival and migration in neural cells by regulating cell surface glycosylation. In the present study, we show that L1 affected cell migration and survival in CHO (Chinese hamster ovary) cell line by modulation of sialylation and fucosylation at the cell surface via the PI3K (phosphoinositide 3-kinase) and Erk (extracellularsignal-regulated kinase) signaling pathways. Flow cytometry analysis indicated that L1 modulated cell surface sialylation and fucosylation in CHO cells. Activated L1 upregulated the protein expressions of ST6Gal1 (ß-galactoside α-2,6-sialyltransferase 1) and FUT9 (Fucosyltransferase 9) in CHO cells. Furthermore, activated L1 promoted CHO cells migration and survival as shown by transwell assay and MTT assay. Inhibitors of sialylation and fucosylation blocked L1-induced cell migration and survival, while decreasing FUT9 and ST6Gal1 expressions via the PI3K-dependent and Erk-dependent signaling pathways. Conclusion : L1 modulated cell migration and survival by regulation of cell surface sialylation and fucosylation via the PI3K-dependent and Erk-dependent signaling pathways.

Role of L1CAM in the Regulation of the Canonical Wnt Pathway and Class I MAGE Genes.

Molecule L1CAM is specific for nerve cells and tumors of various localizations. The expression of L1CAM is significantly higher in melanoma in comparison with benign nevi and correlates with the progress of melanoma and transition from radial to vertical growth. Monoclonal antibodies to L1CAM effectively and specifically attenuate melanoma growth, though stimulates the epithelial-mesenchymal transition. shRNA-mediated knock-down of L1CAM showed the involvement of L1CAM in regulation of activity of the canonical Wnt pathway and expression of genes of class I melanoma-associated antigens (MAGE).

Enzymatic Depletion of the Polysialic Acid Moiety Associated with the Neural Cell Adhesion Molecule Inhibits Antidepressant Efficacy.

Antidepressant drugs are too often ineffective, the exact mechanism of efficacy is still ambiguous, and there has been a paucity of novel targets for pharmacotherapy. In an attempt to understand the pathogenesis of depression and subsequently develop more efficacious antidepressant drugs, multiple theories have been proposed, including the modulation of neurotransmission, the upregulation of neurogenesis and neurotrophic factors, normalizing hypothalamic-pituitary-adrenal reactivity, and the reduction of neuroinflammation; all of which have supporting lines of evidence. Therefore, an ideal molecular target for novel pharmaceutical intervention would function at the confluence of these theories. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) functions broadly, serving to mediate synaptic plasticity, neurogenesis, neurotrophic factor signaling, and inflammatory signaling throughout the brain; all of which are associated with the pathophysiology and treatment of depression. Moreover, the expression of PSA-NCAM is reduced by depression, and conversely enhanced by antidepressant treatment, particularly within the hippocampus. Here we demonstrate that selectively cleaving the polysialic acid moiety, using the bacteriophage-derived enzyme endoneuraminidase N, completely inhibits the antidepressant efficacy of the selective-serotonin reuptake inhibitor fluoxetine (FLX) in a chronic unpredictable stress model of depression. We also observe a corresponding attenuation of FLX-induced hippocampal neuroplasticity, including decreased hippocampal neurogenesis, synaptic density, and neural activation. These data indicate that PSA-NCAM-mediated neuroplasticity is necessary for antidepressant action; therefore PSA-NCAM represents an interesting, and novel, target for pharmacotherapy.

Neurite outgrowth induced by NGF or L1CAM via activation of the TrkA receptor is sustained also by the exocytosis of enlargeosomes.

NGF binding to its protein kinase receptor TrkA is known to induce neurite outgrowth and neural cell differentiation. The plasma membrane expansion, necessary for the process, was shown to be contributed by the VAMP7-dependent exocytosis of endocytic vesicles. Working with wild-type PC12 (wtPC12), a cell model widely used to investigate NGF-induced neurite outgrowth, we found that a few hours of treatment with the neurotrophin (and to a lower extent with basic FGF and EGF) induces the appearance of enlargeosome vesicles competent for VAMP4-dependent exocytosis abundant in high REST-PC12 clones. Both the neurite length assay and the immunocytochemistry of enlargeosomes exocytosis revealed that activation of TrkA is induced not only by NGF, but also by the L1 adhesion protein, L1CAM, whose soluble construct binds the receptor with submicromolar affinity. In the intact wtPC12, the L1CAM construct induced autophosphorylation and internalization of TrkA followed by the activation of the PI3K, MEK, and PKCγ signaling cascades, analogous to the responses induced by NGF. Down-regulation of either VAMP7 or VAMP4 revealed the coparticipation of the two corresponding vesicles to the outgrowth responses induced by NGF and L1CAM. Finally, mixing experiments of wtPC12 cells rich in TrkA with high REST PC12 cells transfected with L1CAM documented the transactivation of the receptor by the adhesion protein surface-exposed in adjacent cells. In view of the known inhomogeneous surface distribution of both L1CAM and TrkA in various neural cells including neurons, their transcellular binding could be restricted to discrete sites, governing local signaling events distinct from those induced by soluble messengers.

Endothelial deficiency of L1 reduces tumor angiogenesis and promotes vessel normalization.

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

The dendritic spines of interneurons are dynamic structures influenced by PSA-NCAM expression.

Excitatory neurons undergo dendritic spine remodeling in response to different stimuli. However, there is scarce information about this type of plasticity in interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate to mediate this plasticity as it participates in neuronal remodeling and is expressed by some mature cortical interneurons, which have reduced dendritic arborization, spine density, and synaptic input. To study the connectivity of the dendritic spines of interneurons and the influence of PSA-NCAM on their dynamics, we have analyzed these structures in a subpopulation of fluorescent spiny interneurons in the hippocampus of glutamic acid decarboxylase-enhanced green fluorescent protein transgenic mice. Our results show that these spines receive excitatory synapses. The depletion of PSA in vivo using the enzyme Endo-Neuraminidase-N (Endo-N) increases spine density when analyzed 2 days after, but decreases it 7 days after. The dendritic spine turnover was also analyzed in real time using organotypic hippocampal cultures: 24 h after the addition of EndoN, we observed an increase in the apparition rate of spines. These results indicate that dendritic spines are important structures in the control of the synaptic input of hippocampal interneurons and suggest that PSA-NCAM is relevant in the regulation of their morphology and connectivity.

The neural plasticity theory of depression: assessing the roles of adult neurogenesis and PSA-NCAM within the hippocampus.

Depression is a devastating and prevalent disease, with profound effects on neural structure and function; however the etiology and neuropathology of depression remain poorly understood. Though antidepressant drugs exist, they are not ideal, as only a segment of patients are effectively treated, therapeutic onset is delayed, and the exact mechanism of these drugs remains to be elucidated. Several theories of depression do exist, including modulation of monoaminergic neurotransmission, alterations in neurotrophic factors, and the upregulation of adult hippocampal neurogenesis, and are briefly mentioned in the review. However none of these theories sufficiently explains the pathology and treatment of depression unto itself. Recently, neural plasticity theories of depression have postulated that multiple aspects of brain plasticity, beyond neurogenesis, may bridge the prevailing theories. The term "neural plasticity" encompasses an array of mechanisms, from the birth, survival, migration, and integration of new neurons to neurite outgrowth, synaptogenesis, and the modulation of mature synapses. This review critically assesses the role of adult hippocampal neurogenesis and the cell adhesion molecule, PSA-NCAM (which is known to be involved in many facets of neural plasticity), in depression and antidepressant treatment.

Photoperiodic variations of the polysialylated form of neural cell adhesion molecule within the hypothalamus and related reproductive output in the ewe.

Cellular mechanisms induced by melatonin to synchronise seasonal reproduction in several species, including sheep, remain unclear. We sought to evaluate the scale and physiological significance of neural plasticity in order to explain the delay between the change of duration of melatonin secretion and the change of reproductive status following a transition from long days (LD, 16 h light/24 h) to short days (SD, 8 h light/24 h) and from SD to LD. Using Western blots in ovariectomised oestradiol-replaced ewes, we evaluated the content of the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a plasticity marker, in the hypothalamus. From day 15 following a transition to SD, most hypothalamic areas showed a decrease of PSA-NCAM level that was particularly significant in the preoptic area (POA). Following a transition to LD, PSA-NCAM content increased at day 15 in most regions except in the premammillary hypothalamic area (PMH) in which a significant decrease was noted. The functional importance of PSA-NCAM variations for seasonal reproduction was assessed for the PMH and POA. PSA-NCAM was degraded by stereotaxic injections of endoneuraminidase N and luteinising hormone (LH) secretion was recorded in treated and control ewes. Degradation of PSA-NCAM in the PMH in SD-treated ewes failed to produce a significant effect on LH secretion, whereas a similar treatment in the POA before a transition to SD delayed activation of the gonadotroph axis in two-thirds of the ewes. Our results suggest that the photoperiod controls variations of the hypothalamic content of a plasticity marker and that these might be important for the regulation of seasonal reproduction, particularly in the POA.

L1CAM and its cell-surface mutants: new mechanisms and effects relevant to the physiology and pathology of neural cells.

The L1 syndrome, a genetic disease that affects 1/30 000 newborn males, is sustained by numerous missense mutations of L1 cell adhesion molecule (L1CAM), an adhesion surface protein active also in transmembrane signaling, essential for the development and function of neurons. To investigate the cell biology of L1CAM, we employed a high RE1-silencing transcription (factor) clone of the pheochromocytoma PC12 line, defective in L1CAM expression and neurite outgrowth. The clone was transfected with wild-type L1CAM and four missense, disease-inducing point mutants encoding proteins distributed to the cell surface. The mutant-expressing cells, defective in adhesion to extracellular matrix proteins and in migration, exhibited unchanged proliferation. The nerve growth factor (NGF)-induced neurite outgrowth was re-established in defective clone cells transfected with the wild-type and the H210Q and I219T L1CAMs mutants, but not in the others. The stimulated outgrowth was confirmed in a second defective PC12 clone over-expressing the NGF receptor TrkA, treated with NGF and/or a recombinant L1CAM chimera. These results revealed a new function of L1CAM, a positive, robust and dose-dependent modulation of the TrkA receptor activated spontaneously or by NGF. The variable effects observed with the different L1CAM mutants suggest that this function contributes to the marked heterogeneity of symptoms and severity observed in the patients affected by the L1 syndrome.

The neural adhesion molecule L1CAM confers chemoresistance in human glioblastomas.

Glioblastoma multiforme (GBM) represents the most common and malignant brain tumor. GBM tissues exhibit elevated expression of the transforming growth factor-beta1 (TGF-ß1) and the adhesion molecule L1CAM. This study investigated the mechanism of L1CAM regulation in GBM cells and its role in the mediation of chemoresistance. L1CAM expression levels varied in GBM cells being highest in A172 cells and low in T98G cells. Inhibition of TGF-ß1 signaling in A172 cells reduced L1CAM expression and vice versa stimulation with exogenous TGF-ß1 led to upregulation of L1CAM in T98G cells. Additionally, TGF-ß1 and L1CAM expression increased during differentiation of glioma stem-like cells. L1CAM expressing GBM cells and differentiated glioma stem-like cells showed a reduced apoptotic response after treatment with the chemotherapeutic drug temozolomide. Accordingly, siRNA-mediated knock-down of L1CAM in A172 cells and differentiated glioma stem-like cells increased chemosensitivity, whereas overexpression of L1CAM in T98G cells and glioma spheroids diminished the apoptotic response. Elevated L1CAM expression caused a diminished expression of caspase-8 in GBM and differentiated glioma stem-like cells. These data show that TGF-ß1 dependent upregulation of L1CAM expression in GBM cells leads to the downregulation of caspase-8 and apoptosis resistance pointing to L1CAM as potential target for improved therapy of GBM patients.

Low prefrontal PSA-NCAM confers risk for alcoholism-related behavior.

The factors underlying vulnerability to alcoholism are largely unknown. We identified in rodents an innate endophenotype predicting individual risk for alcohol-related behaviors that was associated with decreased expression of the neuroplasticity-related polysialylated neural cell adhesion molecule (PSA-NCAM). Depletion of PSA-NCAM in the ventromedial prefrontal cortex was sufficient to render mice unable to extinguish alcohol seeking, indicating a causal role of naturally occurring variation. These data suggest a mechanism of aberrant prefrontal neuroplasticity that underlies enhanced propensity for inflexible addiction-related behavior.

L1CAM: a major driver for tumor cell invasion and motility.

The L1 cell adhesion molecule (L1CAM) plays a major role in the development of the nervous system and in the malignancy of human tumors. In terms of biological function, L1CAM comes along in two different flavors: (1) a static function as a cell adhesion molecule that acts as a glue between cells; (2) a motility promoting function that drives cell migration during neural development and supports metastasis of human cancers. Important factors that contribute to the switch in the functional mode of L1CAM are: (1) the cleavage from the cell surface by membrane proximal proteolysis and (2) the ability to change binding partners and engage in L1CAM-integrin binding. Recent studies have shown that the cleavage of L1CAM by metalloproteinases and the binding of L1CAM to integrins via its RGD-motif in the sixth Ig-domain activate signaling pathways distinct from the ones elicited by homophilic binding. Here we highlight important features of L1CAM proteolysis and the signaling of L1CAM via integrin engagement. The novel insights into L1CAM downstream signaling and its regulation during tumor progression and epithelial-mesenchymal transition (EMT) will lead to a better understanding of the dualistic role of L1CAM as a cell adhesion and/or motility promoting cell surface molecule.

Myofibroblast-induced tumorigenicity of pancreatic ductal epithelial cells is L1CAM dependent.

Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC, are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs, H6c7 cells exhibit a profound resistance against death ligand-induced apoptosis. This apoptosis protection was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7 cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 severe combined immunodeficiency (SCID) mice, whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor outgrowth and metastases resulted from inoculation of L1CAM-overexpressing H6c7 cells in 5/7 and 3/7 SCID mice, respectively, but not from inoculation of mock-transfected H6c7 cells. Treatment of H6c7co tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in 100 and 50%, respectively, of the treated animals. Overall, these data provide new insights into the mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal epithelial cells in early stages of pancreatic tumorigenesis.

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

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

Polysialic acid is required for dopamine D2 receptor-mediated plasticity involving inhibitory circuits of the rat medial prefrontal cortex.

Decreased expression of dopamine D2 receptors (D2R), dysfunction of inhibitory neurotransmission and impairments in the structure and connectivity of neurons in the medial prefrontal cortex (mPFC) are involved in the pathogenesis of schizophrenia and major depression, but the relationship between these changes remains unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-related molecule, may serve as a link. This molecule is expressed in cortical interneurons and dopamine, via D2R, modulates its expression in parallel to that of proteins related to synapses and inhibitory neurotransmission, suggesting that D2R-targeted antipsychotics/antidepressants may act by affecting the plasticity of mPFC inhibitory circuits. To understand the role of PSA-NCAM in this plasticity, rats were chronically treated with a D2R agonist (PPHT) after cortical PSA depletion. PPHT-induced increases in GAD67 and synaptophysin (SYN) neuropil expression were blocked when PSA was previously removed, indicating a role for PSA-NCAM in this plasticity. The number of PSA-NCAM expressing interneuron somata also increased after PPHT treatment, but the percentages of these cells belonging to different interneuronal subpopulations did not change. Cortical pyramidal neurons did not express PSA-NCAM, but puncta co-expressing this molecule and parvalbumin could be found surrounding their somata. PPHT treatment increased the number of PSA-NCAM and parvalbumin expressing perisomatic puncta, but decreased the percentage of parvalbumin puncta that co-expressed SYN. PSA depletion did not block these effects on the perisomatic region, but increased further the number of parvalbumin expressing puncta and increased the percentage of puncta co-expressing SYN and parvalbumin, suggesting that the polysialylation of NCAM may regulate perisomatic inhibition of mPFC principal neurons. Summarizing, the present results indicate that dopamine acting on D2R influences structural plasticity of mPFC interneurons and point to PSA-NCAM as a key player in this remodeling.

ALCAM regulates mediolateral retinotopic mapping in the superior colliculus.

ALCAM [activated leukocyte cell adhesion molecule (BEN/SC-1/DM-GRASP)] is a transmembrane recognition molecule of the Ig superfamily (IgSF) containing five Ig domains (two V-type, three C2-type). Although broadly expressed in the nervous and immune systems, few of its developmental functions have been elucidated. Because ALCAM has been suggested to interact with the IgSF adhesion molecule L1, a determinant of retinocollicular mapping, we hypothesized that ALCAM might direct topographic targeting to the superior colliculus (SC) by serving as a substrate within the SC for L1 on incoming retinal ganglion cell (RGC) axons. ALCAM was expressed in the SC during RGC axon targeting and on RGC axons as they formed the optic nerve; however, it was downregulated distally on RGC axons as they entered the SC. Axon tracing with DiI revealed pronounced mistargeting of RGC axons from the temporal retina half of ALCAM null mice to abnormally lateral sites in the contralateral SC, in which these axons formed multiple ectopic termination zones. ALCAM null mutant axons were specifically compromised in medial orientation of interstitial branches, which is known to require the ankyrin binding function of L1. As a substrate, ALCAM-Fc protein promoted L1-dependent attachment of acutely dissociated retinal cells and an L1-expressing, ALCAM-negative cell line, consistent with an ALCAM-L1 heterophilic molecular interaction. Together, these results suggest a model in which ALCAM in the SC interacts with L1 on RGC axons to promote medial extension of RGC axon branches important for mediolateral axon targeting in the formation of retinocollicular maps.

Cross-talk between stem cells and the dysfunctional brain is facilitated by manipulating the niche: evidence from an adhesion molecule.

In the injured brain, the behavior of neural stem/progenitor cells (NSCs) is regulated by multiple converging factors encountered in the niche, which is composed of several neural and non-neural cell types. Signals emanating from the host influence the migration, survival, distribution, and fate of transplanted NSCs, which in turn can create host microenvironments that favor a return to homeostasis. We tested the hypothesis that overexpression of key facilitatory molecules that define the injury niche might enhance this bidirectional stem cell-host interaction to therapeutic advantage. As proof of concept, we investigated whether conditioning the niche with the neural cell adhesion molecule L1 might enhance recovery in a prototypical neurodegenerative milieu-the MPTP-induced model of Parkinson's disease in aged mice-where cross-talk between NSCs and imperiled host dopaminergic neurons is known to be pivotal in rescuing the function and connectivity of the latter. In lesioned mice (and in unlesioned controls), we overexpressed L1 in the NSCs to be transplanted into the ventral mesencephalon. Several pairwise experimental combinations were tested based on variations of engrafting L1 overexpressing versus nonoverexpressing NSCs into wild-type (WT) versus L1-overexpressing transgenic mice (specifically L1 transcribed from the GFAP promoter and, hence, overexpressed in host astrocytes). Enrichment for L1-particularly when expressed simultaneously in both donor NSCs and host brain-led to rapid and extensive distribution of exogenous NSCs, which in turn rescued (with an efficacy greater than in nonengineered controls) dysfunctional host dopaminergic nigral neurons, even when grafting was delayed by a month. L1 overexpression by NSCs also enhanced their own differentiation into tyrosine hydroxylase-expressing neurons in both WT and transgenic hosts. Graft-host interactions were thus favored by progressively increasing levels of L1. More broadly, this study supports the view that manipulating components of the niche (such as an adhesion molecule) that facilitate cross-talk between stem cells and the dysfunctional brain may offer new strategies for more efficacious neurotransplantation, particularly when treatment is delayed as in chronic lesions or advanced stages of a neurodegenerative disease.

Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60(src).

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1-mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine-based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60(src) as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60(src) activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60(src).