EphB regulates L1 phosphorylation during retinocollicular mapping.

Interaction of the cell adhesion molecule L1 with the cytoskeletal adaptor ankyrin is essential for topographic mapping of retinal ganglion cell (RGC) axons to synaptic targets in the superior colliculus (SC). Mice mutated in the L1 ankyrin-binding motif (FIGQY(1229)H) display abnormal mapping of RGC axons along the mediolateral axis of the SC, resembling mouse mutant phenotypes in EphB receptor tyrosine kinases. To investigate whether L1 functionally interacts with EphBs, we investigated the role of EphB kinases in phosphorylating L1 using a phospho-specific antibody to the tyrosine phosphorylated FIGQY(1229) motif. EphB2, but not an EphB2 kinase dead mutant, induced tyrosine phosphorylation of L1 at FIGQY(1229) and perturbed ankyrin recruitment to the membrane in L1-transfected HEK293 cells. Src family kinases mediated L1 phosphorylation at FIGQY(1229) by EphB2. Other EphB receptors that regulate medial-lateral retinocollicular mapping, EphB1 and EphB3, also mediated phosphorylation of L1 at FIGQY(1229). Tyrosine(1176) in the cytoplasmic domain of L1, which regulates AP2/clathrin-mediated endocytosis and axonal trafficking, was not phosphorylated by EphB2. Accordingly mutation of Tyr(1176) to Ala in L1-Y(1176)A knock-in mice resulted in normal retinocollicular mapping of ventral RGC axons. Immunostaining of the mouse SC during retinotopic mapping showed that L1 colocalized with phospho-FIGQY in RGC axons in retinorecipient layers. Immunoblotting of SC lysates confirmed that L1 was phosphorylated at FIGQY(1229) in wild type but not L1-FIGQY(1229)H (L1Y(1229)H) mutant SC, and that L1 phosphorylation was decreased in the EphB2/B3 mutant SC. Inhibition of ankyrin binding in L1Y(1229)H mutant RGCs resulted in increased neurite outgrowth compared to WT RGCs in retinal explant cultures, suggesting that L1-ankyrin binding serves to constrain RGC axon growth. These findings are consistent with a model in which EphB kinases phosphorylate L1 at FIGQY(1229) in retinal axons to modulate L1-ankyrin binding important for mediolateral retinocollicular topography.

Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells.

Pyramidal cells express various GABA(A) receptor (GABA(A)R) subtypes, possibly to match inputs from functionally distinct interneurons targeting specific subcellular domains. Postsynaptic anchoring of GABA(A)Rs is ensured by a complex interplay between the scaffolding protein gephyrin, neuroligin-2 and collybistin. Direct interactions between these proteins and GABA(A)R subunits might contribute to synapse-specific distribution of GABA(A)R subtypes. In addition, the dystrophin-glycoprotein complex, mainly localized at perisomatic synapses, regulates GABA(A)R postsynaptic clustering at these sites. Here, we investigated how the functional and molecular organization of GABAergic synapses in CA1 pyramidal neurons is altered in mice lacking the GABA(A)R α2 subunit (α2-KO). We report a marked, layer-specific loss of postsynaptic gephyrin and neuroligin-2 clusters, without changes in GABAergic presynaptic terminals. Whole-cell voltage-clamp recordings in slices from α2-KO mice show a 40% decrease in GABAergic mIPSC frequency, with unchanged amplitude and kinetics. Applying low/high concentrations of zolpidem to discriminate between α1- and α2/α3-GABA(A)Rs demonstrates that residual mIPSCs in α2-KO mice are mediated by α1-GABA(A)Rs. Immunofluorescence analysis reveals maintenance of α1-GABA(A)R and neuroligin-2 clusters, but not gephyrin clusters, in perisomatic synapses of mutant mice, along with a complete loss of these three markers on the axon initial segment. This striking subcellular difference correlates with the preservation of dystrophin clusters, colocalized with neuroligin-2 and α1-GABA(A)Rs on pyramidal cell bodies of mutant mice. Dystrophin was not detected on the axon initial segment in either genotype. Collectively, these findings reveal synapse-specific anchoring of GABA(A)Rs at postsynaptic sites and suggest that the dystrophin-glycoprotein complex contributes to stabilize α1-GABA(A)R and neuroligin-2, but not gephyrin, in perisomatic postsynaptic densities.

L1 interaction with ankyrin regulates mediolateral topography in the retinocollicular projection.

Dynamic modulation of adhesion provided by anchorage of axonal receptors with the cytoskeleton contributes to attractant or repellent responses that guide axons to topographic targets in the brain. The neural cell adhesion molecule L1 engages the spectrin-actin cytoskeleton through reversible linkage of its cytoplasmic domain to ankyrin. To investigate a role for L1 association with the cytoskeleton in topographic guidance of retinal axons to the superior colliculus, a novel mouse strain was generated by genetic knock-in that expresses an L1 point mutation (Tyr1229His) abolishing ankyrin binding. Axon tracing revealed a striking mistargeting of mutant ganglion cell axons from the ventral retina, which express high levels of ephrinB receptors, to abnormally lateral sites in the contralateral superior colliculus, where they formed multiple ectopic arborizations. These axons were compromised in extending interstitial branches in the medial direction, a normal response to the high medial to low lateral SC gradient of ephrinB1. Furthermore, ventral but not dorsal L1(Y1229H) retinal cells were impaired for ephrinB1-stimulated adhesion through beta1 integrins in culture. The retinocollicular phenotype of the L1(Tyr1229His) mutant provides the first evidence that L1 regulates topographic mapping of retinal axons through adhesion mediated by linkage to the actin cytoskeleton and functional interaction with the ephrinB/EphB targeting system.

Antibodies directed against L1-CAM synergize with Genistein in inhibiting growth and survival pathways in SKOV3ip human ovarian cancer cells.

Antibodies directed against the L1 cell adhesion protein inhibit growth of SKOV3ip human ovarian cancer cells in vitro and in vivo. Responses of SKOV3ip cells in vitro to anti-L1 mAb chCE7 and Genistein were investigated. Genistein potentiated the anti-proliferative and pro-apoptotic effects of chCE7 in SKOV3ip cells. A combination of mAb chCE7 and Genistein strongly reduced the sensitivity of p44/42 (Erk1,2) kinase, Src kinase and Akt kinase to extracellular stimulation with serum, Epidermal Growth Factor and Hepatocyte Growth Factor. The observed synergy of antibodies directed against L1 with Genistein could lead to a new therapeutic option for ovarian cancer.

CHL1 promotes Sema3A-induced growth cone collapse and neurite elaboration through a motif required for recruitment of ERM proteins to the plasma membrane.

Close homolog of L1 (CHL1) is a transmembrane cell adhesion molecule with unique developmental functions in cortical neuronal positioning and dendritic projection within the L1 family, as well as shared functions in promotion of integrin-dependent neurite outgrowth and semaphorin3A (Sema3A)-mediated axon repulsion. The molecular mechanisms by which CHL1 mediates these diverse functions are obscure. Here it is demonstrated using a cytofluorescence assay that CHL1 is able to recruit ezrin, a member of the ezrin-radixin-moesin (ERM) family of filamentous actin binding proteins to the plasma membrane, and that this requires a membrane-proximal motif (RGGKYSV) in the CHL1 cytoplasmic domain. This sequence in CHL1 is shown to have novel functions necessary for Sema3A-induced growth cone collapse and CHL1-dependent neurite outgrowth and branching in cortical embryonic neurons. In addition, stimulation of haptotactic cell migration and cellular adhesion to fibronectin by CHL1 depends on the CHL1/ERM recruitment motif. These findings suggest that a direct or indirect interaction between CHL1 and ERM proteins mediates Sema3A-induced growth cone collapse as well as neurite outgrowth and branching, which are essential determinants of axon guidance and connectivity in cortical development.