Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts

Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural cell adhesion molecule (NCAM) in the plasma membrane. These complexes are translocated along neurites and trapped at sites of initial neurite-to-neurite contacts within several minutes after initial contact formation. The accumulation of TGN organelles at contacts with NCAM-deficient neurons is reduced when compared with wild-type cells, suggesting that NCAM mediates the anchoring of intracellular organelles in nascent synapses

The 14-3-3ζ Protein Binds to the Cell Adhesion Molecule L1, Promotes L1 Phosphorylation by CKII and Influences L1-Dependent Neurite Outgrowth

BACKGROUND: The cell adhesion molecule L1 is crucial for mammalian nervous system development. L1 acts as a mediator of signaling events through its intracellular domain, which comprises a putative binding site for 14-3-3 proteins. These regulators of diverse cellular processes are abundant in the brain and preferentially expressed by neurons. In this study, we investigated whether L1 interacts with 14-3-3 proteins, how this interaction is mediated, and whether 14-3-3 proteins influence the function of L1. METHODOLOGY/PRINCIPAL FINDINGS: By immunoprecipitation, we demonstrated that 14-3-3 proteins are associated with L1 in mouse brain. The site of 14-3-3 interaction in the L1 intracellular domain (L1ICD), which was identified by site-directed mutagenesis and direct binding assays, is phosphorylated by casein kinase II (CKII), and CKII phosphorylation of the L1ICD enhances binding of the 14-3-3 zeta isoform (14-3-3ζ). Interestingly, in an in vitro phosphorylation assay, 14-3-3ζ promoted CKII-dependent phosphorylation of the L1ICD. Given that L1 phosphorylation by CKII has been implicated in L1-triggered axonal elongation, we investigated the influence of 14-3-3ζ on L1-dependent neurite outgrowth. We found that expression of a mutated form of 14-3-3ζ, which impairs interactions of 14-3-3ζ with its binding partners, stimulated neurite elongation from cultured rat hippocampal neurons, supporting a functional connection between L1 and 14-3-3ζ. CONCLUSIONS/SIGNIFICANCE: Our results suggest that 14-3-3ζ, a novel direct binding partner of the L1ICD, promotes L1 phosphorylation by CKII in the central nervous system, and regulates neurite outgrowth, an important biological process triggered by L1

Polysialylated neural cell adhesion molecule promotes remodeling and formation of hippocampal synapses

Expression of the neural cell adhesion molecule (NCAM) has been shown to promote long-term potentiation (LTP) and stabilization of synapses during early synaptogenesis. Here, we searched for the mechanisms of synaptogenic activity of NCAM, focusing on the role of polysialic acid (PSA), an unusual carbohydrate preferentially associated with NCAM. We show that enzymatic removal of PSA with endoneuraminidase-N (endo-N) abolished preferential formation of synapses on NCAM-expressing cells in heterogenotypic cocultures of wild-type and NCAM-deficient hippocampal neurons. Transfection of NCAM-deficient neurons with either of three major NCAM isoforms (different in intracellular domains but identical in extracellular domains and carrying PSA) stimulated preferential synapse formation on NCAM isoform-expressing neurons. Enzymatic removal of heparan sulfates from cultured neurons and a mutation in the heparin-binding domain of NCAM diminished synaptogenic activity of neuronally expressed PSA-NCAM, suggesting that interaction of NCAM with heparan sulfate proteoglycans mediates this activity. PSA-NCAM-driven synaptogenesis was also blocked by antagonists to fibroblast growth factor receptor and NMDA subtype of glutamate receptors but not by blockers of non-NMDA glutamate receptors and voltage-dependent Na+ channels. Enzymatic removal of PSA and heparan sulfates also blocked the increase in the number of perforated spine synapses associated with NMDA receptor-dependent LTP in the CA1 region of organotypic hippocampal cultures. Thus, neuronal PSA-NCAM in complex with heparan sulfate proteoglycans promotes synaptogenesis and activity-dependent remodeling of synapses

14-3-3ζ binds to CKII-phosphorylated L1ICD more strongly than to nonphosphorylated L1ICD.

<p><b>A. </b><i>Upper panel</i>: Equal amounts (15 µg each) of recombinant His-tagged L1ICD were incubated in the presence or absence of CKII. Where indicated, 5 µM TBB was included to specifically inhibit CKII. To exclude non-specific effects of DMSO in which TBB was dissolved, one reaction took place in the presence of this solvent. After treatment, a GST-14-3-3ζ pull-down was performed to investigate direct binding of L1ICD to 14-3-3. Pull-down eluates were analyzed by Western blotting (WB) with the 74-5H7 anti-L1 antibody. <i>Lower panel</i>, GST and GST-14-3-3ζ were detected in pull-down eluates by Western blotting (WB) with a GST antibody, confirming that comparable amounts of GST or GST-14-3-3ζ were used in each pull-down. <b>B.</b> CKII phosphorylation of L1ICD enhances its association with 14-3-3ζ. Quantification of the lower L1ICD-immunoreactive band in lane 1 (L1ICD only) and the upper L1ICD-immunoreactive band in lane 2 (L1ICD and CKII) was performed by densitometric analysis (<b>A</b>, <i>upper panel</i> shows a representative example). Data represent mean ±SEM of four independent experiments.</p

14-3-3ζ supports CKII-catalyzed L1ICD phosphorylation.

<p><b>A.</b> L1ICD was preincubated in the presence or absence of GST-14-3-3ζ followed by incubation with CKII. At different time points, CKII phosphorylation was stopped by adding SDS loading buffer, and samples subjected to SDS-PAGE. Western blot analysis with the 74-5H7 anti-L1 antibody revealed that the band intensities of phosphorylated L1 (phospho-L1ICD) increased over time when L1ICD was preincubated with GST-14-3-3ζ (compare C). <b>B. </b><i>Upper panel</i>, Comparison of L1ICD phosphorylation by CKII in the presence of GST-14-3-3ζ or GST. The phospho-L1ICD and L1ICD bands at time points 0 and 180 min (representative example given in panel <b>A</b>) were quantified and the ratio of phospho-L1ICD/total L1ICD at t = 0 min and t = 180 min in the presence of GST-14-3-3ζ or GST was calculated. Error bars denote ±SEM based on 3 independent experiments. <i>Lower panel</i>, GST Western Blot analysis of phosphorylation assay samples taken at the indicated time points confirms that equal amounts of GST or GST-14-3-3ζ were present in the reactions. <b>C.</b> L1ICD S1181A mutant was subjected to the same CKII phosphorylation assay as described in <b>A</b>. No band presumably representing newly phosphorylated L1ICD could be observed. <b>D.</b> Samples incubated with CKII for 180 min were subjected to subsequent treatment with λ protein phosphatase 1 (λPP1). Changes in the L1ICD band pattern were analyzed by Western Blot with the 74-5H7 antibody.</p

Ser¹¹⁸¹Ala substitution and RSLESD deletion disrupt binding of L1 to 14-3-3ζ.

<p><b>A.</b> Schematic of recombinant L1ICD constructs. The full-length L1ICD construct contains the RSLESD sequence, a potential 14-3-3 binding motif. L1ICD S1181A has a single amino acid substitution (S1181A) of a serine residue in this motif. Ser1181 can be phosphorylated by CKII. The RSLESD sequence, a potential 14-3-3-binding motif, is deleted in L1ICD ΔRSLESD. Wild-type and mutated L1ICD constructs were recombinantly expressed as 6xHis-tagged proteins, purified from bacterial lysates and used in pull-down experiments. <b>B.</b> Purified mutated L1ICD constructs were analyzed by Western blot using the 74-5H7 anti-L1 antibody. <b>C. </b><i>Upper panel</i>: 6xHis-tagged proteins, purified from bacterial lysates, were subjected to GST-14-3-3ζ pull-down assays after treatment with CKII. GST was used as a control. Pull-down eluates were analyzed by Western blot (WB) with the 74-5H7 anti-L1 antibody. <i>Lower panel</i>, GST and GST-14-3-3ζ were detected in pull-down eluates by Western blot (WB) with an anti-GST antibody, confirming that comparable amounts of GST or GST-14-3-3ζ were used in each pull-down.</p

14-3-3ζ is associated with L1 in endosomes.

<p><b>A. </b><i>Upper panel</i>, Immunoprecipitation (IP) of L1 from vesicle fractions was performed using the anti-L1 monoclonal antibody 557. Proteins were resolved by a SDS-PAGE gradient gel (4–20%) and analyzed by Western blotting (WB) with an anti-L1 antibody (74-5H7), which recognizes full-length L1 and L1 proteolytic fragments containing the L1ICD. Full-length L1 (∼200 kDa), indicated by an arrow, was successfully precipitated. <i>Lower panel</i>, Western blot analysis of the L1 immunoprecipitates was also performed with an isoform-specific anti-14-3-3ζ antibody, revealing the association of 14-3-3ζ with L1 in vesicle fractions. C: crude endosomal preparation; S: cytosolic compounds; P: crude membrane fraction; ctrl: control IgG used for immunoprecipitation. <b>B.</b> To confirm that the isolated fractions are enriched in endosomal markers, equal protein amounts from total brain homogenate and vesicle fractions were analyzed for expression of the early endosome marker Rab 4 (upper panel) and the late endosome marker Rab 9 (lower panel). H: homogenate. In <b>B</b>, the bands shown for the homogenate are from a different blot, but with the same protein amount loaded and the same film exposure time as for the fractions.</p

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter