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

ИНВАЗИВНЫЙ МОНИТОРИНГ СЕРДЕЧНОГО ВЫБРОСА ПО ВРЕМЕНИ ТРАНЗИТА ПУЛЬСОВОЙ ВОЛНЫ ПОСЛЕ АОРТОКОРОНАРНОГО ШУНТИРОВАНИЯ НА РАБОТАЮЩЕМ СЕРДЦЕ

Goal of the study: to evaluate the accuracy of invasive measurement of cardiac output (CO) by pulse wave transit time (PWTT) (esCCO, Japan) compared to transpulmonary thermodilution (TPTD) (PiCCO2 , Germany) after aortocoronary bypass (ACB) without cardiopulmonary bypass (ACB without CPB). Methods. 21 patients with ACB without CPB were enrolled into the study. During early post-operative period CO was simultaneously registered at eight stages basing on PWTT (COPWTT) and TPTD (COTPTD). Statistic analysis included evaluation of congruence of CO absolute values and capability to follow-up changes in CO. Results. In accordance with Bland-Altman analysis the average difference between two methods made 0.3 l/min. with consistency limits of ± 2.1 l/min. and percent error of 40%. Polar chart analysis showed the angular difference of 2.6°, radial consistency limits ± 53.3° and polar concordance of 69%. Conclusion: Lower repeatability of CO measurement by PWTT and insufficient capability to follow the changes in CO after ACB without CPB don not allow recommending this method in its invasive variant for routine practice as an alternative to thermodilution methods.  Цель исследования: оценка точности инвазивного измерения сердечного выброса (СВ) по времени транзита пульсовой волны (ВТПВ) (esCCO, Япония) в сравнении с методом транспульмональной термодилюции (ТПТД) (PiCCO2 , Германия) после аортокоронарного шунтирования (АКШ) без искусственного кровообращения (АКШ без ИК). Методы. В исследование включен 21 пациент после АКШ без ИК. В раннем послеоперационном периоде на восьми этапах выполняли параллельную регистрацию СВ, определенного на основе оценки ВТПВ (СВВТПВ) и ТПТД (СВТПТД). Статистический анализ включал оценку согласованности абсолютных значений СВ и способности отслеживать динамику СВ. Результаты. Согласно анализу Бланда – Альтмана, средняя разница между методами составила 0,3 л/мин с границами согласованности ± 2,1 л/мин и процентной ошибкой 40%. Анализ полярной диаграммы показал угловую разницу 2,6°, радиальные границы согласованности ± 53,3° и полярную конкордантность 69%. Вывод. Низкая воспроизводимость измерения СВ на основе оценки ВТПВ и недостаточная способность отслеживать динамику СВ после АКШ без ИК не позволяют рекомендовать рутинное использование данного метода в его инвазивном варианте в качестве альтернативы термодилюционным методикам.

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking

Serotonin receptors 5-HT1A and 5-HT7 are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT1A and 5-HT7 receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT1A or 5-HT7 receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT7–5-HT7 homodimers, followed by the 5-HT7–5-HT1A heterodimers and 5-HT1A–5-HT1A homodimers. Functionally, heterodimerization decreases 5-HT1A-receptor-mediated activation of Gi protein without affecting 5-HT7-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT1A receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT1A receptor internalization and also enhances the ability of the 5-HT1A receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT7 receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT1A–5-HT7 heterodimers and, consequently, their functional importance undergoes pronounced developmental changes

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