RESUMEN
In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism.
Asunto(s)
Neuronas/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/genética , Sinapsis/fisiología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Regulación de la Expresión Génica , Células HEK293 , Hipocampo/citología , Hipocampo/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis , Neuronas/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de GABA-A/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The marine natural product haliclamide has been synthesized based on macrocyclization by ring-closing olefin metathesis. Using either enantiomer of two of the four building blocks that were employed to assemble the diene precursor for the metathesis reaction, three non-natural isomers of haliclamide were also prepared. On the basis of the comparison of the (1)H and (13)C NMR spectra of the individual stereoisomers with literature data for the natural product, the configuration of the previously unassigned stereocenters at C9 and C20 of haliclamide could be determined to be S for both carbons. The absolute configuration of haliclamide thus is 2S, 9S, 14R, 20S. The antiproliferative activity of synthetic haliclamide against several human cancer cell lines was found to be in the high µM range. The compound showed no antifungal or antibiotic activity.