Recettore NMDA, sistema Neuregulina/ErbB4 e ruolo dei segnali di Calcio nella migrazione dei progenitori stratali ST14A

Neuronal migration is a tightly regulated process in which the orchestrated activity of multiple molecules combines to bring about the appropriate allocation of post mitotic neurons. Previous studies have shown that the Neuregulin1(NRG1)/ErbB4 system is involved in several types of neuronal migration. In addition, intracellular calcium fluctuations, often regulated via Ca2+ permeable membrane channels, play central roles in neuronal motility. In this work we examined the possible interactions between a Ca2+ channel, the NMDA-type glutamate receptor, and the NRG1-ErbB4 system in the migratory process of immortalized neural progenitors (ST14A cells). RT-PCR studies showed that wild type ST14A cells (non-expressing ErbB4), as well as ErbB4(Jma/cyt2)-transfected clones express a restricted number of NMDA receptor (NMDAR) subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form channels characterized by low Mg2+-sensitivity and low Ca2+permeability, and generating small, long-lasting currents. Migration assays showed a non-statistically significant effect of NMDA on basal migration of ErbB4-transfected ST14A cells. However, when NMDA was added to NRG1, we found a significant enhancement of migration compared to NRG1 alone (from 5 to 8 fold increase in migration rate). This strengthening of NRG1-stimulated migration is consistent with cooperation between NMDA and ErbB4 receptors in inducing and/or maintaining a migratory behaviour in ST14A cells. We subsequently asked whether the synergism of NMDAR and NRG1/ErbB4 systems in enhancing ST14A migration could be due to a modulation of calcium signalling. Migration experiments carried out in absence of intracellular Ca2+ showed a highly significant reduction in NMDA+NRG1 stimulated migration, shifting the number of migrated cells approximately to the level of NRG1 alone. This result strongly supports the hypothesis that the mechanism of NMDAR/ErbB4 interaction in ST14A migration involves intracellular Ca2+ fluctuations. We also began to characterize the Ca2+ signalling induced by NRG1 and NMDA in calcium-imaging experiments. Our findings indicate that NRG1 stimulation of ErbB4-transfected ST14A cells exerts an increase in intracellular Ca2+. Stimulation with NMDA led to a small increase in free intracellular Ca2+ reaching a plateau with a maximum value significantly smaller than the one obtained with NRG1. These results are consistent with a lower expression of NMDA compared to ErbB4 receptors in transfected ST14A clones. In preliminary experiments, we did not find an increment in intracellular free calcium when NMDA was added to NRG1 for 10 minutes. Further experiments with longer exposure times are needed to complete the study. In summary, this experimental work suggests for the first time an interaction between NMDA receptors and the NRG1-ErbB4 system in neural precursor migration. This interaction is calcium-dependent and might take place in the course of several minutes or hours. Elucidation of molecular interactions in neuronal migration is fundamental to understand the multifaceted process that leads to the formation of a complex nervous system from a ¿simple¿ pseudo-stratified neuro-ephitelium, and it is important for its numerous pathological implications.