Development of the nervous system Abstract During cerebral cortex development, pyramidal neurons migrate through the intermediate zone and integrate into the cortical plate. These neurons undergo the multipolar—bipolar transition to initiate radial migration. While perturbation of this polarity acquisition leads to cortical malformations, how this process is initiated and regulated is largely unknown. Here we report that the specific upregulation of the Rap1 guanine nucleotide exchange factor, RapGEF2, in migrating neurons corresponds to the timing of this polarity transition. Thus, the specific expression and Cdk5-dependent phosphorylation of RapGEF2 during multipolar—bipolar transition within the intermediate zone are essential for proper neuronal migration and wiring of the cerebral cortex. Download PDF Introduction The migration of neurons from their birthplace to their final destination is fundamental to the architectural formation and functional wiring of the nervous system.

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C3G also plays important roles in angiogenesis, tumor growth, and metastasis through its regulation of the platelet secretome. In addition, C3G contributes to megakaryopoiesis and thrombopoiesis.

C3G-KO platelets showed a significant delay in platelet activation and aggregation as a consequence of the defective activation of Rap1, which resulted in decreased thrombus formation in vivo.

Additionally, we explored the contribution of C3G-Rap1b to platelet signaling pathways triggered by thrombin, PMA or ADP, in the referenced transgenic mouse model, through the use of a battery of specific inhibitors. This phosphorylation was shown to be positively regulated by ERKs through their inhibition of the tyrosine phosphatase Shp2.

Taken together, our data reveal the critical role of C3G in the main pathways leading to platelet activation and aggregation through the regulation of Rap1b. These various receptors trigger signaling cascades that converge in the activation of Rap1b, the most abundant GTPase in platelets.

In platelets, Src and PKC are reciprocally activated by transphosphorylation. Specifically, PKC phosphorylates Src at Ser12, a residue involved in cytoskeletal association and substrate affinity. These results suggest that C3G is the main Rap1 activator under these conditions. The scatter plots represent the bleeding times of 13—15 animals of each genotype. The mean values are shown. The Mann—Whitney U test was performed. The levels of Rap1-GTP were determined by pulldown assay and immunoblotting.

Values are relative to the control value f or to the value for thrombin in wild-type platelets g and were normalized against total Rap1. Two of six C3G-KO mice survived the lethal treatment.

Blood was withdrawn before the injection and 4 min after injection. Thrombi arrowheads appeared as eosinophilic material with borders, with partial or total affectation of the lumen of the small and medium caliber blood vessels located mainly on the periphery of the pulmonary lobes.

Specifically, this pathway participates in the second wave of Rap1 activation induced by thrombin. For this purpose, we used confocal fluorescence techniques, which allow greater sensitivity than western blotting.

As shown in Fig. In addition, both thrombin and PMA induced the phosphorylation of Src at Tyr Tyr in mice , a residue responsible for its activation Fig. All these results suggest that Src is the kinase that phosphorylates C3G during the second wave of thrombin stimulation involving PKC. Upper left: representative immunofluorescence confocal microscopy images of platelets of each genotype under each treatment condition taken at the same exposure time.

T: thrombin. Left: representative immunofluorescence confocal microscopy images of platelets of each genotype under each treatment condition taken at the same exposure time. Previously, we verified the inhibitory effects of these inhibitors and others used in this work on thrombin-induced platelet activation and aggregation Fig. Our hypothesis was that the transgenic expression of C3G would make platelets more dependent on C3G.

Therefore, they would be more sensitive to inhibition of the pathways in which C3G participates, leading to increased inhibition. A similar tendency was observed for the inhibition of P-selectin expression on the surface and the aggregation induced by thrombin Fig. Left panel: representative western blots.

Full size image Inhibition of the ADP receptor P2Y1 also differentially affected tgC3G platelets compared to wtC3G platelets, although to a lesser extent than inhibition of the P2Y12 receptor, since the differences were not significant. This suggests a minor contribution of C3G to this pathway. Notably, tgC3G platelets showed less total Rap1 than their control platelets from an equivalent amount of blood see also Figs.

This is not an effect of C3G on Rap1 expression, since correlative variation between actin levels was observed data not shown. Both inhibitors also produced an almost significant greater inhibition on tgC3G platelet aggregation, compared to wtC3G platelets Fig.

To evaluate whether C3G contributes to the activation of these MAPK pathways, we analyzed their phosphorylation status in platelets from mice of the different genotypes under study treated with thrombin for 1 min in the presence of inhibitors of several platelet signaling pathways Fig. There were no significant differences in the levels of ERK and p38 MAPK phosphorylation between the platelets from mice of the different genotypes stimulated for a short time with thrombin, in concordance with the finding that Rap1 activity does not affect the transient activation of these MAPKs.

S6a , 2 but does not contribute to short-term ERK activation Fig. Platelets were pretreated with U or SB prior to stimulation with thrombin, and the levels of pTyrC3G were analyzed by fluorescence confocal microscopy. Left: representative immunofluorescence confocal microscopy images taken at the same exposure time. Moreover, U did not prevent Src phosphorylation at Tyr Fig. This result indicates that ERKs stimulate C3G phosphorylation at Tyr through inhibiting the phosphatase Shp2, which is responsible for the dephosphorylation of this residue.

All micrographs were taken at the same exposure time. Values are relative to the value for thrombin in wild-type platelets and were normalized against total Rap1. This colocalization was maximal in the presence of U, a condition in which Shp2 activation is increased, and coincided with increased C3G dephosphorylation. This colocalization was partially lost when platelets were pretreated with SHP but recovered when U was also present.

These results indicate that the colocalization of pTyrC3G and Shp2 depends on the activation of Shp2. Figure 6c.


C3G contributes to platelet activation and aggregation by regulating major signaling pathways

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ChemoCentryx Inc (CCXI) Q1 2019 Earnings Call Transcript


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