DCC-mediated Dab1 Phosphorylation Participates In The Regulation Of Radial Migration Of Cerebral Cortical Neurons

The development of the mammalian cerebral cortex consists of a series of closely regulated cytological events.During mammalian embryonic development,neural progenitor cells located in the ventricular zone and subventricular zone of the cerebral cortex undergo symmetrical division and asymmetric division,producing neural progenitor cells and a large number of neurons.These differentiated neuronal cells move to the outermost side of the cortical plate through multipolar migration,radial migration and somotranslocation,and are correctly positioned at the corresponding cortical plate,thereby forming a“inside out”lamellar structure.The radial neuronal migration is an important part of neocortical development,and it is also the basis for the formation and maintenance of neural network function.In the process of radial migration of cortical pyramidal neurons,the multipolar to bipolar morphology transformation is an important process.A large number of scientific research results have shown that the abnormal morphological changes of neurons and the abnormal migration of neurons are closely related to diseases of the nervous system and mental diseases.However,the current research on the mechanisms regulating the radiation migration of neocortical neurons is not very clear.Reelin-Dab1?Disabled 1,Dab1?signaling pathway is the most classical signaling pathway for the regulation of cortical neuronal migration and cortical lamellar localization.Dab1 is a key adaptor protein in this pathway that binds to the transmembrane receptor apolipoprotein E receptor 2?ApoER2?or very low density lipoprotein receptor?VLDLR?,thereby activating the downstream effectors and transferring signals into the cell.The C-terminal of Dab1 is a tyrosine-rich region that contains five tyrosines at positions 185,198/200,220 and 232.In response to Reelin stimulation,these tyrosines are phosphorylated in a specific order,and phosphorylation of Dab1 plays an important role in the development of the cerebral cortex.To date,Dab1 has well been studied in Reelin signaling during brain development.However,in Reelin-null mice,Dab1 tyrosine phosphorylation is decreased but not eliminated,thus suggesting that Reelin-independent tyrosine phosphorylation of Dab1 may exist.Deleted in Colorectal Cancer?DCC?is a transmembrane molecule that belongs to the immunoglobulin superfamily.As one of the receptors of Netrin-1,DCC is involved in regulating neuronal branching,neurite outgrowth,axon pathfinding,neuronal migration and synapse formation during embryonic development.This study shows that Netrin-1/DCC mediates Dab1 phosphorylation and participates in multipolar-to-bipolar transition of migrating neurons.In the present study,the expression patterns of DCC and Dab1 in the developing cerebral cortex during embryonic period were detected by immunoblotting and immunohistochemical staining.Using immunoprecipitation and GST-pull down approaches,we found that DCC and Dab1 formed immunoprecipitate complexes in the case of overexpression or embryonic brain lysate.Furthermore,Netrin-1 stimulates the phosphorylation of Dab1,and this process is regulated by the Src family kinases.In additon,at the peak stage of cortical neuronal migration,we transferred the GFP plasmid into the DCC^Kanga-/-mice by in utero electroporation,or the DCC shRNAs into the wild-type mouse brain to knock-down DCC,and then observed the migration of neurons at different time points.Finally,we carried out rescue experiment using Dab1 and Fyn plasmids to investigate the molecular mechanism of Netrin-1/DCC signaling pathway in regulating cortical neuronal migration.In summary,this study demonstrated that DCC binds to Dab1 through its P3 domain and Netrin-1 can induce Dab1 phosphorylation.Knockdown of DCC or deletion of the P3domain of DCC significantly inhibited neuronal migration and multipolar-to-bipolar transition of migrating neurons.This migration delay and morphological transition defect can be rescued by expressing wild-type Dab1 or constitutively active form of Fyn.In conclusion,DCC-Dab1 was first identified as a functional complex and initially elucidated its function in the migration of cortical neurons during the development of the neocortex.