Study On Function And Mechanism Of Nyap1 In Mouse Corticongenesis

The mammalian isocortex finally forms dense six layers during corticongenesis,which is essential for the physiological function of cerebral cortex.The dysplasia of neocortex may lead to serious neurological diseases,such as epilepsy,cognitive impairment,microcephalies or Hemimegalencephaly and so on.The development of the mammalian neocortex is indispensable to coordinate the proliferation of neural progenitors,neuronal migration,and differentiation,and synaptic connection,etc.The structural formation of six layers of cerebral cortex needs neuronal migration.The late-born neurons move past the early-born neurons and invariably stop beneath the marginal zone（MZ）.After the“inside-out”migration,the six-layer structure is gradually formed.Neuronal tyrosine-phosphorylated adaptor for the phosphoinositide 3-kinase 1（Nyap1）is a member of the Nyap family（NYAPs）of phosphoproteins.The family members of NYAPs include Nyap1,Nyap2,and Myosin16/Nyap3.The NYAP Homology Motif（NHM）contains conserved tyrosine residues and a proline-rich stretch.NYAPs,the major phosphoproteins in the brain,occupy at least three-fourths of PI3K-associated tyrosine phosphorylation.It has been shown that the high expression of NYAPs in cerebral cortex and NYAPs-mediated remodeling of the actin cytoskeleton.On account of neuronal hypotrophy,a clear reduction in the NYAPs-KO brain size was observed,and the most obvious phenomenon in Nyap1-KO brain.Nyap1 is expressed in almost all the cortical plate（CP）zones and begins to express at E10 and peaks at perinatal period,which implies the important role of Nyap1 during corticongenesis.However,it is rare on the report of Nyap1 on neuronal migration.In this study,we targeted at mouse cerebral cortex.The plasmids of overexpression（Nyap1）and knockdown（shNyap1）Nyap1 were constructed,and the tyrosine phosphorylation sites of Nyap1(Nyap1^Y212F,Nyap1^Y257F257F and Nyap1^Y212/257F)were mutaated.We investigate the role and mechanism of Nyap1 during neuronal migration by using in utero electroporation,cell tranfection,Western Blot,immunochemistry and confocal laser photography,etc.Moreover,to investigate the molecular mechanism of Fyn and Nyap1,the shFyn plasmid and tyrosine phosphorylation activated mutation of Fyn（Fyn-CA）were involved in this study.The main findings are as follow:1.The Nyap1 overexpression plasmid was successfully expressed in the NIH3T3cells by constructing Nyap1 overexpression plasmid.Using in utero electroporation to mark migrating neurons.Overexpression of Nyap1 in vivo induced neurite outgrowth and extension and promoted migration of neurons by refining the time of data collection,suggesting that Nyap1 promoted neurons to migrate from intermediate zone（IZ）to CP.2.Nyap1 RNAi vectors were constructed and successfully expressed in the NIH3T3cells.The efficiency of knockdown of Nyap1 was evaluated by Western Blot analysis in HEK293T cells.In the absence of Nyap1,an abnormal morphology of migrating neurons emerged,an appreciable quantity of neurons accumulated in the IZ,and a small number of neurons invaded the CP with a thin leading process.Nyap1 is essential for the multipolar-bipolar transition.3.Overlap-extension PCR was performed to create the mutants of Nyap1(Nyap1^Y212F,Nyap1^Y257F257F and Nyap1^Y212/257F),and the plasmids could express in the NIH3T3 cells.When two different mutants that changed the level of Nyap1 phosphorylation(Nyap1^Y212F and Nyap1^Y257F)were introduced,the phenotypes caused by Nyap1 were weakened.The inhibiting effect of neuronal migration appeared even when both phosphorylation sites were mutated(Nyap1^Y212/257F).More neurons extended two leading processes in the IZ-CP border when Nyap1^Y212/257F was introduced.These findings indicated that Nyap1 may regulate neuronal migration through these two phosphorylation sites.4.To illuminate the molecular mechanism of Nyap1,we performed a rescue experiment by co-electroporation of Nyap1 and shFyn.Co-electroporation of Nyap1 with the Fyn shRNA corrected the abnormal accumulation of Fyn-silenced cells in the IZ.A reverse rescue was performed by co-electroporation of Fyn-CA and shNyap1-1,the effective rescue was not displayed.The results of rescuing effect of Nyap1^Y212/257F to shFyn showed that Nyap1^Y212/257F could not completely rescue the abnormal neuronal migration by shFyn.All together,these results demonstrated that Nyap1 mediates the phosphorylation signal of Fyn,and that Nyap1 regulated the crucial step of the multipolar-bipolar transition in the cerebral cortex downstream of Fyn during neuronal migration.Overall,the tyrosine phosphorylation of Nyap1 is crucial to regulate neuronal migration,and Nyap1 activation during cortical development could be phosphorylated by Fyn.