N4bp1 Participates In The Early Development Of Mouse Cerebral Cortex By Regulating Notch Signaling Pathwa

The mammalian brain is the most complex of all animal organs,producing a variety of motor and social behaviors,including the most human behaviors such as abstract thinking,art and sense of humor,by processing information from all sensory modalities and integrating it with previously stored information.These higher behaviors result from the exceptionally developed human cerebral cortex,and this complex organization of the cerebral cortex is acquired through a complex developmental process.This process involves the proliferation of a limited number of neural stem cells,which also give rise to numerous neurons as well as glial cells.In addition,as new cortical neurons are formed,these neurons and glial cells must move from their birth site to their final location near the surface of the brain,and the process of migration is very strictly genetically regulated.Disturbances in any of these steps can lead to significant tissue abnormalities,resulting,for example,in severe learning deficits,cognitive impairment,and intractable epilepsy.Therefore studying and understanding the regulatory mechanisms of cortical development is an important proposition in developmental biology.The Notch signaling pathway is an evolutionarily extremely conserved pathway that determines the fate of numerous cells during the development of multicellular organisms.Decades of research have shown that Notch plays an important role in embryonic brain development and is the specification and basis for neuronal development.Pre-laboratory work confirmed that N4bp1(NEDD4 binding protein1)is highly expressed in the cerebral cortex and acts as a novel Notch signaling regulator to promote neuritogenesis.It was also demonstrated that N4bp1 antagonizes Notch signaling by downregulating the protein level of NICD,and it was also demonstrated that the degradation of NICD by N4bp1 is nondependent on the PEST structural domain.However,there are still unresolved questions,such as which structure of N4bp1 plays a key role in NICD degradation,which structure of NICD N4bp1 depends on for its degradation,which degradation pathway N4bp1 uses for NICD degradation,which E3 ubiquitin ligase N4bp1 uses for NICD degradation,and the phenotypic analysis of knockout mice.To address the above questions,we used immunoprecipitation technique to demonstrate that N4bpl and Notchl can bind in vivo,and further verified that there is a regulatory relationship between them;using segmental cloning technique,we confirmed that N4bp1 degrades NICD through RAM structural domain,and indeed non-dependent on PEST structural domain degradation;to further verify this conclusion,we used GST-pull down to confirm that N4bp1 binds to the RAM structural domain in vitro;using the proteasome inhibitor MG 132 to treat cells and assay the stability of NICD,we confirmed that N4bp1 degrades NICD via the ubiquitin-proteasome pathway.in addition,we also explored the functional structural domain of N4bp1.Using molecular cloning-related techniques,deletion mutations and point mutations were performed on N4bp1,and the results showed that deletion of the CUE-like structural domain or 862-863(FP)mutation resulted in the absence of N4bp1’s NICD degradation function,demonstrating that the key structural domain of N4bp1 for NICD degradation is CUE-like,and the key site of its structural domain is 862-863(FP).In addition,we demonstrated that knocking down the endogenous E3 ubiquitin ligase Trim21,which has an inhibitory effect on the function of N4bp1 for NICD degradation,using immunoprecipitation technique,proving that Trim21 is involved in the degradation of NICD by N4bp1.To further comprehensively understand the function of N4bpl,we constructed N4bp1 gene mice and used N4bp1 conditional knockout mice as a model.We found that after N4bpl knockout in the cerebral cortex,stem cells in the germinal area were more aggregated in the bottom layer and did not migrate upward,while stem cells in the wild type were more dispersed and had a tendency to migrate upward;while P3 conditional knockout mice compared with the wild type There were no significant changes in neurons at all levels,but the cortical thickness was thinner,and the reason for this has not been analyzed.In the future,we will conduct more detailed analysis of the N4bp1 knockout mouse model to refine the data.