Exploration Of New Mechanisms Of Cyclin-dependent Kinase 5 Involved In Neuronal Apoptosis

Alzheimer's disease(AD)is a kind of neurodegenerative disease featured with memory loss and cognitive function impairment.Brain atrophy caused by neuronal loss is considered as the direct reason for AD.Cyclin-dependent kinase 5(CDK5)is a critical kinase involved in the process of neuronal apoptosis.p35,the activator of CDK5,could be cleaved to p25 in the pathogenesis of AD or neurons stimulated by neuronal toxicants.The generation of p25 overactivates CDK5 and induces phosphorylation of substrates like tau to promote neuronal apoptosis.In order to get further understanding of the role of CDK5 in neuronal apoptosis,we identified some new substrates of CDK5 and explored their functions in neuronal apoptosis.We found that Chloridion intracellular channel 4(CLIC4)is a novel substrate of CDK5.CDK5 phosphorylates CLIC4 at serine 108 and promotes CLIC4 protein stability.In the process of neuronal apoptosis,overactivation of CDK5 is paralleled with increased level of CLIC4.Overexpression of CLIC4 in primary neurons promotes neuronal apoptosis,while knockdown of CLIC4 by genetic approach or inhibition of CLIC4 by IAA94 attenuates neuronal apoptosis induced by H2O2.These results indicate that CLIC4 is involved in the process of neuronal apoptosis induced by CDK5 overactivation.Cell-cycle exit and neuronal differentiation 1(CEND1)is another substrate of CDK5 identified by us.CDK5 phosphorylates CEND1 at serine 10 and facilitates its degradation.CEND1 was down-regulated in neuronal apoptosis paralleled with CDK5 overactivation,while knockdown of CEND1 in neurons promotes neuronal apoptosis.We observed that CEND1 interacts with Epidermal Growth Factor Receptor(EGFR)at the tyrosine kinase domain.Binding of EGFR by CEND1 does not affect the kinase activity of EGFR,while CEND1 could decrease ubiquitination of EGFR to promote its stability.EGFR signaling has been documented as a key regulator to maintain neuronal survival,thus CEND1-mediated downregulation of EGFR may induce neuronal apoptosis.This study elucidated a new mechanism underlying the induction of neuronal apoptosis by CEND1 deficiency.CEND1 could be phosphorylated by CDK5 at serine 10 and that leads to CEND1 degradation,and loss of CEND1 protein promotes neuronal apoptosis by facilitating ubiquitination and degradation of EGFR.Glycogen synthase kinase 3?(GSK3?)and CDK5 are both critical protein kinases involved in the pathogenesis of AD.They induce hyperphosphorylation of tau,which generates neurofibrillary tangles(NFT).We found that p25,the activator of CDK5,could bind and activate GSK3?.GSK3? and p25 produce more severe impairment than CDK5 and p25.Compared with CDK5 siRNA,GSK3?siRNA could relieve the neurodegeneration more effectively.These data suggest that the neuronal toxity of p25 is mainly mediated by GSK3? but not CDK5.This uncovers the relationship of GSK3P and CDK5,which both closely related to AD,and provides theoretical basis for pathogenic mechanism and therapeutic targets for AD.As a critical kinase involved in AD pathogenesis,CDK5 exerts its function maily through regulating its substrates.This article focuses on two noval substrates of CDK5,CLIC4 and CEND1,and their function in neuronal apoptosis,which provides us more understanding of CDK5.We also find that p25 could regulate the activity of both CDK5 and GSK3?.In conclusion,these studies help us more comprehensively understand the pathogenesis of AD,and provide theoretical and experimental foundation of developing new medical targets of AD.