The Regulation Of Drosha By Cdk5 Participates In The Excitotoxicity-related Nerve Injury

Background and Objective:Excitotoxicity is one of the main pathological mechanisms of common neurological diseases,such as trauma brain injury,stroke,epilepsy,neurodegeneration and so on.Studies have shown that excitotoxicity-related ion disorder,caused by the action of excitatory neurotransmitters such as glutamate on its corresponding receptors,is the pathologic basis of subsequent neuronal injury and degeneration.However,the molecular mechanism remains unclear.Inhibition of the excitatory receptors or remodeling of the ion homeostasis usually results in a wide range of side effects,which impedes its clinical application.Therefore,it has become the focus to study the specific mechanism of neuronal injury induced by excitotoxicity and find out the potential therapeutic targets.Cdk5 is one of the cyclin-dependent kinase(CDK)and highly expressed in central nervous system.It is involved in multiple important processes such as neuron migration and development physiologically.Recent studies have found that Cdk5 is abnormally activated in excitotoxicity-related diseases and phosphorylates multiple pathogenic substrates.Our previous study found that Drosha,the key enzyme for miRNA biosynthesis,is the potential substrate of Cdk5.Since the change of Drosha under stress condition is closely related to neuronal death,we carried out a series of experiments to figure out whether and how the abnormally activated Cdk5 participates in the regulation of Drosha under excitotoxicity.Further,we explored the clinical transformation potential of this regulation mode.and want to provide new ideas for clinic treatment in excitotoxicity related diseases.Methods:In this study,the small RNA sequencing data of TBI and the existing small RNA-seq data of MCAO in GEO database were analyzed to evaluate the disturbance of miRNA biosynthesis in excitotoxicity related diseases.Then the change of key enzymes Drosha and Dicer in the injured and surrounded tissues in TBI,MCAO and ICH were detected by western blotting.The phosphorylation of Cdk5’s classical substrate,Tau,was also detected to estimate the activity of Cdk5 indirectly.We up-regulated or down-regulated the kinase activity of Cdk5 in HEK-293 T and used immunoprecipitation,immunoblotting and in vitro kinase experiment to confirm that kinase Cdk5 can directly phosphorylate Drosha.Further,we overexpressed different truncated mutants and single/multiple site mutants of Drosha to find out the exact amino acid residue for Cdk5 phosphorylation on Drosha by immunoprecipitation,immunoblotting and in vitro kinase experiment.The effects of the Cdk5-Drosha pathway on neuronal survival in primary cultured cortical neurons and nerve injury in Drosha conditioned overexpression transgenic mice were verified by immunofluorescence staining,cell viability detection,Nissl staining and behavioral tests.In the end,combined with Cdk5 inhibitors that have undergone clinic trial,we explored the therapeutic potential for Cdk5 inhibition in Drosha protection,neuronal survival and neuro-function protection in TBI by using western blotting,morphological staining,and behavioral tests.Results:Experiment 1:Dysregulation of miRNA biosynthesis in the injured and surrounding brain tissues in excitotoxicity related nerve injury mice.(1)Analyzation of the miRNA level of TBI and MCAO mice showed that the miRNA biosynthesis was down-regulated as a whole in the injured and surrounding brain tissue.(2)The key enzymes for miRNA biosynthesis in the injured and surrounded tissues in TBI,MCAO and ICH mice were detected.The results showed that the protein level of Drosha and Dicer were decreased,accompanied with the increased activity of kinase Cdk5.(3)The expression of Drosha in different cell types in nerve tissue was detected.The results showed that Drosha was highly expressed in primary cultured neurons,HT22 cells and HEK-293 T cells.(4)We further evaluated the correlations between Cdk5 and Drosha.The results showed that the increase of Cdk5 activity promoted the degradation of Drosha.Experiment 2:Cdk5 phosphorylates Drosha specific amino acids residue directly and promotes Drosha degradation(1)The sequence analysis of Drosha indicated six potential phosphorylation sites for Cdk5.Then,we confirmed that Cdk5 could bind and phosphorylate Drosha directly by co-immunoprecipitation and in vitro kinase reaction.(2)We used multiple Drosha mutant to figure out the Cdk5 specific phosphorylation sites on Drosha.The results showed that Ser221,Ser255,Thr274,Ser300 and Ser355 on Drosha RS rich domain were identified as Cdk5 specific phosphorylation sites.(3)We found that the Cdk5-promoted Drosha degradation in HEK-293 T was related to its phosphorylation modification.Experiment 3:Maintaining Drosha protein levels alleviated nerve damage during excitatory stress(1)Western blotting showed that the inhibition of Cdk5 activity in primary cultured cortical neurons protected Drosha protein level.(2)CCK8 test and Tunel staining showed that the maintenance of Drosha protein level protected primary cultured cortical neurons survival.(3)In transgenic mice,it was found that the inhibition of Drosha reduction could alleviate neuronal death after TBI injury.Experiment 4:Maintaining Drosha protein levels improves the prognosis of excitotoxicity related nerve injury(1)Modified neurological function score,wire grip and motion test,limb symmetry test and open field test showed that the maintenance of the level of Drosha in the injured and surrounding brain tissue promoted the repair of motor function in the early stage of TBI mice.(2)Localized navigation test and space exploration test showed that the maintenance of the level of Drosha in the injured and surrounding brain tissue alleviated the cognitive impairment in the middle and late stage of the TBI mice.Experiment 5:Exploration of the therapeutic potential for Cdk5-Drosha pathway in neuronal survival and neurofunction protection in TBI(1)Western blotting showed that the intraperitoneal injection of Cdk5 inhibitor protected the level of Drosha in the injured and surrounding brain tissue after TBI modeling.The protective time window was: Roscovitine ≤ 30 min and Dinaciclib≤ 1 h.(2)Morphology and behavior tests showed that the intraperitoneal injection of Cdk5 inhibitor after TBI modeling could reduce neuronal death,promote the early motor function restore and alleviate the cognitive impairment in the middle and late stage of the TBI mice.Conclusion:This study found that the Cdk5 kinase activity increased abnormally in the injured and surrounding brain tissue in excitotoxicity related nerve injury,accompanied with the decrease of Drosha and the imbalance of miRNA expression.It was confirmed that CDK5 could phosphorylate Drosha specific residues directly and promote Drosha degradation.Five sites for Cdk5 phosphorylation were identified in Drosha RS rich domain,namely Ser221,Ser255,Thr274,Ser300,and Ser355.In the primary cultured cortical neurons and Drosha conditioned overexpression transgenic mice,we found that the maintenance of the level of Drosha could protect the nerve survival under excitatory stress,promote the early motor function repair and also reduce the cognitive impairment in TBI mice.Further,we explored the time window for the maintenance of Drosha levels under excitatory stress by Roscovitine and Dinaciclib,the small molecule inhibitors of CDK5 that have entered clinical trials.The results showed that the intraperitoneal injection of Roscovitine or Dinaciclib within 30 minutes after injury could protect neuronal survival and improve neurological impairment.This study confirmed the importance of Cdk5-Drosha pathway in excitotoxicity related nerve injury,and illumed its clinic prospects,which may provide a new idea for the treatment of excitotoxicity related diseases.