The Effect Of Clk1 Gene On Aβ-induced Autophagy In HT22 Cells And Its Mechanism

The Clk1 gene was first discovered in Caenorhabditis elegans and is considered to be a gene related to longevity.The mutation of Clk1 gene in Caenorhabditis elegans will prolong its lifespan.Similarly,the lifespan of Clk1^+/-mice was also significantly prolonged in mice,accompanied by an increase in hypoxia-inducible factor-1α（HIF-1α）.Therefore,it is speculated that Clk1 gene may be related to age-related diseases.Alzheimer’s disease（AD）is an age-related neurodegenerative disease characterized by progressive memory loss,cognitive dysfunction,behavioral changes,and personality changes.It has been reported in the literature that after intracerebroventricular injection of adeno-associated virus r AAV-HIF-1αin AD model rats,HIF-1αcan be highly expressed in the hippocampus and inhibit the apoptosis of hippocampal neurons.At the same time,iron chelating agent,as an inducer of HIF-1αexpression,has neuroprotective effect.When Clk1 gene is deleted,the level of HIF-1αis increased,so it is suggested that Clk1 may be closely related to the occurrence and development of age-related diseases including AD.One of the main pathological features of AD is senile plaques formed by the deposition of amyloidβ-protein（Aβ）.Our previous study showed that the learning and memory ability of elderly Clk1^+/-mice was improved compared with wild-type mice.The mechanism may be to reduce the production of BACE1 and reduce the content of Aβin hippocampus.On this basis,further studies have found that the AD model Clk1^+/-mice injected with Aβin the lateral ventricle also have better learning and memory ability than the wild-type model mice,but the specific mechanism is still unclear.Recent studies on autophagy have shown that abnormally regulated autophagy may be related to the development of neurodegenerative diseases such as AD.Clk1 gene deletion can inhibit autophagy by regulating mTOR.Therefore,this experiment will explore the possible mechanism of Clk1 gene deletion to improve AD cognitive dysfunction from the perspective of autophagy.In this study,HT22 cells were cultured with different concentrations of Aβ_25-35for 24 h,and the cell viability was observed.The optimal concentration of Aβ_25-35cell injury was screened by MTT assay.The transcription process of Clk1 gene in cells was interfered by si RNA transfection technique.After 24 h,the level of Clk1 in cells was detected by real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）and Western blotting,and the best Clk1 interference sequence was screened.After silencing Clk1 gene for 24 h,normal medium and medium containing Aβ_25-35were added respectively.Cell viability was detected by MTT assay.The expression of autophagy marker microtubule-associated protein 1 light chain 3（LC3）was observed by immunofluorescence.Western blotting was used to detect the expression of PI3K,p-AKT/AKT and p-mTOR/mTOR proteins.The expression of p-AKT and p-mTOR was observed by immunofluorescence.Western blotting and RT-q PCR were used to detect the expression of autophagy-related factors Beclin1,LC3,p62 protein and m RNA.The results showed that 20μmol·L^-1Aβ_25-35could significantly reduce the viability of HT22 cells for 24 h（P<0.01）.The expression of Clk1m RNA and protein was significantly decreased by si RNA transfection,while the expression of HIF-1αprotein was increased.MTT results showed that the viability of HT22 cells in NC+Aβgroup was significantly lower than that in NC+NS group（P<0.01）,while the viability of si Clk1+Aβgroup was significantly higher than that in NC+Aβgroup（P<0.01）.Immunofluorescence results showed that the expression of LC3 in NC+Aβgroup was significantly higher than that in NC+NS group（P<0.01）,and the expression of LC3 in si Clk1+Aβgroup was significantly lower than that in NC+Aβgroup（P<0.01）.Western blotting results showed that compared with NC+NS group,the expression of PI3K,p-AKT/AKT and p-mTOR/mTOR protein in NC+Aβgroup was significantly decreased（P<0.01）.Compared with NC+Aβgroup,the expression of PI3K,p-AKT/AKT and p-mTOR/mTOR protein in si Clk1+Aβgroup was significantly increased（P<0.01）.The change trend of p-AKT and p-mTOR was detected by immunofluorescence,and it was found that the change of p-AKT and p-mTOR fluorescence intensity was consistent with the change of protein.Compared with NC+NS group,the expression of Beclin1 and LC3-II/I protein in NC+Aβgroup was significantly increased（P<0.01）,and the expression of p62 protein was significantly decreased（P<0.01）.Compared with NC+Aβgroup,the expression of Beclin1 and LC3-II/I protein in si Clk1+Aβgroup was significantly decreased（P<0.01）,and the expression of p62 protein was significantly increased（P<0.01）.The addition of LY294002 and YC-1 reversed the effects of silencing Clk1 on Beclin1,LC3-II/I,p62,PI3K,p-AKT/AKT,and p-mTOR/mTOR proteins.RT-q PCR results showed that there was no significant difference in Beclin1,LC3 and p62 m RNA levels between si Clk1+Aβ+YC-1 group and si Clk1+Aβgroup（P>0.05）.The changes of Beclin1,LC3 and p62 m RNA and protein levels were consistent among the other groups.Silencing Clk1 expression can alleviate the decrease of cell survival rate caused by excessive autophagy in HT22 cells induced by Aβ_25-35,and its mechanism may be related to PI3K/AKT/mTOR signaling pathway and HIF-1α.