To Investigate The Regulatory Effects Of DAPK1-mediated CRMP1 In Parkinson’s Disease Cell Model

Objectives:Parkinson’s Diseases(PD)is a common neurodegenerative disease,and selective apoptosis of neurons is one of the main pathological changes of PD,and its pathogenesis is closely related to axonal degeneration of dopaminergic neurons.It is known that DAPK1 protein plays a role in regulating apoptosis,while CRMP1 plays a key role in mediating the growth of neuronal protrusions.This study focused on the protein expressions of DAPK1 and CRMP1 in PD cell model and the relationship between them,as well as the effects of DAPK1 on axonal degeneration and apoptosis in PD cell model.Methods:(1)Primary cortical neurons were cultured in vitro for different time periods(1d,3d,5d,7d,14d,and 21d),and the time expression of DAPK1 and CRMP1 proteins at cell level was detected by Western blot.Primary neurons were treated with MPP~+for 8h to establish PD cell model.Western blot and immunofluorescence(IF)were used to detect the expression of tyrosine hydroxylase(TH)protein,and the PD model was successfully established.The expression levels of DAPK1 and CRMP1 in PD cell model were detected by Western blot.(2)The p RK5-DAPK1-flag and p CMV-CRMP1-HA plasmids were co-transfected in HEK293 cells,and the relationship between DAPK1and CRMP1 was detected by immunoprecipitation(CO-IP).The immunofluorescence method was used to detect the co-localization of DAPK1 and CRMP1 in primary neurons.The cerebral cortex proteins of C57BL/6J mice in different days after birth(1d,3d,5d,7d,14d,21d,and28d)were extracted,the time expression patterns of DAPK1 and CRMP1proteins at the animal level were detected by Western blot,and the mutual binding of DAPK1 and CRMP1 at the animal level was detected by CO-IP.(3)The p RK5-DAPK1-flag plasmid was transfected into the SH-SY5Y conventional cell line to overexpress DAPK1,and Western blot was used to detect the expression of CRMP1.(4)The DAPK inhibitor was used to inhibit the expression of DAPK1 in normal primary cultured neurons,and Western blot was used to detect the expression of CRMP1.The lentivirus interfering plasmid p LKO.1-sh DAPK1 was constructed by ourselves with p LKO.1-TRC as the vector.sh DAPK1lentivirus was packaged by HEK293 cells and infected with SH-SY5Y cells,which were screened and passaged by puromycin,and the SH-SY5Y stably transfected cell line(sh DAPK1-SH-SY5Y)with DAPK1 knockdown was constructed.Western blot was used to detect the expression of CRMP1 in the sh DAPK1-SH-SY5Y cell line.The sh DAPK1 lentivirus was used to infect the normally cultured primary neurons,and Western blot was used to detect the expression of CRMP1.At the same time,the lentivirus sh CRMP1 was used to infect normal neurons,and Western blot was used to detect the expression of DAPK1.(5)In the PD cell model,the infection of sh DAPK1 lentivirus inhibited DAPK1 expression,while Western blot was used to detect CRMP1expression.(6)In the PD cell model,lentivirus infects sh DAPK1,and the axonal rupture and swelling of neurons are observed by immunofluorescence.After being treated with MPP~+for 24h in a SH-SY5Y stably transfected cell line with lentivirus knockout DAPK1(sh DAPK1),Western blot was used to detect the expression levels of Bax,Bcl-2 and Caspase-3 apoptosis-related proteins,and flow cytometry was used to detect the apoptosis.The PD cell model was infected with lentivirus sh DAPK1,and the expression level of apoptosis-related proteins was detected by Western blot.Results:(1)In the primary cortical neurons cultured in vitro,Western blot results showed that the expression of DAPK1 was high on days 3d,5d,and 7d,and the expression of CRMP1 was high on days 5d,7d,and 14 d.The expression of TH protein was decreased by Western blot and IF detection,and the results both proved that the PD cell model was successfully established.In the PD cell model,the DAPK1 and CRMP1 proteins detected by Western blot were significantly higher than those in the control group.(2)The overexpression of DAPK1 and CRMP1 in HEK293 cells revealed that they could bind to each other by CO-IP.The results of IF detection in primary neurons showed that DAPK1 and CRMP1 were co-located in neurons.In the cerebral cortex tissues of mice of different ages,Western blot results showed that the expression levels of DAPK1 and CRMP1 were high on the 5th and 7th day,and significantly decreased on the 14th day.CO-IP results showed that DAPK1 and CRMP1 also had interaction at the animal level.(3)DAPK1 was overexpressed in the SH-SY5Y conventional cell line,and Western blot results showed that the CRMP1 protein increased with the increase of DAPK1.(4)In normal primary cultured neurons where DAPK inhibitor was used to inhibit DAPK1 expression,Western blot results showed that the expression of CRMP1 protein was decreased.Western blot showed that the expression of DAPK1 in lentivirus-infected SH-SY5Y cells was decreased,indicating that the sh DAPK1-SH-SY5Y stably transfected cell line was successfully constructed.Western blot results showed that CRMP1 expression was decreased in both the sh DAPK1-SH-SY5Y cell line and the primary cultured neurons infected with sh DAPK1 lentivirus.However,lentivirus sh CRMP1 inhibited CRMP1 expression in normal primary neurons but did not affect DAPK1protein expression.(5)In the PD cell model,lentivirus sh DAPK1 inhibits DAPK1 expression and then down-regulates the expression of CRMP1protein.(6)In the PD cell model,lentivirus inhibits the expression of DAPK1 in neurons,and the immunofluorescence results show that the incidence of neuronal axonal rupture or swelling is significantly decreased.At the same time,in the SH-SY5Y stably transfected cell line with sh DAPK1 knock-down,Western blot and flow cytometry tests indicated that the incidence of apoptosis was decreased significantly.Lentiviruses in the PD cell model inhibited DAPK1,and Western blot results showed that the expression of apoptosis-related protein Bcl-2 was increased,while the expression of Bax and Caspase-3 was decreased.Conclusion:(1)The expressions of DAPK1 and CRMP1 proteins in the PD cell model were increased;(2)There was an interaction between DAPK1 and CRMP1,and DAPK1 could regulate the expression of CRMP1 in PD cell model.(3)In PD cell model,inhibition of DAPK1 can alleviate neuronal axonal degeneration and apoptosis.