The Regulatory Effect Of RCAN1 On BACE2 And TMP21 In Alzheimer's Disease And Underlying Mechanisms

Background:Alzheimer's disease(AD)is the most common form of dementia.It is a neurodegenerative disease characterized by neuritic plaques,neurofibrillary tangles and neuronal loss.Clinical manifestations of AD are characterized by progressive memory loss and cognitive impairment.The ?-amyloid protein(A?)is the main component of senile plaque,which is formed from ?-amyloid precursor protein by sequential cleavage of ?-and ?-secretase.Abnormal A? accumulation in brain is considered to be the key part of AD pathology.BACE2 is a conditional ?-secretase,and TMP21(transmembrane protein,21KD)regulates the activity of ?-secretase.The previous research has demonstrated that as the major isoform of RCAN1(regulator of calcineurin 1),abnormal increased expression of RCAN1.1L was detected in the brain of AD patient.However,the regulatory effect of RCAN1.1L on the expression of BACE2 and TMP21 has not been elucidated.Therefore,we will determine the regulatory effects of RCAN1.1L on the levels of BACE2 and TMP21 and underlying mechanisms in this thesis,providing a novel insight in the pathogenesis of AD.Objective:To determine the regulatory effects of RCAN1 on levels of BACE2 and TMP21 and mechanismsMethods:1.Degradation pathway of BACE2 protein?HEK293 cells transfected with plasmid BACE2-mycHis,as well as HEK293 cells and mouse cortical neurons were treated with CHX.Western Blot was performed to measure the level of exogenous or endogenous BACE2 protein and determine the half-life of exogenous or endogenous BACE2;?HEK293 and SH-SY5Y cells transfected with plasmid BACE2-mycHis,as well as HEK293 cells and mouse cortical neurons were treated with lysosomal inhibitors and proteasomal inhibitors,respectively.Western Blot was performed to measure the level of exogenous or endogenous BACE2 protein and determine the degradation pathway of BACE2.2.The regulatory effect of RCAN1 on BACE2?Semi-quantitative PCR was performed to measure the difference of BACE2 mRNA between HEK293 and HRNLM cells(stable overexpressing RCAN1.1L-mycHis in HEK293);?HEK293 and HRNLM cells were transfected transiently with plasmid BACE2-mycHis.Western Blot was performed to measure the difference of exogenous BACE2 protein;?HEK293 and HRNLM cells transiently transfected with plasmid BACE2-mycHis were treated with CHX,and the difference of the half of exogenous BACE2 was examined;?HEK293 and HRNLM cells transiently transfected with plasmid BACE2-mycHis were treated with lysosomal inhibitor and proteasomal inhibitor.Western Blot was performed to measure the difference of the level of BACE2 protein and determine the regulatory effect of RCAN1 on BACE2 degradation.3.The regulatory effect of RCAN1 on TMP21Semi-quantitative PCR and Western Blot were performed to measure the difference of the level of TMP21 mRNA and protein in HEK293 and HRNLM cells.Results:1.BACE2 is degraded by the lysosome pathway and the proteasome pathway?The half-life of exogenous BACE2 in HEK293 cells is approximately 6h,and the half-life of endogenous BACE2 in HEK293 cells and primary cortical neurons is approximately 4h;?Both exogenous and endogenous BACE2 is degraded by the lysosome pathway and the proteasome pathway in different types of cells.2.RCAN1 inhibits the degradation of BACE2?There is no difference of endogenous BACE2 mRNA between HEK293 and HRNLM cells;?The level of exogenous BACE2 protein is up-regulated in HRNLM cells,compared to that in HEK293 cells;?The half life of BACE2 protein is longer in HRNLM cells than that in HEK293 cells;?RCAN1 inhibits the proteasome pathway-mediated BACE2 degradation.3.RCAN1 increases the expression of TMP21The levels of TMP21 mRNA and protein are significantly up-regulated in HRNLM cells,compared to that in HEK293 cells.Conclusions:1.BACE2 is degraded by both the lysosome pathway and the proteasome pathway;2.RCAN1 inhibits the proteasome pathway-mediated BACE2 degradation;3.RCAN1 promotes the expression of TMP21 by increasing its transcription,but the effect of RCAN1 on TMP21 expression may not be mediated by the Calcineurin/NFAT pathway,and the specific mechanism is still unclear.