Effect Of The Loss Of Presenilin1on Motochondrial PARL-OPA1Apoptotic Pathway

Loss of presenilins (PS) causes progressive neurodegeneration independent of amyloid-β (Aβ) deposition. Mitochondrial abnormalities are common events in Alzheimer’s disease. We attempted to explore whether loss of PS would exert influence on mitochondrial construction and function and further to reveal the underlying mechanism of mitochondria dependent apoptosis. In this study, we ablated presenilin1using small-interfering RNA (si-RNA) in SHSY5Y cell and further to verify the cell model in RNA and protein levels including selecting the preferable siRNA and performing time screen of RNA interference. Our findings showed that the expression of optic atrophy typel (OPA1) is responsible for cristae remodeling plus membrane integrity and its upstream protein presenilins-associated rhomboid-like (PARL) were down-regulated. In addition, apoptosis factor cytochrome c (cyt-c) was releasing from mitochondria in PS1interference SHSY5Y cell. Together, active caspase3was also found in PS1interference SHSY5Y cell. The TUNEL assay that detects the DNA fragment also indicated the apoptosis in PS1interference SHSY5Y cell. So we come to the conclusion that loss of PS1influences mitochondrial function and induces mitochondria dependent apoptosis mediated by PARL-OPA1pathway. The apoptotic experiments also used short hairpin RNA interfere (sh-RNAi) SHSY5Y cell model established by others.1. The establishment and verification of PS1down-regulation SHSY5Y modelPresenilins (PS1and PS2) play the major causative role of familial Alzheimer’s disease and182FAD mutations have been links to PS1gene (4). Mice lacking both PS1and PS2in the forebrain showed progressive neurodegenerative symptoms including age-dependent memory decline, forebrain degeneration and progressive impariment in cognitive abilities such as problem solving and language. There is no Aβ deposition in the brain of this transgenic model, the reason and the mechanism of the pathological phenomena are not clarified yet. There may exit underlying mechanism of AD pathogenesis results from loss function of PS. Furthermore, the deficiency of PS causes a series of neuronal apoptosis alternations such as neuronal loss, astrogliosis and caspase3mediated apoptosis. We wanted to investigate whether PS1exerts direct influence on neurodegeneration and other functions of PS gene. Down-regulation of the specific gene and detecting some possibly relative phenomenon is a good way to uncover the function of a gene. So we established a cell model losing of PS1using RNAi technology. We designed three siRNA sequences and all these siRNA down regulated PS1expression dramaticly. We selected the best one to conduct the further research. The selected siRNA showed significant down-regulation for24h,36h,48h and60h after the transfection experiments and the best time is48h.2. Loss of presenilinl causes apoptosis related to mitochondrial PARL-OPA1 pathwayWe detected the apoptosis factor OPA1concerning on cristae remodeling and mitochondria membrane potential in both of the transient and constant transfection SHSY5Y cell. The alteration of up-stream protein the presenilins-associated rhomboid-like (PARL) was also detected. Knock down of PS1resulted in lower expression of OPA1and PARL both on RNA and protein levels.Because caspase3is a key down-stream protease essential for apoptosis, we examined whether the PS deficiency displays cleavage of caspase3, a reliable sign of the activation of apoptosis. Our result indicates that PS1alteration seems to cause apoptosis by activation of caspase3dependent mechanism, we found that the decrease of pre-caspase3and the increase of the active caspase3.Regarding mitochondrial dependent apoptosis, mitochondrial outer membrane is breached, numerous proteins of the mitochondrial intermembrane region came tumbling out especially cytochrome c and it effectively signs the death indicator for the cell. For PS1knock down SHSY5Y cell, we observed the releasing of cytochrom c from mitochondria both in sh-RNAi group and si-RNA group.To investigate how apoptosis appears in the absence of presenilinl, we performed TUNEL analysis to mark apoptotic SHSY5Y cell after dealing with H2O2of1mM for4h. Significant increase in TUNEL+SHSY5Y cell were shown in PS interference groups.