Influence Of Puromycin-sensitive Aminopeptidase On Apoptosis Induced By Tau^P301L In Cells

BackgroundAlzheimer’s disease was the Neurodegenerative diseases which occurred in the cerebral cortex mostly in the elderly.The main clinical features included Progressive deterioration of memory and cognitive function, impairment of daily living function. Epidemiological studies have found that the prevalence of dementia was5%in the elderly over65. The percentage of people with Alzheimer’s disease doubles every five years after age65, The prevalence of dementia is increasing with the percentage of older people increasing each year. It not only brings severe pain to patients, but also brings a heavy burden to our families and society. So AD has become a serious social problem and has caused widespread concern.Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and profound memory loss. Brains affected by AD display widespread neuronal loss and gross atrophy of the cortex and hippocampus. AD is characterized by lesions throughout the brain that are caused by deposition of amyloid beta peptide (Ab) to form plaques, and aggregation of highly phosphorylated Tau protein to form neurofibrillary tangles. These pathological features continue to develop over the course of the disease tangle formation, rather than amyloid plaque deposition, is better correlated with the cognitive decline observed in AD patients. Interestingly neuronal loss is not found in preclinical AD cases and in cognitively intact persons with substantial numbers of amyloid-(A) plaques. The precise molecular mechanisms by which neurons are dying in AD are not yet fully elucidated. There is growing evidence for apoptosis as a major mechanism contributing to neuronal loss in AD. However, the closest correlate may be neurons density, since analyses of AD brains revealed that the extensive neurons loss that occurs in disease strongly correlates with cognitive impairment. Much of the work related to synapse loss in AD has focused on pathological Tau. West has found that the loss of neurons is closely related to the pathological Tau.PSA/NPEPPS (EC3.4.11.14), originally described as MP110, is a conservative aminopeptidase that hydrolyzes N-terminal amino acids and belongs to the M1metalloprotease family. PSA/NPEPPS is mainly a cytoplasmic protein that is most abundant in the brain protein puromycin-sensitive aminopeptidase (PSA/NPEPPS). Indeed, PSA/NPEPPS may have other functions in the central nervous system (CNS). For example, it was demonstrated that PSA/NPEPPS is a major peptidase responsible for the degradation of polyglutamine repeats, implicating this enzyme in the pathogenesis of polyQ diseases, including Huntington disease. Another recent report links PSA/NPEPPS function to autophagy and demonstrates its ability to facilitate removal of other neurotoxic substrates such as polyQ-expanded huntingtin exon-1, ataxin-3, mutant a-synuclein and SOD1. Karsten found that PSA protected against Tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. he further show that human PSA directly proteolyzes Tau in vitro. Kudo found that double-transgenic animals for hPSA and TauP301L transgenes demonstrated a distinct trend for delayed paralysis and showed significantly improved motor neuron counts, no gliosis and markedly reduced levels of total and hyperphosphorylated Tau in the spinal cord, brain stem, cortex, hippocampus and cerebellum of adult and aged animals when compared with TauP301L mice. Therefore he concluded that increasing PSA/NPEPPS activity may be a feasible therapeutic approach to eliminate accumulation of unwanted toxic substrates such as Tau. However the specific molecular mechanism remains unclear. Objective:To explore the effect of puromycin-sensitive aminopeptidase (PSA) on apoptosis induced by TauP301Lin SH-SY5Y cells and PC12cells.Methods:1. Construction the expression vector of pcDNA3.1-TauP301L and transfection it into SH-SY5Y cells and PC12cells.2. MTT method was used to detect the proliferation of SH-SY5Y cells and PC12cells treated with TauP301L24h,48h and72h.3. Morphological changes were detected by fluorescence microscopes after staining with Hoechst33342.4. In order to detect the effect of over expression PSA on cells, PC12cells were randomly divided into four groups and treated with pcDNA3.1, TauP301L, PSA, PSA+TauP301L, respectively. In order to detect the effect of knock down PSA on cells, SH-SY5Y cells were randomly divided into five groups and treated with pcDNA3.1, TauP301L, control RNA, PSA-siRNA, PSA-siRNA+TauP301L, respectively..The expression of Caspase-3, PSA and TauP301L were detected by Western blotting, the activity of Caspase-3were detected with Microplate reader. and the percentage of apoptosis was determined by flow cytometer.Results:1. Proliferation of SH-SY5Y cells and PC12cells were inhibited after TauP301L transfection48h (P<0.01).2. The observed morphological changes indicated that cells apoptosis were induced by TauP301L.3. Compared with pcDNA3.1group, the activity and expression of Caspase-3were increase (P<0.01) in TauP301L group, as well as the apoptotic rate. The apoptotic rate induced by TauP301L was declined by over expression PSA in PC12cells (P<0.01)and the activation of Caspase-3induced by TauP301L was declined (P<0.01)by over expression PSA in PC12cells, the activity of Caspase-3was decrease(P<0.01). The apoptotic rate and the activation of Caspase-3induced by TauP301L was increased (P<0.01) by PSA-siRNA transfection in SH-SY5Y cells, the activity of Caspase-3was enhanced (P<0.01). Conclusion:TauP301L could induce apoptosis in SH-SY5Y cells and PC12cells after transfection. The apoptosis induced by TauP301L was declined by over expression PSA in PC12cells while the apoptosis induced by TauP301L was enhanced by interfering PSA expression in SH-SY5Y cells. PSA has a protective effect on nerve cells by inhibiting apoptosis induced by TauP301L.