The Preliminary Study Of Molecular Mechanism Of Beta-amyloid-induced Human Neuroblastma Cell Apoptosis

Amyloid protein(Aβ)is a 42 amino acid peptide,derived fromβ-amyloid precursor protein that accumulates as an insoluble deposit around neurons in Alzheimer's disease(AD).Increased deposition of Aβis thought to contribute to the pathophysiology of AD by inducing neuronal cell death,consequences of which include alterations in AD patients's behaviour and cognition.It has been reported that neuronal apoptosis,calcium dysregulation and oxidative stress are implicated in Aβneurotoxicity.However,the mechanisms,factors and genes involved in this neurotoxic effect have not been yet clearly identified.In the present study,functional genomics, proteomics and pharmacogenomics approach such as high-density microarray, fluorescence real-time quantitative PCR(QPCR),two-dimensional liquid chromatography,RNAi and western blotting techniques were applied to analyze the mechanism ofβ-amyloid-induced apoptosis in cultured SH-SY5Y neuroblastoma cells,a well-characterized cell line that has been extensively used as a neuronal model in AD-related research.1.Gene expression profile in Aβ_1-42-treated SH-SY5Y neuroblastoma cells SH-SY5Y cells were treated with Aβ_1-42and cell viability was measured by CCK-8 kit.Apoptosis was detected by Hoechst33342 staining and Annexin V-Cy5/Calcein staining.The gene expression profile has been determined using the Agilent GeneChip Human 1A(V2).Aβ_1-42inhibited cell growth in dose-dependent manner and induced neuronal apoptosis.The expression of fourty-six genes was altered including Caspase-2,Caspase-3,Bax,TP53 and TRAF1.2.Proteomic analysis of SH-SY5Y exposed to Aβ_1-42In order to provide a more complete picture of mechanism in Aβ_1-42-induced neuronal apoptosis,a proteomic analysis was carried out with Aβ_1-42treatment in SH-SY5Y cells.ProteomeLab^TMPF 2D Protein Fractionation System was used to compare the levels of proteins in cell lysates from SH-SY5Y cells exposed Aβ_1-42 (10μM)for 24h to those in control incubation.Seventy-two proteins were differentially expressed.Three proteins had been identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Aβ_1-42 exposure of SH-SY5Y cells led to increased histone protein and decreased eIF5A and PDIA6.3.The role of Caspase-2 and TP53 in Aβ_1-42-induced neuronal apoptosis In order to investigate the role of Caspase-2 and TP53 in Aβ_1-42-induced SH-SY5Y cell apoptosis,exogenous Caspase-2 or siRNA for Caspase-2 was transfected into SH-SY5Y cells respectively to overexpress Caspase-2 or inhibit the expression of endogenous Caspase-2;Pifithrin-a was applied to block its transcription activity and siRNA for TP53 was transfected for reducing the TP53 expression. Overexpression of Caspase-2 caused apoptosis and exhibited no enhanced effect on apoptosis by Aβ_1-42.Apoptosis-induced by Aβ_1-42was not affected by reduction of Caspase-2.Apoptosis-induced by Aβ_1-42was also not repressed by pifithrin-αor siRNA for TP53.The ratio of Bax/Bcl-xl and the expression level of Caspase-3 was increased by Aβ_1-42.However,the effects of Aβ_1-42was abolished by pifithrin-αcombined with siRNA-mediated knockdown of Caspase-2,or by siRNA-mediated knockdown of Caspase-2 and TP53,demonstrating that both Caspase-2 and TP53 were required for Aβ_1-42-hiduced apoptosis.4.Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by Aβ_1-42A therapeutic target for AD is to block the cascade reaction induced by Aβ.It has been demonstrated that Glucagon-like peptide-1(GLP-1),which is an endogenous insulinotropic peptide secreted from the gut,binds to its receptor in the brain and possesses neuroprotective effects.Using site-directed mutagenesis and gene recombination techniques,we generated a mutated recombinant human Glucagon-Like Peptide-1(mGLP-1)which has longer half-life as compared with native GLP-1.This present work aims to examine whether mGLP-1 attenuates Aβ_1-42-mediated cytotoxicity in SH-SY5Y cells and to explore the possible mechanisms.Our data indicate that≥0.02ng/mL of mGLP-1 facilitated cell proliferation and 0.1ng/ml and 0.5ng/ml of mGLP-1 rescued SH-SY5Y cells from Aβ_1-42-induced apoptosis.Moreover,Aβ_1-42treatment dramatically stimulated the release of Ca²⁺from internal calcium stores in SH-SY5Y cells,while mGLP-1 helped to maintain the intracellular Ca²⁺homeostasis.Aβ_1-42also significantly increased the expression level of TP53 and Bax genes which are involved in apoptotic pathways, and mGLP-1 decreasedAβ_1-42-induced up-regulation of TP53 and Bax.Since mGLP-1 treatment elevated cytosolic cAMP concentration in SH-SY5Y cells,we postulate that mGLP-1 may exert its influence via binding to GLP-1 receptors in SH-SY5Y cells and stimulating the production of cAMP.These results suggest that mGLP-1 exhibited neuronal protection properties,and could potentially be a novel therapeutic agent for intervention in Alzheimer's disease.5.Effects of lysophosphatidylcholine onβ-amyloid-induced neuronal apoptosisDysfunctional lipid homeostasis has a significant causative impact on the initiation and progression of AD.It has been known thatβ-amyloid can induce lipid peroxidation.However,the relationship between the lipid peroxidation and Aβ_1-42 induced neuronal apoptosis remains to be elucidated.We have investigated the effects of lysophosphatidylcholine(LPC),a product of lipid peroxidation,on Aβ_1-42-induced SH-SY5Y cell apoptosis.Our results showed that long-term exposure of SH-SY5Y cells to LPC augmented the neurotoxicity of Aβ_1-42.Furthermore,the Bax/Bcl-xl ratio and the expression levels as well as the activity of Caspase-3 were elevated,while the expression level of TRAF1 was reduced after LPC treatment.Since LPC was reported as a specific ligand for an orphan G-protein coupled receptor G2A,we investigated LPC-mediated changes of calcium level in SH-SY5Y cells.Our results demonstrated that LPC could enhance Aβ_1-42-induced elevation of intracellular concentration of calcium.Interestingly,Aβ_1-42could significantly increase the expression of G2A in SH-SY5Y cells,while the knockdown of G2A using siRNA could reduce the effects of LPC on Aβ_1-42-induced neurotoxicity.These results suggested that the effects of LPC on Aβ_1-42-induced apoptosis could be through the signal pathways of the orphan G-protein coupled receptor.