αv And β₁ Integrins Mediate The Aβ-induced Neurotoxicity To Primary Cultured Hippocampal Neurons Via FAK Signal Pathway

Part I Primary hippocampal neurons cultured, identified and the vitro Ap-induced neurotoxicity model establishedObjective To separate and culture the rat hippocampal neurons in vitro and identify the purity of neuronal cells. To establish a robust A(3 neurotoxicity model in vitro.Methods The hippocampus from 1 d SD rats were isolated and the hippocampal neurons were cultured as previously described by literature. The purity of neuronal cells were analyzed by microtubule-associated protein-2 (MAP-2) immunocytochemical analysis and the neurons viability treated by aged fibrillar Aβof different dose and time was detected utilizing the colorimetric 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to explore the optimal A(3 concentration and treating time to establish a AP neurotoxicity model in vitro.Results The isolated and cultured cells showed the characteristic manifestations of neuron. The purity of cells was quantificated over 95% identified through MAP-2 immunostaining which indicated it can be use in the subsequent experiments. The survival of the neurons treated with aged fibrillar Aβdemonstrated a dose-and time-dependent manner. Compared with the control neurons, the cell viability reduced to 63.5% and 46.9% treated with 10μM Aβfor 3 d and 4 d, respectively.Conclusion The primary hippocampal neurons can be successfully cultured according the literature. The Aβ-induced neurotoxicity disclosed a dose-and time-dependent feature and 10μM Aβtreated the neurons for 3 d was used as a standard induction in the subsequent studies basis on the experiment design. Part II Construction and screening an effective anti-FAK Of hippocampal neurons RN Ai lentivirus vectorObjective To construct FAK gene-targeted small interfering RNA (siRNA) and its lentivirus vector, and to screen out the most efficient pair of siRNA following transfecting the primary hippocampal neurons with the vector.Methods Three pairs of siRNA sequences (S1,S2,S3) anti-FAK of hippocampal nerrons were designed, synthesized, enzyme digested, transformed, identified by PCR, DNA sequenced and then packed by lentivirus. After transfecting the neurons by the lentivirus, the rate of transfecting was detected by fluorescence microscope and the expressions of FAK gene and protein were investigated by real-time PCR and Western blotting, respectively.Results Three siRNA sequences were constructed and transfected into hippocampal neurons. The rate of transfection was over 90% when the MOI was 10 and 5μg/mL polybrene was added. Compared to S2 and S3, S1 sequence had more significantly efficient which can silence 65% of FAK gene.Conclusion Screen out the most efficient transfecting condition and the siRNA sequence S1, which will provide experimental basis for researching the role of integrin in the progress of AD. PartⅢav andβ1 integrins mediated Aβ-induced neurotoxicity to hippocampal neurons and the mechanism was investigatedObjective To investigate the effect of av andβ1 integrins mediating the neurotoxicity induced by Aβto primary hippocampal neurons and to detect the underlying mechanism.Methods 1) The hippocampal neurons were treated by 2.5μg/mL 17E6 and 5μg/mL ab58524, the specific antibodies of av integrin andβ1 integrin, respectively, for 24 h prior to 10μM Aβtreatment. The MTT cell viability assay, flow cytometry and TUNEL immunostaining were determined to detect cellular events that have undergone cell apoptosis due to the Aβ-induced neurotoxicity. 2) Small interfering RNA was utilized to silence focal adhesion protein (FAK), a downstream target gene of integrin. The condition of siRNA was obtained that the siRNA sequence was T1, the MOI was 10, and 5μg/mL polybrene was added. The neurons were transfected according with the condition above and the apoptosis of variety groups'cells was detected. 3) Using real-time PCR and Western blotting, the expression level of FAK, pFAK genes and proteins were investigated to explore the underlying mechanism and the signaling pathway by which av andβ1 integrins mediated the Aβ-induced neurotoxicity to hippocampal neurons.Results 1) MTT assay showed that both 17E6 and ab58524 significantly increased the cell viability compared to the AP treated neurons (P<0.01, P<0.05, respectively). However, the protective effect markedly attenuated after transfection to siFAK. 2) Compared to Aβtreated cells, the TUNEL immunostaining and the flow cytometry indicated that both 17E6 and ab58524 significantly protected hippocampal neurons against apoptosis induced by Aβ(P<0.05). Nevertheless, the protective effect was revoked by siFAK. 3) The gene and protein level of FAK both increased after AP treatment. Interesting, compared with the gene and protein level of FAK decreased, the pFAK protein expression markedly raised. Moreover, the gene and protein expression of FAK and pFAK both significantly degraded.Conclusion Both av and (31 integrins interfered in the neurotoxicity induced by A(3 to hippocampal neurons, and the mechanism may be partly contributed to the activation of Itg-FAK signaling pathway.