The Neuroprotective Effect Of Neprilysin In Neuronal Apoptosis Induced By Amyloid-beta Peptide

Objective: The deposition of amyloid-beta peptide (Abeta), formed after sequentialcleavage of amyloid precursor protein (APP), is the key pathological features ofAlzheimer's disease. Neprilysin, one of known zinc metallopeptidases that mightplay a role in amyloid metabolism, may have a remarkable effect on Abetadegradation. Disruption of NEP gene can cause elevation of Abeta levels in mousecortex and hippocampus. In my study we overexpressed NEP in cultured rat corticalneurons, observed the protective effect NEP on neuron apoptosis induced by Aβ_25-35at both mRNA and protein levels and tried to find a new way of genetic interventionfor the neurodegenerative disease.Methods:(1) The plasmid pcDNA3.1 (+)-NEP was transformed into Escherichia coli JM109and amplified in the bacteria. The plasmid was extracted and purified fromthebacteria using the conventional method.(2)Plasmids were cut by enzymes and evaluated by electrophoresis to confirm theirintegrity.(3) Primary culture of Neuronal cells: Cerebral tissue from fetal Wistar rats was isolated and digested. We cultured the primary cortical neurons with Ara-C supplementing method to impress the growth of neuroglia, observed systematically the morphological properties of neurons in different stages. We assessed the purity of primary neurons by immunocytochemistry using anti-MAP2 antibody.(4) Cell injuries induced by Aβ_25-35: examined the cells survival rate with MTT and made the proper conditions (Aβ_25-35 15uM, for 24 hours).(5) Cell transfection: neurons were transfected with the plasmids by Lipofectamine^TM2000.(6) The changes of cells apoptosis were measured by MTT assay and flow cytometric DNA analysis. The expression of target mRNA and proteins were measured by RT-PCR and Western Blot.Results: Exposure of primary neurons to Aβ_25-35 resulted in changes of cell apoptosis. MTT assay showed that cell activity decreased remarkably (P<0.01) and cytometric DNA analysis showed an increase in the apoptosis rate. While in the neurons transfected with NEP genes, there were no such remarkable changes when treated with Aβ_25-35. Considerable expression of NEP could decrease APP in RT-PCR and Western Blot, and the expression of Bax decreased while Bcl-xl increased after transfection.Conclusion: Our hypotheisis is that the increasing level of Bcl-xl gene and decreasing level of Bax may indicate that NEP expression maybe neuroprotective against Abeta-evoked neuronal cell death and antagonize the pathological process of Alzheimer's disease. These studies provide evidence for further investigation how NEP gene regulating neuron apoptosis induced by Abeta.