Dissertation > Medicine, health > Pharmacy > Pharmacology

Effects and Mechanism of Nicotinergic Compounds on β-Amyloid in SH-EP1-α4β2 nAChR-hAPP695 Cell Strain

Author NieHuiZhen
Tutor YinMing
School Shanghai Jiaotong University
Course Pharmacology
Keywords Alzheimer’s disease α4β2 nicotinic acetylcholine receptors SH-EP1 cell promoter activity analysis
Type PhD thesis
Year 2008
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Amyloid hypothesis of Alzheimer’s disease indicates that amyloidβ-peptide is the major causative agent of the disease process. Amyloid precursor protein (APP) is processed following two different pathways, the so-called amyloidogenic and non-amyloidogenic processing, there is no release of intact Ab peptide, and the generated stubs do not aggregate and have no amyloidogenic activity. In Alzheimer’s disease pathology, the production of b-amyloid peptide is the result of APP amyloidogenic processing. This involves the activity first ofβ- and then ofγ-secretase. When abnormal mutations occur in APP genes, the amyloidogenic processing prevails, which results in increase of Aβ. Aβaggregate into oligomers, which are toxic to neurons, and induce inflammation, oxidation stress, hyperphosphorylstion of tau protein, and tangle of fiber.To date, the primary treatments for Alzheimer’s disease with proven efficacy have been acetylcholinesterase inhibitors and NMDA receptor antagonist memantine. Although these agents have some benefit in alleviating cognitive impairment, they have limited clinical utility because of insufficient efficacy and marginal tolerability. Within the last decade, there has been much experimental supports for the use of therapeutics that directly target nicotinic acetylcholine receptors (nAChRs) to improve cognitive function and slow neurodegenerative disease progression. There is abundant evidence that nAChR modulators have the potential to alleviate cognitive impairment in demented states. In addition to improving cognitive function, a large body of research implicates a role for nAChRs in neuroprotection, suggesting potential for disease modification. These findings have spurred considerable research efforts to develop ligands selective for nAChRs.nAChRs are ligand-gated ion channels with a pentameric structure formed by a combination of five different subunits (α,β,γ,δandε), each encoded by different genes. The most widely spread subtype in the central nervous system isα4β2, which are highly expressed in brain regions that develop Alzheimer’s disease neuropathology, thus implicating these receptors in the pathogenesis of this dementia. Other data showed that there is a decrease in the number of brain nAChRs, particularlyα4-containing receptors, in the absence of a general decrease in the number of neurons.α4β2 nAChRs is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human APP695 gene into SH-EP1 cells which have been transfected with human nicotinic receptorα4 subunit andβ2 subunit gene, to observe the effects ofα4β2 receptor activation onβ-amyloid, expecting to provide a new cell line for drug research. Additional, gene promoter activity analysis was conducted to fined the effective method of drug screening in the cell line.Liposome transfection was used to express APP695 in SH - EP1 -α4β2 nAChR cells. Function of the transfectedα4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selectiveα4β2 nicotinic receptor agonist) onβ-amyloid were detected by Western blotting and ELISA. Effects of nicotine and epibatidine on APP 695,β-secretase BACE1,γ-secretase PSEN1subunit, ERK1, NFκB P65 subunit mRNA level was measured using real-time PCR. Luciferase reporter gene assay was used to analysis promoter activity of PSEN1 and BACE1.Another objective of this study was to find a prompt and economical method of highthrough screening in the constructed SH-EP1- 4β2 nAChR-APP695 cell line. Dual-luciferase reporter gene was used to measure gene promoter activity in experiment. pGL3-BASIC plasmids containing PSEN1 and BACE1 promoter gene were constructed for dual-luciferase analysis , to observe effects of nicotine on PSEN1 and BACE1 promoter activity.Human APP695 gene was stably expressed in SH-EP1-α4β2 nAChR cells; Neomycin was used to screen cells that expressing neo gene. Limiting dilution assay was applied to get single clones and 36 single clones were selected totally. APP695 mRNA expression were confirmed in 16 of the 36 single clones by RT-PCR.α4 subunit gene andβ2 subunit mRNA expression were confirmed in 4 of the 16 single clones that expressing APP695 mRNA. APP695 protein expression was detected by western blotting, and cell clone numbered 1C8 had the highest expression among the 4 cell clones. Patch clamp was used to confirm function ofα4β2 nAChR in 1C8 cell line and the result showed that when treated with 100μM nicotine,α4β2 nAChR was activated and introvert current was induced. With highly expression of APP695 and normal function ofα4β2 nAChR, 1C8 was selected as the cell line for further chemical compounds experiments. SH - EP1-α4β2 nAChR - APP 695 cell line, which co-expressingα4β2 nAChR and APP695, were constructed successfully.SH-EP1-α4β2 nAChR-APP695 were treated with nicotinic agonist nicotion, selectiveα4β2 subtype agonist epibatidine and selectiveα4β2 subtype antagonist DHβE. Nicotine (0.1μM, 1μM) and epibatidine (0.1μM) decreased intracellular and secretedβ-amyloid in the cells; and activation ofα4β2 receptors did not affect APP695 mRNA level, but decreased mRNA level ofβ-secretase BACE1,γ-secretase PSEN1, ERK1and NFκB P65 subunit.pGL3-BASIC plasmids containing PSEN1 and BACE1 promoter gene, pGL3-PSEN1-918, pGL3-BACE1-757, pGL3-BACE1-1876, and pGL3-BACE1-2000 were constructed with Bgl II and Hind III restriction enzyme. Plasmids were transfected into SH-EP1-α4β2 nAChR-APP695 cells by liposome. Luciferase activity of cells transfected with pGL3-BACE1-1876 and pGL3-BACE1-2000 was decreased with nicotine. BACE1 promoter activity was decreased by activation ofα4β2 nAChR.In conclusion:1. The cell line, expressing both APP695 gene and human nicotinic receptorα4 subunit andβ2 subunit gene, was constructed successfully. Activation ofα4β2 nAChR decreased incellular and secretedβ-amyloid level in SH-EP1-α4β2 nAChR-APP695 cell line. The results indicated that activation ofα4β2 nAChR might play positive role in prevention and therapy of Alzheimer’s disease.2. Alteration ofβ-amyloid level induced by activation ofα4β2 nAChR in our study occurs at a post-translational level. Nicotine inhibits BACE1 and PSEN1 expression and its activity through the MAPK and NFκB pathways in cell model.3. The constructed SH-EP1-α4β2 nAChR-APP695 cell line might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease and other central nervous diseases. BACE1 promoter activity analysis might be useful for drug screen as a high throughput screening method in SH-EP1-α4β2 nAChR-APP695 cell line .

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