P4-241: Pathogenesis of intracellular amyloid beta-protein 42 and P53: A novel therapeutic target in Alzheimer’s disease

Abstract

Recent major therapeutic strategies of Alzheimer's disease (AD) are to inhibit amyloid beta–protein (Abeta) generation and aggregation, and to promote extracellular Abeta removal, because extracellular Abeta, especially oligomeric form, induces neurodegeneration. Intracellular Abeta42 is also suggested to intimately cause neurodegeneration in AD because, i) the pathogenic Abeta42 is accumulating in the neurons at early stage in AD and mouse model; ii) intracellular Abeta42 causes neuronal apoptosis at quite low levels; and iii) neuronal loss but not plaque formation correlates with the severity of dementia. Recently, we have reported that intracellularly accumulating Abeta42 activates p53 promoter resulting in p53 mRNA overexpression and apoptosis in vitro (Ohyagi et al., FASEB J 19: 255–257, 2005). Also, intracellularly accumulating Abeta42 has recently been reported to induce mitochondrial and synaptic damage. Since cytosolic Abeta42 is degraded by proteasome, activation of proteasome may decrease the cytosolic Abeta42 levels and reduce the risk of neuronal apoptosis. To find new drugs to promote cytosolic Abeta degradation and to protect neurons from apoptosis, we intended to establish an in vitro assay system using cultured cells. Synthetic Abeta40 peptide was utilized because of more water solubility. At first, human neuroblastoma cells (SH–SY5Y) were incubated with ∼200 μg/ml Abeta40 peptide in the hyperosmotic medium for 10 min. The cells were then incubated in hypo–osmotic medium for 2 min. After that, we studied decreasing process of intracellular Abeta40 from 0 to 30 min by immunocytochemistry and Western blot. Furthermore, we treated the cells with a proteasome inhibitor, MG132, to inhibit Abeta degradation and some drugs to improve Abeta degradation efficacy. Also, intracellular p53 protein levels were checked by Western blot; and cell viability was evaluated by the Cell Titer Blue method similar to MTT assay. At present, we have identified a reagent that proves to markedly enhance cytosolic Aβ degradation/p53 degradation and protect cells from apoptosis. Such a drug may be a newcomer as candidate drugs to inhibit neurodegeneration in AD.