Cellular effects of APOE4: Implications for Alzheimer's disease

Abstract

The dominant risk factor for Alzheimer's disease (AD) is the epsilon-4 allele of apolipoprotein E (ApoE4). This allele confers increased risk for sporadic as well as familial AD disease. Individuals with two copies of the ApoE e4 allele have an approximately eight-fold increased risk of AD and have a significantly lower age of onset compared to AD patients not carrying this allele. Recent data indicate that the greater risk of AD associated with the ApoE4 isoform might relate to ApoE's susceptibility to proteolysis and neurotoxicity. However, the exact molecular mechanisms underlying ApoE and amyloid precursor protein (APP) interactions sub serving the risk-factor effect of ApoE4 remain unclear. The amyloid precursor protein, APP, has been shown to function as a molecular switch: cleavage at the beta, gamma, and caspase sites results in the production of four peptides-sAPPb (from which N-APP is derived), Ab, Jcasp, and C31-that mediate neurite retraction, synaptic reorganization, and ultimately programmedcell death. In contrast, cleavage at the alpha site produces the trophic peptide sAPPa and the inhibitor of APP gamma-site cleavage, aCTF. The decision between these two proteolytic pathways is governed at least in part by ligand binding: interaction with the axon guidance and trophic factor netrin-1 increases a-site cleavage, whereas interaction with the anti-trophin Ab inhibits alpha-site cleavage and increases net production of the four neurite-retractive peptides. Since this system exhibits positive feedback (e.g., Ab begets more Ab via APP processing, whereas aCTF inhibits Ab production), it is by definition prionic. Therefore, it is of interest to determine whether ApoE isoforms impact this trophic-anti-trophic peptide balance differentially, and, if so, by what mechanism. HEK-293T cells, B103-APP (rat neuroblastoma cells stably expressing human wild-type APP), 7W CHO cells (stably transfected with human APP751) and A172 human glioblastoma cells were used to determine whether ApoE, and in particular ApoE4, triggered alternative cellular APP processing that would provide a basis for understanding the cellular toxicity that has been reported previously. Transient transfection of cells was performed with Lipofectamine 2000. Whole-cell extracts were prepared and typically, 100mg of cell extract was loaded for SDS-PAGE and Western blot analysis. A total of 150-200 μg protein was also subjected to immunoprecipitation using specific antibodies to pull down the desired protein. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. The sAPPa, sAPPb, and Ab levels were obtained using the PerkinElmer (PE) AlphaLisa assay, and measurements were performed on an AlphaLisa reader. Immunocytochemistry was performed on cells 24 hrs after transfection with APP, ApoE, or the two in combination. The ApoE expression constructs were a kind gift from Dr. Yadong Huang of the Gladstone Institute. Cells were probed with primary antibodies and stained with an AlexaFluor secondary antibody (either 488 or 555). APP transcriptional activity was performed using the Gal4-APP system with a luciferase readout. Cells were harvested 48 h after transfection and the luciferase and b-galactosidase activities were determined with the Promega luciferase assay kit and the Promega b-galactosidase assay kit, respectively. ApoE3, ApoE4, and its proteolytic product ApoE4delta (ApoEdelta272) co-immunoprecipitated with full-length APP both in cell lysates and conditioned media. Interaction of APP with ApoE4 or ApoE4delta was accompanied by decreased sAPPa secretion and reduced sAPPa/Aß and sAPPa/sAPPß ratios as compared to ApoE3. Both ApoE4 and ApoE4δ reduced interaction between APP and Fe65 and also suppressed Fe65-mediated intracellular APPs.