Abstract
Susceptibility to the development of late-onset Alzheimer's disease is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Although several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment is unknown. Our previous studies showed that a physiologically relevant cell-derived apoE3 particle stimulates neurite outgrowth in an isoform-specific manner. In an attempt to delineate the biochemical mechanism responsible for the stimulatory effects of apoE3 on neurite outgrowth, we performed a detailed physical characterization of cell-derived apoE3 and apoE4 particles. Immunoaffinity chromatography followed by SDS-PAGE illustrated homogeneity in protein content (apoE >95%). The affinity-purified particles contained phospholipid and 1 mol of cholesterol per mole of apoE but no core lipids. Nondenaturing gradient gel electrophoresis identified two major particle populations with hydrated diameters of 8.0 and 9.2 nm. Neurite outgrowth assays performed with the affinity-purified particles resulted in similar isoform-specific differences as seen previously, apoE3 stimulatory and apoE4 neutral. Interestingly, we did not observe a reduction in apoE medium concentrations over the duration of the neurite outgrowth assays, suggesting little or no endocytic uptake. Ligand blot analysis demonstrated that the affinity-purified apoE particles bind to several Neuro-2a membrane proteins. Western blots of the Neuro-2a membrane proteins indicated that the LDL receptor, gp330, and LR8B might be involved in the apoE-binding event. These results discriminate against the lipid delivery hypothesis and suggest that the biological activity of the phospholipid apoE3 particles may be due to cell surface signaling.—DeMattos, R. B., L. L. Rudel, and D. L. Williams. Biochemical analysis of cell-derived apoE3 particles active in stimulating neurite outgrowth.
Highlights
Susceptibility to the development of late-onset Alzheimer’s disease is increased for individuals harboring one or more apolipoprotein E4 alleles
Compositional or size differences between the affinity-purified apoE3 or apolipoprotein E4 (apoE4) particles were not detected, the apoE3specific enhancement of neurite outgrowth was still retained in the immunoaffinity-purified particles and in particles active in binding to heparin
Analysis of the isolated particles demonstrated the absence of core lipids, and no difference in cholesterol content between apoE3 and apoE4 particles
Summary
The stimulatory effects of apoE in each of these studies could be demonstrated only after lipoproteins were further enriched with exogenous human apoE, despite the fact that each lipoprotein already contained abundant apoE protein (rabbit apoE for the -VLDL studies and human apoE for the cerebrospinal fluid studies) These studies did not identify the stimulatory mechanism of apoE in neurite outgrowth, the results were consistent with the lipid delivery hypothesis. Preliminary analysis of the cellderived apoE isoform by density gradient ultracentrifugation showed it to be in a poorly lipidated particle with a density between 1.19 and 1.26 g/ml These results demonstrated that the biological activity of apoE3 did not depend on either an interaction of apoE3 with exogenous lipid sources or independent actions of apoE3 and -VLDL.
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