Biochemical analysis of cell-derived apoE3 particles active in stimulatingneurite outgrowth

Susceptibility to the development of late-onset Alzheimer's disease is incr eased for individuals harboring one or more apolipoprotein E4 (apoE4) allel es. Although several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment i s 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 f or the stimulatory effects of apoE3 on neurite outgrowth, we performed a de tailed physical characterization of cell-derived apoE3 and apoE4 par tides. 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 p opulations with hydrated diameters of 8.0 and 9.2 nm. Neurite outgrowth ass ays performed with the affinity-purified particles resulted in similar isof orm-specific differences as seen previously apoE3 stimulatory and apoE4 neu tral. Interestingly, we did not observe a reduction in apoE medium concentr ations over the duration of the neurite outgrowth assays, suggesting Little or no endocytic uptake. Ligand blot analysis demonstrated that the affinit y-purified apoE particles bind to several Neuro-2a membrane proteins. Weste rn 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 bi ological activity of the phospholipid apoE3 particles may be due to cell su rface signaling. - DeMattos, R. B., L. L. Rudel, and D. L. Williams. Bioche mical analysis of cell-derived apoE3 particles active in stimulating neurit e outgrowth.