Figure 3.
Synthetic ADDLs, but not low molecular weight species, bind neurons analogously to AD-derived species. Primary hippocampal neurons were incubated with synthetic ADDLs fractionated by a Centricon filter (A, B) or biotinylated ADDLs separated by size exclusion chromatography (E, F) for 30 min [as described in Chromy et al. (2003)]. After removing unbound species by washing with fresh media, cell-bound ADDLs were assessed under nonpermeabilized immunolabeling conditions using M94 and Alexa 488-conjugated anti-rabbit secondary antibody (A, B) or Alexa 488-conjugated streptavidin (E, F). Confocal images demonstrate that oligomer immunoreactivity is at the plasma membrane of neurons and predominantly within dendritic arbors, although some binding to cell bodies is also evident. Punctate binding is reminiscent of receptor clusters and analogous to that of Alzheimer's Aβ oligomers (Fig. 2). As with fractionated soluble oligomers from AD brain, incubation of hippocampal neurons with synthetic ADDL species ranging from 100 to 10 kDa shows immunoreactive puncta (A), where as species ≤ 10 kDa, which would contain monomer and dimer, do not (B). Inset, Western blot of ADDL species present in the culture media after a 6 hr incubation with hippocampal neurons demonstrated that ADDLs are stable and contain no species with molecular weight >100 kDa (C). Lanes represent two separate culture media. Separation of biotinylated ADDLs (∼14 nmol of a 70 μm ADDL preparation) by SEC on Superdex 75 yielded two peaks (D). Histogram of elution volume versus absorbance at 280 nm depicted peak 1 at 8.1 ml with an absorbance at 16.9 mAU and peak 2 at 13.9 ml with an absorbance at 11.7 mAU. Fractions B1 and D6 with respective protein concentrations of 6.5 and 4 μm were incubated for 1 hr with mature hippocampal cells at a final concentration of 500 nm in parallel to an SEC-control fraction (taken between peaks 1 and 2). Binding of biotinylated species was detected with Alexa Fluor 488-conjugated streptavidin. Hot spots of fluorescence were observed exclusively with peak 1 fraction B1 (E), consistent with species of molecular weight >50 kDa such as 12-mers. No fluorescence was seen with the peak 2 fraction D6 (F) or the control fraction (data not shown). Confocal images were acquired with constant confocal microscope settings (laser power, filters, detector gain, amplification gain, and amplification offset). Images are representative of three different experiments. Scale bar, 40 μm.