Figure 2

Synaptic fractionation demonstrates that low molecular weight Aβ oligomers associate with the PSD in AD but not NDAN hippocampal specimens. (A) PSD95, a postsynaptic marker, and synaptophysin (Synphys), a presynaptic marker, were probed in the total homogenate (Hom), synaptosomal (Syn), synaptic vesicles (SV), and postsynaptic density (PSD) fractions isolated from a frozen human hippocampal specimen from a control, non-demented aged individual. Enrichment of the different fractions is shown by the presence/absence of synaptic markers. The fractionation was successful for all case types (control, AD and NDAN) as shown by the purity of PSD fractions in (B). The isolated PSD fraction from hippocampal samples from different cases from each group (aged matched controls, AD, and NDAN) were immunoblotted together (representative blot shown; 40 μg protein each lane of a 10-20% tris-glycine gradient gel) and probed using 6e10 (C) showing LMW Aβ only associates with AD samples. (D) Densitometric analyses of each of the Aβ species (monomer/dimer = 4–8.5 kD band, trimer = 12 kD, and tetramer = 16 kD) demonstrates that all Aβ species are increased in AD. The results are expressed as the mean ± SEM using propagation of error and normalized with AD = 100%. The asterisk denotes the values significantly higher than control (monomer/dimer, p = 0.012, trimer, p = < 0.001, and tetramer, p = 0.014; ANOVA; a Bonferroni correction was required for the monomer/dimer and trimer density values). Hippocampus PSD fractions (80 μg) from age-matched controls, AD, and NDAN samples were immunoblotted and probed using NU4 (E). A lane is included at the end showing synthetic (Synt) Aβ oligomers probed by NU4 for comparison. The densitometric analysis (F) of the dimer/trimer band shows that AD cases are significantly higher than control and NDAN (both groups are essentially the same as background), while the tetrameric species was not found to be significantly different across the groups (p = 0.67). The asterisk denotes significance compared to control, p < 0.05, ANOVA. All Western blots shown are representative of 9–10 individual cases assayed in each group and experiments were repeated 3 – 4 times