Figure 6.

Role of hAPP/Aβ in the accumulation of hSYN in vivo and in vitro. (a–c) Sections of temporal cortex from 12-month-old hSYN/hAPP (a and c) or hAPP (b) mice were double-labeled with antibodies against hSYN (red) and hAPP (green) (a and b) or against hSYN (red) and Aβ (green) (c) and analyzed by confocal microscopy. Note the colabeling for hAPP and hSYN (yellow) of the neuron (arrow) containing a dense hSYN accumulation (a) and the close association of Aβ deposits (green granules) with intracellular hSYN accumulations (red) (c). All images were obtained at a magnification of ×930. The image in c was magnified electronically to better visualize Aβ-immunoreactive granules. (d) Recombinant hSYN (10 μM final concentration) was or was not mixed with freshly solubilized synthetic Aβ1–40, Aβ1–42, or control peptide of reverse sequence (Aβ42–1) (each at a final concentration of 10 μM) in 20 μl of 100 mM Tris⋅HCl buffer (pH 7.5). All samples were incubated at 37°C for 24 h and then subjected to SDS/15% PAGE. After blotting of samples onto nitrocellulose membrane, the membrane was blocked with Tris-buffered saline (TBS; 20 mM Tris⋅HCl, pH 7.5/150 mM NaCl) containing 3% BSA, followed by incubation with anti-hSYN (1:1000) in TBS containing 1% BSA. The membrane was then incubated with ¹²⁵I-labeled protein A (ICN), followed by autoradiography. (e–j) Neuronal GT1–7 cells stably transfected with SYN cDNA (e–g) or control plasmid (h–j) as described (27), were left untreated (e and h) or exposed for 48 h to Aβ1–40 (f and i), Aβ1–42 (g and j), or Aβ42–1 (not shown) at 2 μM final concentration, followed by immunoperoxidase staining with anti-SYN (27).