Figure 8

Oligomeric and fibrillar Aβ42 increase levels of APPsβsw in Tg2576 transgenic mouse primary astrocytes. Tg2576 or non-transgenic (non-Tg) littermate mouse primary astrocyte cultures were treated with 10 μM oligomeric or fibrillar Aβ42. Following treatment, cells were harvested for APP and APPsβsw immunoblots. Treatment times are indicated. Olig., Aβ42 oligomer treatment; Fibr., Aβ42 fibril treatment; OC, oligomer vehicle control; FC, fibril vehicle control. The numbers in parentheses indicate primary astrocytes cultured from different Tg2576 mouse pups. Immunosignals identified by the anti-APP antibody 22C11 clearly demonstrated that Tg2576 astrocytes overexpress transgene-derived APPsw (A-C, upper row). Note that Tg2576 APPsw migrates faster than non-Tg astrocytic APP on SDS-PAGE because the Tg2576 transgene expresses the 695 amino acid form of APP, while endogenous APP expressed by astrocytes is the 751 amino acid form. Aβ42 oligomers and fibrils caused both transgenic APPsw and endogenous astrocytic APP to become elevated at 24 and 48 h (A, B). The APPsw increase largely returned to control levels by 72 h for Tg2576 astrocytes (C). Most importantly, oligomeric and fibrillar Aβ42 treatment dramatically induced β-secretase processing of APPsw to generate robust levels of APPsβsw in Tg2576 astrocytes (A-C, center row), as indicated by immunosignals produced by an antibody that recognizes the BACE1-cleaved C-terminal neo-epitope of APPsβ containing the Swedish mutation. The APPsβsw elevation remained high for the duration of the experiment. GFAP immunosignals served as loading controls (A-C, lower row).