Fig. 3. Effects of Aβ treatment on APP and BACE1 5′–flanking sequence activity.

A. A 1.2 kb fragment of the APP 5′–flanking region (Song and Lahiri, 1998) was cloned into the pGL3 luciferase expression vector as described in the text. The APP fragment contains a putative site for Aβ binding. B. A 3.3 kb fragment of the human BACE1 5′–flanking region (Ge et al., 2004b) was cloned into the pGL3 luciferase expression vector as described in the text. The BACE1 fragment contains a confirmed site for Aβ binding, determined herein. Primary rat cerebrocortical neuronal (PRCN) cultures were transiently transfected with a Renilla luciferase control vector and C. APP/pGL3 firefly luciferase fusion clone or D. BACE1/pGL3 luciferase fusion clone. Transfected cells were left untreated or treated with Aβ1–28, 1–40, or 1–42, all 1 μM. Cells were harvested and activities of firefly and Renilla luciferases were measured. Firefly luciferase activity within each treatment was normalized to corresponding Renilla luciferase activity. All within each cell type was then normalized to the average firefly/Renilla ratio for untreated APP/luciferase fusion activity. Letters above data bars indicate general linear mixed model multiple range categories. Samples sharing a letter do not significantly differ at p < 0.05. Thus, no differences in APP promoter activity were found between any treatment group, while there were significant increases in BACE1 promoter activity in the Aβ1–28 and Aβ1–42 treatment groups relative to untreated cells and Aβ1–40 was similar to vehicle.