ChIP and PCR were carried out as described in the text. A. ChIP for Aβ binding a 200 bp region containing the predicted APP −3833 AβID. B. ChIP for Aβ binding a 200 bp region containing the BACE1 −119 AβID. C. Precipitated, cross–linked chromatin was probed for presence of Aβ peptide (lanes 4, 8), SP1 (lanes 5, 9), and the N–terminus of the APP protein (lane 6) within the indicated region of the APP promoter. D. Precipitated, cross–linked chromatin was probed for presence of Aβ peptide (lanes 4, 9), SP1 (lanes 5, 10), and β–galactosidase (lanes 6, 11) within the indicated region of the BACE1 promoter. Treatment conditions in the cells and other appropriate controls in the assay were as indicated in the figure.

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.

Polymorphic promoter/enhancer CAT expression clones and pCAT3P vector plasmid were transfected into PC12 cells and the transfected cells were treated with H₂O₂ and Aβ, singly or in combination, as described in the main text. Reporter CAT ELISA signal was normalized to total cell protein. A. Both of the clones tested (A and G) had significantly higher activity than the vector, and the G allele was significantly more active than the A allele. B and C. Both Aβ and H2O2 treatment significantly increased activity of this site, independent of the polymorphism. Results are presented as proportional to untreated pCAT3P vector transfected cells. Letters above data bars indicate generalized linear mixed model multiple range categories. Samples sharing a letter do not significantly differ at p < 0.05. For Aβ treatment and H₂O₂, “*” indicates significant difference at p < 0.05.

A. Non–cytotoxic pathway. Aβ levels are insufficient to significantly activate apoptotic genes such as TP53 and the “cell death pair response” of upregulated ASCL1 and downregulated OLIG2 through their AβID sequences. These levels maintain APP and BACE1 at non–pathogenic levels. B. Cytotoxic pathway. Aβ levels and resulting APP and BACE1 production are stimulated by conditions such as metal–induced oxidation (ROS), which ultimately results in increased production of Aβ peptides. The levels of Aβ are sufficient to significantly increase levels of p53 and induce the “cell death pair response” of upregulated ASCL1 and downregulated OLIG2. Additional Aβ is produced beyond what is required for physiological function, and the excess Aβ becomes deposited as amyloid plaque.

Human SK–N–SH neuroblastoma cells were treated with FITC–Aβ1–40 in presence or absence of H₂O₂ as described in the text. Cells were incubated with these treatments for 48 hours and visualized with Hoechst 33342 dye, and EtHD, corresponding to all cell nuclei and dead nuclei, respectively. Cells were fluorescently imaged at appropriate wavelengths for FITC, Hoechst treatment, and EtHD treatment. A and E. with all nuclei labeled with Hoechst 33342. B and F. FITC–Aβ1–40 labeled nuclei. C and G. Dead cells labeled with EtHD. D and G. Composite of all three fluorescent signals overlaid on a phase contrast image of the cells.

Polymorphic promoter/enhancer CAT expression clones and pCAT3P were transfected into PC12 cells and the transfected cells were treated with H₂O₂ and Aβ, singly or in combination, as described in the main text. Reporter CAT ELISA signal was normalized to total cell protein. Results are presented as proportional to untreated pCAT3P vector transfected cells. Letters above data bars indicate general linear mixed model multiple range categories. Statistical significance is indicated by letters above the data bars, with samples sharing a letter do not significantly differ at p < 0.05, and conversely, samples not sharing a letter are significantly different at p < 0.05.

Polymorphic promoter/enhancer CAT expression clones and pCAT3P were transfected into PC12 cells and the transfected cells were treated with H₂O₂ and Aβ, singly or in combination, as described in the main text. CAT ELISA signal was normalized to total cell protein. Results were then normalized to adjusted signal for untreated pCAT3P transfected cells and expressed as marginal means estimated by holding a single effect (clone, Aβ treatment, or H₂O₂ treatment) constant. A. Aβ treatment increased transcriptional activity of the G but not the A clone. Both clones were significantly more active than the pCAT3P vector. B. H₂O₂ treatment increased the activity of both clones, indicating that this effect is not sequence–specific. C. Aβ treatment in the absence of H₂O₂ increased promoter activity, and H₂O₂ increased promoter activity significantly both in the presence or absence of Aβ. Letters above data bars indicate generalized linear mixed model multiple range categories. Samples sharing a letter do not significantly differ at p < 0.05. Although the −H₂O₂/−Aβ treatment had a significant difference from combinations that were treated with H₂O_2,, Aβ, or H₂O₂ + Aβ, when examining Aβ × H₂O₂, the interaction was not significant overall by Type III test.