Figure 6

Female 5XFAD mice of either BACE1 ^+/+ or BACE1 ^+/− genotypes have higher steady-state transgenic APP levels than males. (A) Representative transgenic APP immunoblot (6E10) and Ponceau S staining of brain homogenates from 4 month old male (M) and female (F) mice. Note the high level of APP in the 5XFAD/BACE1^−/− lane. (B-D) Relative quantifications of transgenic APP immunoblot signals. At all ages, steady-state transgenic APP levels are higher in 5XFAD/BACE1^+/− and 5XFAD/BACE1^+/+ females than in corresponding males. Additionally, there is a trend toward higher transgenic APP levels in 5XFAD/BACE1^+/− compared to 5XFAD/BACE1^+/+ mice that becomes significant at 9 months, which could result from less BACE1 cleavage in the BACE1^+/− genotype. (E) Representative APP immunoblot (C-terminal APP antibody) and Ponceau S staining of brain homogenates from 6 month old 5XFAD/BACE1^+/+ (+) and non-Tg/BACE1^+/+ (−) mice to measure both human and mouse APP. (F) Relative quantifications of APP immunoblot signals in (E) demonstrates that while non-Tg males and females have equal levels of endogenous APP, 5XFAD females have significantly higher APP levels than 5XFAD males, a result that is consistent with the 6E10 immunoblot data showing elevated transgenic APP levels in 5XFAD females. n = 9–14 mice per sex per genotype. (G) Schematic diagram of murine Thy-1 promoter transgene cassette with 5’ upstream sequence showing estrogen resonse element (ERE). The APP or PS1 coding regions are inserted into the XhoI site. Thy-1 gene exons are shaded blue. Base-pair (bp) numbering is relative to the transcriptional start site for Thy-1 exon 1a [32]. The ERE is located from −1116 bp to −1104 bp in the 5’ upstream regulatory region of the Thy-1 promoter. The one bp deviation of the Thy-1 ERE at position +6 could be overcome by the flanking A-T rich sequence, which enhances ERE transcriptional potency in vivo[33].