Figure 4.

The Bri2–23 peptide is required for the anti-amyloidogenic effect of the BRI2 protein in vivo and is present in human CSF. A, Schematic of BRI2 and BRI2del244–266 constructs. BRI2del244–266 construct does not encode the Bri2–23 peptide. P0 TgCRND8 mice were transduced by intracerebroventricular injection of rAAV1-BRI2del244–266. B, C, Cortical sections (magnification, 200×) of 3-month-old TgCRND8 mice were immunostained with anti-Aβ1–16 antibody (33.11; B), and amyloid plaque burdens were quantified (C). D, Total-brain Aβ levels were analyzed by Aβ end-specific ELISA 3 months after transduction. E, Western blot analysis of steady-state levels of the rAAV1-delivered transgenes. Three lysates representing the typical range of expression are shown for the BRI2, BRI2-Aβ1–40, and BRI2del244–266 transgenes. Anti-β-actin is used as a loading control, and a lysate from transgenic BRI2-Aβ40 mice is used as a positive control (TgBRI2-Aβ40). F, HPLC/MS detection of Bri2–23 in cells transfected with BRI2. Synthetic Bri2–23 yielded a peak that was in excellent agreement with its predicted mass-to-charge ratio (m/z) of 2630. Spectra from the conditioned media of cells transiently transfected with pcDNA3-BRI2 yielded a peak absent from both the blank and conditioned media from cells transfected with pcDNA3 BRI2del-244–266. The peak has an assessed mass-to-charge ratio within 1 unit of the Bri2–23 peptide standard. G, HPLC/MS detection of Bri2–23 in human CSF. Spectra from the CSF of three normal subjects each yielded a peak absent from the blank with an assessed mass-to-charge ratio nearly identical to the Bri2–23 peptide standard. yo, Years old; F, female; M, male.