Figure 8.
NRBF2 regulates APP-CTF degradation via CCZ1-MON1A-mediated RAB7 activation. (A-D) NRBF2 overexpression reduces APP-CTFs and intracellular Aβ1-40, Aβ1-42 levels in primary neuron culture. Primary cortical neurons isolated from 3xTg AD mouse were transfected with Recombinant lentivirus packed GFP-NRBF2 or GFP, the expression of NRBF2, FL-APP, APP-CTFs were examined by immunoblotting. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (E and F) ELISA results demonstrated that NRBF2 overexpression significantly reduced intracellular Aβ. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (G-K) Silencing Nrbf2 increased APP-CTFs and intracellular Aβ. Primary cortical neurons were transfected with lentiviral Nrbf2 shRNA or nontargeting shRNA. After knocking down (KD) Nrbf2, the level of FL-APP, APP-CTFs were detected by immunoblotting. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (L and M) ELISA results demonstrated that Nrbf2 KD significantly increased intracellular Aβ level. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (N) N2a cells were transfected with mCerulean-Nrbf2, RFP-Lc3 and CTFβ-His under basal or CQ-treated conditions. CTFβ-His was stained with anti-His antibody and the colocalization of mCerulean-NRBF2, RFP-LC3 and CTFβ-His were visualized under confocal microscope. Scale bar: 7.5 μm. (O and P) GTP-RAB7 in N2 S and nrbf2-/- N2 S cells were determined by GTP-beads affinity-isolation assay, we used empty agarose beads as equivalent control. The empty agarose beads cannot pull down RAB7. The “empty agarose” indicated affinity isolation with empty agarose beads, and the “pull-down” indicated affinity isolation with GTP-beads. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control