Figure 6.

Ritonavir up-regulates neuronal BACE1 expression via translation-dependent control. A: Rat neurons were exposed to indicated treatments for 16 hours. BACE1 mRNA was determined using the ΔΔCt method relative to TATA-Box binding protein (TBP). Quantitative RT-PCR assays were performed in triplicate (one-way analysis of variance, Dunnett post hoc). B: Rat neurons were either treated with ritonavir alone or pretreated with anisomycin or actinomycin D for 12 hours before ritonavir. Fast green was used as a loading control. C: Compared to ritonavir-only treated neurons, anisomycin pretreatment resulted in decreased BACE1 expression (one-way analysis of variance, Newman-Keuls post hoc). D: Rat neurons were treated for 16 hours, and cytoplasmic and nuclear fractions were analyzed by immunoblotting. E: Densitometric analysis revealed a significant increase in activating transcription factor 4 (ATF4) and BACE1 (one-way analysis of variance, Dunnett post hoc). F: Fourteen DIV rat cortical cultures were transfected with pcDNA3.1Zeo⁺ vector containing full human BACE1 coding region, including the BACE1 5′ UTR but missing the 3′ UTR (+5′ UTR), or pcDNA3.1Zeo⁺ vector containing human BACE1 coding region, including the 3′ UTR but missing the 5′ UTR (-5′ UTR). Forty-eight hours later, cultures were treated with dimethyl sulfoxide or 10 μmol/L ritonavir for 16 hours, and immunoblotted for BACE1. Representative blots are shown. G: Quantification of BACE1 was normalized to actin (one-way analysis of variance, Newman-Keuls post hoc). H: Rat neurons were treated with 10 μmol/L ritonavir, 1 μmol/L saquinavir, or 10 μmol/L lactacystin for 16 hours. Whole cell lysate (10 μg) from each condition was used to determine 20S proteasome activity (one-way analysis of variance, Dunnett post hoc). n = 4 (A, C, and E); n = 3 (B); n = 2 (H). ^∗P < 0.05, ^∗∗P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UT, untreated.