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. Author manuscript; available in PMC: 2009 Dec 26.
Published in final edited form as: Neuron. 2008 Dec 26;60(6):988–1009. doi: 10.1016/j.neuron.2008.10.047

Figure 1. Glucose deprivation increases BACE1 protein levels in BACE1-293 cells via a post-transcriptional mechanism.

Figure 1

(A) BACE1-293 cells were incubated for 3, 6, 12, 24, 36, and 48 hrs in either normal DMEM with glucose (25mM; CON) or DMEM without glucose (NG). Cells were lysed at the end of the treatment periods and 5μg of total protein per lane was used for immunoblot analysis of BACE1. β-actin was used as a loading control. (B) BACE1 immunosignals in (A) were quantified by phosphorimager, normalized to β-actin, and expressed as percentage of control (CON) for each time-point. BACE1 protein levels were significantly elevated compared to CON in response to glucose deprivation from 6 through 48 hrs (mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001). An upward trend for a BACE1 increase was present at 2 hrs of NG treatment (n=3 per group). (C) Triplicate wells of BACE1-293 cells were incubated for 12 hrs in regular DMEM with glucose (CON), DMEM without glucose (NG), DMEM with glucose containing 1.7μg/mL actinomycin D (ActD), or DMEM without glucose containing 1.7μg/mL actinomycin D (ActD + NG). Cells were lysed at the end of the treatment period and 5μg of total protein was used for immunoblot analysis of BACE1. β-actin was used as a loading control. (D) BACE1 immunosignals in (C) were quantified by phosphorimager, normalized to β-actin, and expressed as percentage of control (CON). BACE1 protein levels were significantly elevated in response to 12 hrs of NG treatment either in the presence or absence of ActD, compared to CON (*, p<0.05), demonstrating that BACE1 gene transcription was not required for the BACE1 increase. BACE1 protein levels were significantly decreased with 12 hrs of ActD treatment alone, compared to CON (mean ± SEM; #, p<0.05; n=3 per group) (E) Parallel BACE1-293 cell cultures were treated as in (C), except that total mRNA was isolated and levels of BACE1 mRNA measured via the TaqMan real-time PCR relative quantification method and expressed as percentage of control (CON; n=6). There were no differences in BACE1 mRNA levels between cells treated in media with or without glucose, verifying that NG treatment did not increase BACE1 gene transcription or mRNA stability. Note that ActD performed as expected and produced a robust decrease of BACE1 mRNA in cells treated with or without glucose, compared to CON (mean ± SEM; ##, p<0.01). (F) BACE1-293 cells were pulse-labeled in media containing 35S-methionine/cytseine and chased for up to 24 hrs in normal DMEM with glucose (CON), DMEM without glucose (NG), or DMEM containing 50mM 2-deoxyglucose (2-DG). Radiolabeled BACE1 was immunoprecipitated from cell lysates at 0, 3, 6, 12, and 24 hrs post-label, separated via SDS-PAGE, and visualized by autoradiography. Note that immaturely glycosylated BACE1 is ~50kDa, while maturely glycosylated BACE1 is ~70 kDa. There were no apparent differences between the half-lives of BACE1 protein under CON, NG, and 2DG conditions, indicating that BACE1 increases are not due to BACE1 protein stabilization. In histograms, error bars represent S.E.M. (G) HEK-293 cells were transiently transfected with pcDNA3.1Zeo(+) vector containing the entire human BACE1 coding region (~1.5kb) plus the BACE1 5’UTR (+5’UTR) or pcDNA3.1Zeo(+) vector containing only the human BACE1 coding region (-5’UTR; (Lammich et al., 2004). Following overnight recovery, cells were incubated for 24 hrs in normal DMEM with glucose (CON) or DMEM without glucose (NG). Cell lysates were prepared and 5μg of total protein per lane was used for immunoblot analysis of BACE1 and β-actin. Note that transfection with the -5’UTR construct caused the accumulation of immature ~60kDa BACE1, in addition to mature ~70kDa BACE1 (Haniu et al., 2000). (H) BACE1 immunosignals in (G) were normalized to β-actin and expressed as percentage of +5’UTR control (CON, +5’UTR). The glucose deprivation-induced BACE1 increase did not occur in the absence of the BACE1 5’UTR, implicating a BACE1 5’UTR-dependent translational control mechanism. (n=3 per group; **, comparison between CON and NG, +5’UTR; p < 0.01; mean ± SEM). Error bars = S.E.M. in all histograms.