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. 2008 Dec 26;60(6):988-1009.
doi: 10.1016/j.neuron.2008.10.047.

Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis

Tracy O'Connor  1 , Katherine R Sadleir, Erika Maus, Rodney A Velliquette, Jie Zhao, Sarah L Cole, William A Eimer, Brian Hitt, Leslie A Bembinster, Sven Lammich, Stefan F Lichtenthaler, Sébastien S Hébert, Bart De Strooper, Christian Haass, David A Bennett, Robert Vassar
Affiliations

Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis

Tracy O'Connor et al. Neuron. .

Abstract

beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimer's disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

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Figures

Figure 1
Figure 1. Glucose deprivation increases BACE1 protein levels in BACE1-293 cells via a post-transcriptional mechanism
(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.
Figure 2
Figure 2. Glucose deprivation increases eIF2α phosphorylation and activates a specific set of stress-response signaling pathways in BACE1-293 cells
(A) BACE1-293 cells were incubated for 2 or 24 hrs in either normal DMEM with glucose (4,500 mg/L; CON) or DMEM without glucose (NG; n=3 wells per group). Cells were lysed and 10μg of total protein per lane was used for immunoblot analysis of BACE1, phospho-eIF2α (Ser51), total eIF2α, phospho-c-Jun(Ser63), total c-Jun, phospho-JNK(Thr183/Thr185), total JNK (54kD and 46kD isoforms), phospho-eIF4E(Ser209), and β-actin (see Supplement for list of antibodies used). ~10μg of lysate from UV-treated 293 cells was used as a positive control (+) for induction of stress-response pathways. (B) Immunosignals in (A) were quantified by phosphorimager and normalized in the following ways: BACE1, eIF2α, JNK, and eIF4E-P were normalized to β-actin; eIF2α-P was normalized to total eIF2α ; c-Jun-P was normalized to c-Jun, and JNK-P was normalized to JNK. Values are expressed as percentage of control (CON) for each time-point (error bars = S.E.M.). After 2 hrs of NG treatment, eIF2α-P, c-Jun-P, JNK-P(46kD), and JNK(46kD) were all significantly elevated. At 24 hrs NG, eIF2α-P, c-Jun-P, and JNK-P(46kD) remained elevated, and BACE1, c-Jun, and JNK-P(54kD) became significantly increased as well. eIF4E phosphorylation was unaffected by NG treatments at either time-point. These results show that eIF2α-P is temporally increased before BACE1, as expected if eIF2α-P regulates BACE1 translation. (mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001).
Figure 3
Figure 3. Direct manipulation of eIF2α phosphorylation affects the BACE1 increase in BACE1-293 cells
(A) BACE1-293 cells were incubated for 24 hrs in normal DMEM with glucose (25mM; CON), DMEM without glucose (NG), or DMEM containing 100μM salubrinal (SAL). Cell lysates were prepared and 10μg of total protein per lane was used for immunoblot analysis of phosphorylated eIF2α and total eIF2α. A parallel immunoblot was prepared with 5μg of cell lysate per lane for analysis of BACE1 and β-actin. The arrow identifies the authentic eIF2α-P band (the upper band is non-specific). (B) Immunosignals in (A) were quantified by phosphoimager, normalized, and expressed as percentage of control (CON). BACE1 immunosignals were normalized to β-actin, and eIF2α-P was normalized to total (T) eIF2α. Levels of BACE1 and eIF2α-P/eIF2α-T were significantly elevated in response to both glucose deprivation and salubrinal treatment, compared to control (mean ± SEM; *, p<0.05; ***, p<0.001; n = 2 per group). Note that salubrinal treatment caused direct increases of eIF2α-P and BACE1 levels in the absence of glucose deprivation. These salubrinal-induced increases were similar in size to those caused by no-glucose treatment. (C) BACE1-293 cells were transiently transfected with GADD34 control vector (CON, 12h NG) or GADD34ΔN (NG + GADD34ΔN), the constitutively active PP1 regulatory subunit. Following overnight recovery, cells were incubated for 12 hrs in normal DMEM with glucose (CON) or DMEM without glucose (12h NG, NG + GADD34ΔN). Cell lysates were prepared and 10μg of total protein per lane was used for immunoblot analysis of BACE1, P-eIF2α (Ser51), total eIF2α, and β-actin. “+” lane: 10μg of lysate from UV-treated 293 cells was used as a positive control for eIF2α-P. (D) Immunosignals in (C) were quantified by phosphoimager, normalized, and expressed as percentage of control (CON). BACE1 immunosignals were normalized to β-actin, and eIF2α-P was normalized to total eIF2α. Glucose deprivation-induced increases in levels of BACE1 and eIF2α-P/eIF2α-T were completely prevented by overexpression of GADD34ΔN, demonstrating that eIF2α phosphorylation was responsible for the BACE1 increase. (n=4; comparison between CON and 12h NG: *, p < 0.05; ***, p < 0.001; comparison between 12h NG and NG + GADD34ΔN: ##, p < 0.01; ###, p < 0.001; mean ± SEM). (E) BACE1-293 cells were transiently transfected with pcDNA3 empty vector (CON, NG) or dominant negative PERK (PERKDN). Following overnight recovery, cells were incubated for 24 hrs in normal DMEM with glucose (CON) or DMEM without glucose (NG, NG + PERKDN). Cell lysates were prepared and 10μg of total protein per lane was used for immunoblot analysis of phosphorylated eIF2α and total eIF2α. A parallel immunoblot was prepared with 5μg of cell lysate per lane for immunoblot analysis of BACE1 and β-actin. (F) BACE1 immunosignals in (E) were quantified by phosphoimager and expressed as percentage of control (CON). Glucose deprivation-induced increases in BACE1 levels were completely prevented by overexpression of PERKDN, demonstrating that PERK was the eIF2α kinase responsible for eIF2α phosphorylation and the BACE1 increase. (n=3 per group; **, comparison between CON and NG, p < 0.01; #, comparison between NG and NG + PERKDN, p < 0.05; mean ± SEM). (G) BACE1-293 cells were transiently transfected with pcDNA3 empty vector (CON, NG) or dominant negative GCN2 (GCN2DN). Following overnight recovery, cells were incubated for 24 hrs in normal DMEM with glucose (CON) or DMEM without glucose (NG, NG + GCN2DN). Cell lysates were prepared and 10μg of total protein per lane was used for immunoblot analysis of phosphorylated eIF2α and total eIF2α. A parallel immunoblot was prepared with 5μg cell lysate per lane was used for immunoblot analysis of BACE1 and β-actin. (H) BACE1 immunosignals in (G) were quantified by phosphoimager and expressed as percentage of control (CON). The glucose deprivation-induced increase in BACE1 level was not prevented by overexpression of GCN2DN, demonstrating that GCN2 was not the eIF2α kinase responsible for eIF2α phosphorylation and the BACE1 increase. (n=3 per group; *, comparison between CON and NG, p < 0.05; mean ± SEM). Error bars = S.E.M. in all histograms.
Figure 4
Figure 4. eIF2α phosphorylation causes post-transcriptional increases of BACE1 and promotes Aβ production in cultured primary neurons
(A) Primary cortical C57/BL6 neurons were cultured for 7 DIV and then incubated for 36 hrs in media containing 20mM glucose (CON) or no glucose (NG). Cell lysates were prepared and 10μg of total protein per lane was used for immunoblot analysis of BACE1, P-eIF2α(Ser51), total eIF2α (eIF2α-T), and β-actin. (B) Immunosignals in (A) were quantified by phosphorimager, normalized, and expressed as percentage of control (CON). BACE1 immunosignals were normalized to β-actin, and eIF2α-P was normalized to eIF2α-T. Levels of endogenous neuronal BACE1 and eIF2α-P/eIF2α-T were significantly elevated in response to glucose deprivation (mean ± SEM; *, p < 0.05; n=3), and the sizes of the increases were similar to those observed in BACE1-293 cells. (C) Parallel C57/BL6 primary neuron cultures were treated as in (A), except that total mRNA was isolated and levels of endogenous BACE1 mRNA measured via the TaqMan real-time PCR relative quantification method and expressed as percentage of control (CON; n=3). Unexpectedly, neuronal BACE1 mRNA levels were significantly reduced in cultures treated with NG-media compared to glucose-containing media (CON) (#; p < 0.05; mean ± SEM), clearly demonstrating a post-transcriptional mechanism for the BACE1 protein increase. (D) Primary cortical Tg2576 neurons were cultured for 7 DIV and then incubated for 48 hrs in normal media with glucose (CON), glucose-containing media with 50μM salubrinal (Sal), or glucose-containing media with 80μM Sal. Cell lysates were prepared and 10μg of total protein per lane was used for immunoblot analysis of BACE1, phosphorylated eIF2α, total eIF2α, and β-actin. 10μg of lysate from UV-treated 293 cells was used as a positive control (+) for eIF2α-P. (E) Immunosignals in (D) were quantified by phosphorimager, normalized, and expressed as percentage of control (CON). BACE1 was normalized to β-actin, and eIF2α-P was normalized to eIF2α-T. BACE1 levels and eIF2α-P/eIF2α-T ratios were significantly increased in Tg2576 neurons treated with both concentrations of Sal compared to control (CON; n=4; *, p<0.05; **, p<0.01; ***, p<0.001; mean ± SEM), demonstrating that direct induction of eIF2α phosphorylation causes BACE1 levels to rise. (F) Conditioned media collected from 48hr salubrinal-treated Tg2576 neurons in (D) were analyzed using a human Aβ40 ELISA. Values are expressed as ng of Aβ40 in the media per mg of total protein in the corresponding cell lysate. Aβ40 levels were significantly elevated in Tg2576 neurons treated with both 50μM and 80μM salubrinal compared to control (CON; n=4; *, p<0.05; **, p<0.01; mean ± SEM), demonstrating that direct induction of eIF2α phosphorylation led to an increase in Aβ production.
Figure 5
Figure 5. Elevated BACE1 levels are correlated with increased eIF2α-P and amyloidogenesis in a chronic model of energy deprivation in vivo
(A) Nine-month old Tg2576 mice were administered i.p. injections of saline (VEH), 1g/kg 2-deoxyglucose (2DG), or 80mg/kg 3-nitropropionic acid (3NP) once a week for 3 months (8-9 mice/group). One week after the final injection, hemibrains from each mouse were harvested and prepared for immunoblot and immunohistochemical analyses. Brain homogenates were prepared and 10μg of total protein per lane were used for immunoblot analysis of total BACE1 protein and β-actin. “+” lane: 5μg of BACE1-293 cell lysate as a BACE1 positive control.. “-“ lane: 10μg of BACE1-/- mouse brain homogenate as a BACE1 negative control. (B) 15μg of total protein per lane of brain homogenate from VEH, 2DG, 3NP treated Tg2576 mice were used for immunoblot analysis of eIF2α-P, eIF2α-T, and β-actin. “+” lane: 10μg of lysate from UV-treated 293 cells as a positive control for eIF2α-P. (C) Immunosignals in (A) and (B) were quantified by phosphorimager and expressed as percentage of vehicle (VEH). BACE1 levels and eIF2α-P/eIF2α-T ratios were significantly elevated in 2DG and 3NP treated Tg2576 mice compared to VEH (mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001). (D) Guanidine-extracted brain homogenates from VEH, 2DG, and 3NP treated Tg2576 mice were analyzed using a human Aβ40 ELISA. Values were expressed as ng of Aβ40 per mg of total protein. A clear trend toward elevation of Aβ40 level was observed in 2DG and 3NP treated mice, although values did not reach statistical significance. Increases in Aβ40 levels tended to parallel the elevations of eIF2α-P and BACE1 for 2DG and 3NP treatments (compare C and D). (E) Fixed hemibrains of each mouse treated with VEH, 2DG, and 3NP were sectioned and stained with anti-Aβ antibody 4G8. The total number of 4G8-immunopositive plaques was counted in a set of eight evenly spaced parasagittal sections that spanned the entire medial-lateral dimension of each hemibrain. A clear trend toward an increase in plaque number was observed for both 2DG and 3NP treatments compared to VEH. Plaque numbers for the different treatments paralleled the relative levels of Aβ40, as expected (compare to 5D), as well as the levels of eIF2α-P and BACE1 (compare to 5C). (F) Representative parasagittal brain sections from VEH, 2DG, and 3NP treated Tg2576 mice were immunostained with 4G8 and micrographed at 4x. 4G8-positive plaques in the brains of 2DG and 3NP treated mice tended to be larger and more numerous than those in VEH treated mice.
Figure 6
Figure 6. Chronic energy deprivation causes post-transcriptional increases of BACE1 in vivo
(A) Nine-month old wild-type C57/BL6 mice were administered i.p. injections of saline (VEH), 1g/kg 2-deoxyglucose (2DG), or 80mg/kg 3-nitropropionic acid (3NP) once a week for 3 months. One week after the final injection, hemibrains from each mouse were harvested and prepared for immunoblot and TaqMan quantitative real-time PCR analyses. Brain homogenates were prepared and 10μg of total protein per lane were used for immunoblot analysis of BACE1, full-length APP, and β-actin. “+” lane: 5μg of BACE1-293 cell lysate as a BACE1 positive control. “-“ lane: 10μg of BACE1-/- mouse brain homogenate as a BACE1 negative control. (B) Immunosignals in (A) were quantified by phosphorimager and expressed as percentage of vehicle (VEH; n = 4-6 per group). BACE1 levels were significantly elevated in the brains of both 2DG and 3NP treated C57/BL6 mice compared to VEH (mean ± SEM; *, p<0.05; ***, p<0.001). (C) Total mRNA was isolated from the hemibrains of VEH, 2DG, and 3NP treated C57/BL6 mice and levels of endogenous BACE1 and APP mRNAs measured via the TaqMan real-time PCR relative quantification method and expressed as percentage of vehicle (VEH; 9-12 mice per group). Strikingly, both BACE1 and APP mRNA levels were significantly decreased in 2DG and 3NP treated C57/BL6 mice compared to VEH (mean ± SEM; **, p<0.01; ***, p<0.001).
Figure 7
Figure 7. eIF2α-P levels are elevated in 5XFAD transgenic mouse and human AD brains and positively correlate with BACE1 levels and amyloid loads
(A) Brain homogenates were prepared from 6-month old 5XFAD transgenic (5XFAD) mice and non-transgenic littermates (Non-Tg), and 10μg of total protein per lane was used for immunoblot analysis of BACE1, full-length APP (FLAPP), eIF2α-P, eIF2α-T, and β-actin. “+” lane: 10μg of lysate from UV-treated 293 cells was used as a positive control for eIF2α-P. (B) Immunosignals in (A) were quantified by phosphorimager, normalized, and expressed as percentage of non-transgenic littermate levels. BACE1 and eIF2α-T immunosignals were normalized to β-actin, and eIF2α-P was normalized to eIF2α-T. “-”: non-transgenic littermates; “+”: 5XFAD transgenic mice. BACE1 levels and eIF2α-P/eIF2α-T ratios were significantly elevated in 5XFAD mice compared to Non-Tg (n=5 mice/group; *, p<0.05; **, p<0.01; mean ± SEM). (C) Human post-mortem frontal cortex samples from AD patients (AD) and age-matched non-demented controls (N) were homogenized and prepared for immunoblot analysis as described (Materials and Methods). 15μg of total protein per lane were analyzed by immunoblot for BACE1, eIF2α-P, eIF2α-T, and β-actin. 10μg of lysate from UV-treated 293 cells was used as a positive control for eIF2α-P (+). (D) Immunosignals in (C) were quantified by phosphorimager, normalized, and expressed as percentage of non-demented (ND) control. BACE1 and eIF2α-T immunosignals were normalized to β-actin, and eIF2α-P was normalized to eIF2α-T. Both BACE1 level and eIF2α-P/eIF2α-T ratio were significantly elevated in AD brains compared to ND (n=9 AD, 13 ND; *, p<0.05; **, p<0.01; mean ± SEM). (E-G) BACE1 levels and eIF2α-P/eIF2α-T ratios determined in (D) and amyoid loads (% area; Materials and Methods) were correlated by linear regression analyses of BACE1 level vs. eIF2α-P/eIF2α-T ratio (E), amyloid load vs. eIF2α-P/eIF2α-T ratio (F), and amyloid load vs. BACE1 level (G) for individual AD and ND frontal cortex samples. Amyloid load was expressed as the average percent area of AD or ND frontal cortex section occupied by plaques immunostained with total anti-Aβ antibody. Statistically significant correlations were found for BACE1 level vs. eIF2α-P/eIF2α-T ratio (p < 0.05), amyloid load vs. eIF2α-P/eIF2α-T ratio (p < 0.01), and amyloid load vs. BACE1 level (p < 0.05).
Figure 8
Figure 8. Models for role of eIF2α phosphorylation in regulation of BACE1 mRNA translation and sporadic Alzheimer’s disease pathogenesis
(A) eIF2α phosphorylation determines the concentration of ternary complex and consequently the rate of translation initiation. Under normal or post-stress conditions (left), levels of phosphorylated eIF2α are kept at low levels in the cell due to expression of regulatory subunits of the phosphatase PP1 (GADD34 and CReP), which complex with PP1 and direct PP1 phosphatase activity to eIF2α. The drug salubrinal is a selective inhibitor of the PP1 complex and prevents dephosphorylation of eIF2α-P (Boyce et al., 2005). eIF2B, which catalyzes the exchange of GDP for GTP on the γ subunit of eIF2, freely binds to and dissociates from unphosphorylated eIF2α in the eIF2 complex. In its GTP form, eIF2 assembles into a ternary complex along with methionine-charged tRNA (eIF2 GTP Met-tRNAi). The ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is then capable of initiating translation at an AUG sequence of the appropriate context on an mRNA transcript. Recognition of the start codon catalyzes GTP to GDP on eIF2, which is then released from the 40S subunit for another cycle of translation initiation. When eIF2α-P levels are low, ternary complex concentration is high and normal mRNA transcripts with short 5’ UTRs are translated with high efficiency. However, transcripts like BACE1 with long, uORF-containing, secondary structure-rich 5’UTRs are inefficiently translated. Under stress conditions (right), eIF2α is phosphorylated by one of four different kinases in response to various stimuli. In this case, eIF2B does not readily dissociate from phosphorylated eIF2α in the eIF2α complex, inhibiting the exchange of GDP for GTP on the γ-subunit of eIF2 and effectively reducing the concentration of ternary complex. In this case, the translation of normal mRNA transcripts is inhibited, while transcripts with long, uORF-containing, structured 5’UTRs are paradoxically translated with increased efficiency (de-repression). (B) Model for eIF2α phosphorylation-dependent de-repression of BACE1 mRNA translation, based on yeast GCN4 mRNA translational regulation (Schroder and Kaufman, 2006). The BACE1 5’ UTR is 453 nucleotides long, has three uORFs (numbered boxes), and has predicted stable secondary structure with a free energy of -215.3 kcal/mol (Lammich et al., 2004). uORF2 has been shown to be the major uORF that inhibits BACE1 mRNA translation under normal conditions (Mihailovich et al., 2007; Zhou and Song, 2006). A stable stem-loop structure (indicated by the hair-pin) is predicted downstream of uORF 2 (Fig. S5B). The AUGs of the three ORFs are in favorable contexts, and reports indicate that they are translated (De Pietri Tonelli et al., 2004; Mihailovich et al., 2007; Zhou and Song, 2006). In our model, ribosomes would bind to the BACE1 mRNA cap, scan down the 5’ UTR, and initiate translation first at uORF1. After translation of uORF1 is terminated and the 60S subunits dissociate, a proportion of 40S subunits remain attached and continue to scan along the BACE1 mRNA 5’ UTR. Under normal conditions, levels of phosphorylated eIF2α are low (left) and ternary complex concentrations are high. In this case, 40S subunits are efficiently reloaded with ternary complexes, 43S preinitiation complexes are rapidly formed, and translation reinitiation occurs at uORF2. A rare proline codon at the 3’ end of uORF2 (boxed P; Fig. S5A) and downstream stable secondary structure (Fig. S5B) increase dissociation of ribosomes from the BACE1 mRNA, reducing the proportion of 40S subunits that scan through the BACE1 mRNA 5’ UTR and reach the authentic BACE1 start codon for reinitiation. As a consequence, translation of the BACE1 ORF is inefficient under normal conditions. In contrast, under cellular stress conditions, phosphorylated eIF2α levels are high (right) and ternary complex concentrations are low. Translation initiation is still predicted to occur first at uORF1. Following termination of uORF1 translation, a proportion of 40S subunits remain attached and continue scanning the 5’ UTR, as in low eIF2α-P conditions. However, when eIF2α-P is high, decreased ternary complex availability causes the 40S subunit to spend more time scanning before becoming reloaded to form the 43S preinitiation complex. As a result, a higher proportion of 40S subunits scan through uORF2-3 and reach the authentic BACE1 AUG to reinitiate translation at the BACE1 ORF. Thus, BACE1 mRNA translation is more efficient under stress conditions than normal. (C) Common risk factors for Alzheimer’s disease such as age, high cholesterol, cardiovascular disease, traumatic brain injury, and ApoE4 genotype may lead to a state of impaired energy metabolism in the brain. Reduced energy availability activates the eIF2α-P stress-response pathway in neurons. Phosphorylation of eIF2α augments the translation of specific stress response proteins such as BACE1. Increased production of these stress-response proteins presumably enhances the ability of neurons to survive under low-energy conditions. However, if the stress persists, eIF2α phosphorylation and BACE1 levels remain chronically elevated, leading to increased Aβ production. Even a small increase in Aβ generation may have a significant impact on amyloid accumulation over the many years apparently required for AD development. Elevated Aβ levels may, in turn, cause neuronal dysfunction and stress, feeding back and further activating the eIF2α-P stress-response pathway. Over time, and in combination with other exacerbating factors such as impaired Aβ clearance/degradation, Aβ begins to accumulate in the brain and plaques form. Aβ accumulation may trigger downstream pathology, (e.g., tau hyperphosphorylation), ultimately leading to neurodegeneration associated with sporadic Alzheimer’s disease. (UPR = unfolded protein response).
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