Energy Deprivation Increases BACE1 Levels by a Translational Mechanism
Previously, we observed increased BACE1 protein levels in the absence of increased BACE1 mRNA levels following acute energy inhibitor treatment in mice, suggesting a post-transcriptional mechanism (Velliquette et al., 2005
). To investigate the molecular mechanism responsible for raising the BACE1 level in response to energy inhibition, we used HEK-293 cells that stably overexpress the entire 2.5kb human BACE1 transcript, including the complete 5’ and 3’ UTRs, driven by the CMV promoter (BACE1-293 cells). BACE1-293 cells are ideal for investigating BACE1 post-transcriptional mechanisms because the cell line expresses BACE1 mRNA from a constitutive heterologous promoter that is transcriptionally stable under a wide range of conditions, and it expresses BACE1 mRNA with the endogenous 5’ and 3’ UTRs that are required for the normal translational regulation of BACE1 mRNA. We treated BACE1-293 cells for 2 to 48 hrs in media containing 25mM glucose (CON) or lacking glucose (NG) and analyzed BACE1 levels in lysates by immunoblot (). Energy deprivation slowed cell growth but was otherwise non-toxic, as indicated by failure to activate caspase 3 (Fig. S2
). BACE1 levels in NG-treated cells showed a clear upward trend at 2 hrs that became statistically significant at 6 hrs and peaked around 12 hrs (; p < 0.001). The glucose deprivation-induced BACE1 increase plateaued at ~150-170% of control (CON) levels and remained high for the duration of the experiment. Importantly, the early increase in BACE1 levels following glucose deprivation suggested a post-transcriptional mechanism.
Glucose deprivation increases BACE1 protein levels in BACE1-293 cells via a post-transcriptional mechanism
Although the constitutively active CMV promoter made it unlikely that increased BACE1 gene transcription was the cause of the glucose deprivation-induced BACE1 elevation, we sought to definitively exclude this possibility by treating BACE1-293 cells with the transcriptional inhibitor actinomycin D (ActD) in the presence or absence of glucose (). First, it is relevant to note that 12 hrs of NG treatment of BACE1-293 cells on its own did not increase steady-state BACE1 mRNA levels relative to control treated cells (, two left bars), even though BACE1 protein levels were significantly elevated (, two left bars). Most importantly, ActD treatment failed to prevent BACE1 levels from increasing in cells incubated in NG medium. BACE1 was increased to the same level in NG-treated cells regardless of whether they were exposed to ActD or not (). This is a striking result, given that ActD dramatically reduced BACE1 mRNA levels in cells either incubated with or without glucose, as determined by BACE1 primer-specific TaqMan quantitative real-time PCR (, two right bars). These results conclusively demonstrate that the BACE1 increase induced by energy deprivation in BACE1-293 cells was the result of a post-transcriptional mechanism and was not due to either elevated BACE1 transgene mRNA synthesis or increased BACE1 mRNA stability.
Recent studies have shown that BACE1 protein stability can be modulated by the lysosomal and ubiquitin-proteasomal degradation pathways (Koh et al., 2005
; Qing et al., 2004
; Tesco et al., 2007
). To determine whether increased BACE1 protein stability was the post-transcriptional mechanism underlying the BACE1 increase in response to energy deprivation, we used pulse-chase 35
S-metabolic radiolabeling to measure the half-life (t1/2
) of BACE1 protein in BACE1-293 cells incubated under normal chase conditions compared to cells chased in NG media or in media containing 2-deoxyglucose (2DG), which competes with glucose for catabolism by hexokinase. We observed that the normal t1/2
of BACE1 is ~12 hours, similar to previous reports (Haniu et al., 2000
; Huse et al., 2000
). Importantly, under NG or 2DG chase conditions, we found that the t1/2
of BACE1 was similar to control (), indicating that increased BACE1 protein stability is not the post-transcriptional mechanism responsible for elevated BACE1 levels in response to energy deficiency.
Taken together, our results thus far excluded gene transcription, mRNA stability, and protein stability as causes of the energy deprivation-induced BACE1 increase, suggesting that the BACE1 increase was likely to occur by a translational mechanism. In support of this notion, recent studies have shown that translation is inhibited by the BACE1 mRNA 5’ UTR, which is long (453 nts), GC-rich (77%), is predicted to have extensive secondary structure, and contains 3 uORFs (De Pietri Tonelli et al., 2004
; Lammich et al., 2004
; Mihailovich et al., 2007
; Rogers et al., 2004
). These characteristics of the BACE1 5’UTR are typical of translationally-regulated stress response mRNAs (Clemens, 2001
), suggesting that the BACE1 transcript may be a target of translational control by one or more stress-activated pathways. However, previous studies had not addressed whether BACE1 was regulated by stress-induced translational pathways, and if so, whether BACE1 translational control could be relevant to AD pathogenesis. To initially test these hypotheses, we investigated whether the BACE1 mRNA 5’UTR was required for the energy deprivation-dependent BACE1 increase. We transiently transfected HEK-293 cells with CMV promoter-driven expression vectors encoding BACE1 either with (+5’UTR) or without (-5’UTR) the BACE1 5’UTR (Lammich et al., 2004
) and then treated cultures in normal or NG media for 24 hrs (). Similar to our BACE1-293 cell experiments, transfection with the BACE1 +5’UTR construct followed by BACE1 immunoblot analysis revealed a ~150% increase of BACE1 levels when cells were incubated in NG media, compared to control glucose-containing media (, left). Removal of the BACE1 5’UTR caused an increase in BACE1 levels in transfected cells (, right), as previously reported (Lammich et al., 2004
). In addition to the mature ~70kDa BACE1 species, a significant proportion of the BACE1 in cells transfected with the BACE1 -5’UTR construct migrated at ~60kDa on immunoblots. Since the ~60kDa BACE1 species also labeled with an anti-BACE1 C-terminal antibody (not shown), it was full-length BACE1 rather than a truncated BACE1 fragment and therefore was likely to be immaturely glycosylated BACE1 (Haniu et al., 2000
). Apparently, the increased expression of BACE1 from the BACE1 -5’UTR construct overloaded the ER, causing accumulation of immature BACE1. Importantly, transfection with the BACE1 -5’UTR construct followed by glucose deprivation failed to cause BACE1 levels to increase above those observed in BACE1 -5’UTR transfected cells incubated in glucose-containing media (, right). As expected, glucose deprivation did not affect levels of BACE1 mRNA transcribed from BACE1 +5’UTR and -5’UTR expression vectors, as measured by TaqMan RT-PCR analysis (not shown). Although unlikely, at this point we cannot exclude the possibility that the glucose deprivation-induced BACE1 increase was masked by the large rise in BACE1 expression from the BACE1 -5’UTR construct. However, our results strongly suggest that the BACE1 mRNA 5’ UTR is necessary for the energy deprivation-dependent increase of BACE1 and further indicate that a translational control mechanism is at play.
Energy Deprivation Increases eIF2α Phosphorylation
Control of translation initiation is considered to be of prime importance for translational regulation. It is well established that diverse cellular stresses affect translation at the level of initiation (Clemens, 2001
). Since our experiments demonstrated that the BACE1 mRNA 5’ UTR was required for the energy deprivation-induced BACE1 increase, and that the BACE1 5’UTR exhibited characteristics of translationally-regulated stress response mRNAs, we investigated whether BACE1 levels might be regulated by stress-induced translational control pathways during energy deprivation. Initially, we screened cell lysates from NG-treated and control BACE1-293 cells by immunoblot for phosphorylation of proteins involved in translational regulation following stress (, S1
). Using this screen we found that phosphorylation of serine 51 of the α subunit of eukaryotic translation initiation factor 2 (eIF2α-P(Ser51)) was dramatically increased in response to NG treatment, implicating this component of the translation initiation complex in the energy deprivation-induced BACE1 elevation. Strikingly, 24 hrs of NG treatment increased the ratio of eIF2α-P(Ser51) to total eIF2α (eIF2α-T) to ~250% of cells grown in normal glucose-containing media (; p < 0.01). Notably, eIF2α-P levels were increased to ~200% of control at only 2 hrs of NG treatment, a time when BACE1 levels had just begun to rise but were not yet significantly elevated. Therefore, NG-induced eIF2α phosphorylation preceded the BACE1 increase, as expected for a cause-and-effect relationship.
Glucose deprivation increases eIF2α phosphorylation and activates a specific set of stress-response signaling pathways in BACE1-293 cells
We found no evidence of alterations in the phosphorylation state of the other major factor that is involved in the control of translation initiation during stress, phosphorylated eukaryotic translation initiation factor 4E (eIF4E-P; ), indicating that energy deprivation specifically increases phosphorylation of eIF2α. In addition, treatment of BACE1-293 cells with rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), had no effect on BACE1 levels (Fig. S1
). mTOR is the kinase that regulates eIF4E binding protein (4E-BP), eukaryotic elongation factor 2 (eEF2) kinase, and the ribosomal subunit protein S6 kinase. S6 and eEF2 are in turn phosphorylated and regulated by S6 kinase and eEF2 kinase, respectively. Therefore, the negative rapamycin results further exclude a role for eIF4E and indicate lack of involvement of eEF2 and S6 in translational control of BACE1.
We also found no evidence of caspase-3 activation in glucose-deprived BACE1-293 cells, indicating that apoptosis is not involved in the BACE1 increase (Fig. S2
). We did observe increased phosphorylation of the stress-activated proteins c-Jun and c-Jun N-terminal kinase (JNK) in response to glucose deprivation (), which may simultaneously lead to transcriptional activation of other stress response proteins. Interestingly, we also observed increased levels of total c-Jun protein, the structural and functional homolog of the yeast protein GCN4 (Struhl, 1987
; Vogt et al., 1987
) which is a well-established translational target of the eIF2α-P pathway (; (Cigan et al., 1993
; Vazquez de Aldana et al., 1993
). Although it is unknown whether eIF2α-P regulates translation of c-Jun mRNA, several studies report that c-Jun mRNA translation is increased under different conditions of stress or injury (Gietzen et al., 2004
; Polak et al., 2006
; Rao, 2000
; Spruill et al., 2008
; Vardimon et al., 2006
). Taken together, these results demonstrate that eIF2α phosphorylation is specifically increased as a result of energy deprivation and is correlated with the BACE1 increase.
Selective Inhibition of eIF2α Dephosphorylation Increases BACE1 Levels
Phosphorylation of eIF2α on serine 51 is a major mechanism that regulates initiation of translation in response to various cellular stresses, including virus infection, nutrient deprivation, iron deficiency, and accumulation of unfolded proteins in the ER (Clemens, 2001
; Proud, 2001
). Depending on the specific cellular stress, eIF2α is phosphorylated by at least 4 different kinases, including double-stranded RNA-activated kinase (PKR), general control nonderepressible 2 kinase (GCN2), heme-regulated inhibitor kinase (HRI), and PKR-like ER kinase (PERK). Following stress-induced phosphorylation of eIF2α, translation of normal cellular mRNAs is repressed, while the translational initiation of select mRNAs involved in stress response is stimulated. eIF2α-P is dephosphorylated by protein phosphatase-1 (PP1) complexed with its regulatory subunit, growth arrest and DNA damage-inducible protein 34 (GADD34). Importantly, the PP1/GADD34 complex is inhibited by the small molecule drug salubrinal (Sal), which selectively blocks dephosphorylation of eIF2α-P but not other PP1 substrates (Boyce et al., 2005
If eIF2α phosphorylation stimulates the translation of BACE1 mRNA, then directly raising levels of eIF2α-P by selective inhibition of eIF2α-P dephosphorylation with Sal should increase BACE1 levels in the absence of energy deprivation. To test this hypothesis, we treated BACE1-293 cells for 24 hrs with Sal (100μM), NG medium, or glucose-containing medium. Like NG treatment, incubating BACE1-293 cells with salubrinal caused both eIF2α-P(Ser51) and BACE1 levels to increase to ~150-200% of control values (; p < 0.05). Therefore, PP1/GADD34 inhibition raised eIF2α-P levels and directly caused the BACE1 increase without the need of glucose deprivation, demonstrating that BACE1 is a translational target of the stress-activated eIF2α-P(Ser51) pathway.
Direct manipulation of eIF2α phosphorylation affects the BACE1 increase in BACE1-293 cells
Activation of the eIF2α Kinase PERK is Necessary for the Energy-Deprivation Induced BACE1 Increase
Of the four known eIF2α kinases, the two most likely to be induced by energy metabolism stress are PERK and GCN2, which are activated by the ER stress/unfolded protein response (UPR) and amino acid deprivation, respectively. PKR and HRI are less likely to play a role in the energy deprivation-induced BACE1 increase because they are activated by virus infection and heme deficiency in erythrocytes, respectively (Clemens, 2001
). Therefore, we sought to determine whether PERK or GCN2 was responsible for phosphorylating eIF2α and causing the BACE1 increase following glucose deprivation. To accomplish this, we used kinase-dead forms of PERK and GCN2 that had a C-terminal deletion (PERK; Harding et al., 1999
) or were mutated at the critical lysine 618 residue in the conserved kinase domain of the enzyme (GCN2; Sood et al., 2000
), thus producing dominant-negative molecules that block eIF2α phosphorylation. We transiently transfected BACE1-293 cells with constructs encoding dominant negative PERK (PERKDN) or GCN2 (GCN2DN), allowed cells to recover overnight, and then treated cells for 12 hrs in NG or glucose-containing media. Expression of GCN2DN in combination with glucose deprivation failed to prevent eIF2α phosphorylation or BACE1 elevation in BACE1-293 cells (), indicating that GCN2 is not the kinase that phosphorylates eIF2α under glucose-deficient conditions. In contrast, expression of PERKDN in combination with NG treatment completely blocked the increases of both eIF2α-P and BACE1 (), demonstrating that PERK is the specific kinase responsible for phosphorylating eIF2α and controlling the energy deprivation-induced BACE1 elevation.
To provide further support for the role of PERK in BACE1 translational control, we performed two additional experiments using a cellular inhibitor of PERK, P58IPK
(Yan et al., 2002
), and the compound tauroursodeoxycholic acid (TUDCA), which alleviates ER stress (Ozcan et al., 2006
). The unfolded protein/ER stress response induces the expression of P58IPK
, a molecular chaperone that is thought to bind to the kinase domain of PERK and inhibit its activity (Yan et al., 2002
). Transient transfection of BACE1-293 cells with an expression vector encoding P58IPK
prevented the BACE1 increase following glucose deprivation (Fig. S3A, B
), mirroring our previous results with PERKDN transfection. Similarly, TUDCA treatment of BACE1-293 cells also blocked the BACE1 increase caused by incubation in NG medium (Fig. S3C, D
). Taken together, our experiments with PERKDN, GCN2DN, P58IPK
, and TUDCA strongly suggest that PERK is the eIF2α kinase activated by energy deprivation, and that it is ultimately responsible for the eIF2α-P induced translational increase of BACE1.
Selective Inhibition of eIF2α Dephosphorylation Increases BACE1 Level and Aβ production in Tg2576 Primary Neurons
If eIF2α phosphorylation increases the translation of BACE1 mRNA in neurons as it does in BACE1-293 cells, then directly raising levels of neuronal eIF2α-P by selective inhibition of eIF2α-P dephosphorylation with salubrinal treatment should elevate BACE1 levels in the absence of energy deprivation. Moreover, the resulting rise in BACE1 level should cause an increase in neuronal Aβ generation as well. To test these hypotheses, we cultured primary cortical neurons from Tg2576 transgenic mice that overexpress human APPsw (Hsiao et al., 1996
) and treated the cultures with salubrinal. Since the effects of salubrinal on primary neurons had not yet been published, we chose two salubrinal concentrations (50μM and 80μM) that we determined from a previous study (Boyce et al., 2005
) would be likely to inhibit PP1/GADD34 with minimal neuronal toxicity. Tg2576 primary neurons from E15.5 embryos were cultured for 7 DIV, treated with 50μM or 80μM salubrinal for 48 hrs, and conditioned media and neuronal lysates collected. As we observed in BACE1-293 cells, immunoblot analysis revealed that salubrinal treatment increased levels of both eIF2α-P(Ser51) and endogenous BACE1 protein in Tg2576 neurons (). Furthermore, these results together with the glucose deprivation experiments establish that the same mechanism of BACE1 increase occurs in both BACE1-293 cells and primary neurons.
Next, we determined whether salubrinal treatment of the Tg2576 neuron cultures had increased Aβ production. To do so, we used a human Aβ40
-specific ELISA to measure levels of the 40-amino acid isoform of Aβ (Aβ40
) secreted into the conditioned media of control, 50μM, and 80μM Sal-treated neuron cultures. Changes in BACE1 activity affect the generation of all isoforms of Aβ equally (Vassar et al., 1999
is the most abundant Aβ isoform produced in cells and tissues, and it is a gauge of BACE1 activity in cells. In addition, Aβ40
is the major isoform of Aβ produced in Tg2576 transgenic mice. As expected, Aβ40
levels were significantly elevated in conditioned media from salubrinal-treated neurons, as compared to control (; CON = 13.25 ± 0.33 ng Aβ40
/mg protein; 50μM Sal = 15.88 ± 0.63, p < 0.05; 80μM Sal = 27.19 ± 1.61, p < 0.01).
The Aβ40 increase for the 50μM Sal-treated Tg2576 neurons was less than expected, based on the level of phosphorylated eIF2α (). The reason for this is unclear, but we suspect that BACE1 levels increased at a lower rate in 50μM Sal compared to 80μM Sal, thereby delaying the Aβ40 increase. It is likely that Aβ40 levels in 50μM Sal treated cultures would have eventually caught up with those of 80μM Sal. Regardless, it is important to note that the Aβ40 increase in 50μM Sal-treated Tg2576 neurons was significant, and that the Aβ40 increase for 80μM Sal was ~2-fold over control. In addition, our results demonstrate that selective inhibition of eIF2α dephosphorylation directly causes BACE1 levels to increase in neurons in the absence of energy deprivation. Taken together, the salubrinal-treated Tg2576 neuron cultures confirm that BACE1 is a translational target of eIF2α-P(Ser51) in neurons and demonstrate that direct induction of eIF2α-P increases Aβ production, which has important implications for AD pathogenesis.
Chronic Energy Inhibition Increases eIF2α Phosphorylation, BACE1 Levels, and Amyloidogenesis in Tg2576 Mice
Thus far, our results demonstrated that energy deprivation caused increases in eIF2α phosphorylation, BACE1 translation, and Aβ production in vitro
, conditions that we predicted would exacerbate amyloid deposition in vivo
. To test this, we designed a chronic in vivo
energy deprivation paradigm based on our previous acute study in which we treated Tg2576 mice with inhibitors of energy metabolism that caused increases in BACE1 level and Aβ production (Velliquette et al., 2005
). In that acute study, we gave 2 month-old Tg2576 mice single intraperitoneal (i.p.) injections of compounds that produced or mimicked hypoglycemia, inhibited ATP synthesis, or caused neuronal over-excitation which depleted ATP stores: insulin, 2-deoxyglucose (2DG) and 3-nitropropionic acid (3NP), and kainic acid (KA). Since our in vitro
studies primarily focused on the effects of glucose deprivation on eIF2α phosphorylation and BACE1 level elevation, in the chronic in vivo
study we chose to only use the two compounds that would specifically interfere with glucose energy metabolism at the level of glycolysis and the Krebs cycle: 2DG and 3NP. 2DG is a glucose analog that is a competitive inhibitor of hexokinase, while 3NP inhibits the Krebs cycle and electron transport chain enzyme succinate dehydrogenase. Insulin and KA were not used in the current study because we reasoned that these two compounds were more likely to cause pleotrophic physiological effects in vivo
, especially under conditions of chronic dosing, thus potentially complicating the interpretation of results.
For our chronic in vivo
energy deprivation experiment, 9 month-old Tg2576 and C57/BL6 (wild-type) mice were treated once per week with i.p. injections of 2DG (1g/kg), 3NP (80mg/kg), or vehicle for a duration of 3 months. In our acute study, BACE1 and Aβ levels stayed elevated for at least a week following a single dose of 2DG or 3NP (Velliquette et al., 2005
), thus providing the rationale for single weekly injections in the current study. Treatments were started at 9 months of age, when Tg2576 mice were on the cusp of amyloid plaque formation (Hsiao et al., 1996
), and concluded when mice were 12 months old. The doses of 2DG and 3NP were chosen based on a pilot study showing that they were able to increase BACE1 to levels similar to those observed in our acute study (not shown). These doses were well-tolerated by the mice, no mortality was observed, and treatments caused only temporary lethargy lasting 30-60 min. Cresyl violet staining and GFAP immunohistochemistry of brain sections from 2DG or 3NP treated mice revealed no significant neurodegeneration or gliosis associated with treatment (Fig. S4
Following 3 months of treatment, hemibrains of each Tg2576 mouse were analyzed for levels of eIF2α-P, BACE1 (by immunoblot) and Aβ40
(ELISA), and amyloid plaque number (by anti-Aβ immunohistochemistry and Thioflavin-S staining). C57/BL6 hemibrains were analyzed in a similar manner as those of Tg2576, except one hemibrain from each C57/BL6 mouse was processed for TaqMan mRNA quantification rather than histology. As expected, chronic experimental glucose deprivation caused a significant increase of BACE1 level in the brains of Tg2576 () and C57/BL6 () mice. 2DG and 3NP-treated mice exhibited BACE1 increases of ~120-130% (p < 0.05) and ~160-170% (p < 0.001), respectively, in comparison to vehicle control treatment. Importantly, the BACE1 increases following chronic treatment were similar in magnitude to those observed under conditions of acute energy deprivation in vivo
(Velliquette et al., 2005
) and in vitro
Elevated BACE1 levels are correlated with increased eIF2α-P and amyloidogenesis in a chronic model of energy deprivation in vivo
Chronic energy deprivation causes post-transcriptional increases of BACE1 in vivo
Based on our in vitro studies with BACE1-293 cells and primary neurons, we predicted that the BACE1 increase following chronic in vivo energy deprivation would be post-transcriptional. To test this, we isolated mRNA from hemibrains of C57/BL6 mice chronically treated with 2DG, 3NP, and vehicle and measured levels of endogenous mouse BACE1 mRNA by TaqMan quantitative real-time PCR analysis. Indeed, BACE1 mRNA levels were not increased upon 2DG or 3NP treatment, and in fact were significantly decreased compared to vehicle (). Although we do not fully understand the reason for this mRNA decrease, we speculate that it may be a result of global down-regulation of transcription caused by ATP depletion. In any case, our results clearly demonstrate that the BACE1 elevations in response to chronic in vivo energy deprivation were not the result of either increased BACE1 gene transcription or enhanced BACE1 mRNA stabilization.
Since our previous results showed that eIF2α phosphorylation and enhanced BACE1 translation were responsible for the energy deprivation-induced BACE1 increase in vitro, we wanted to determine whether increased eIF2α phosphorylation might account for the BACE1 elevation in vivo. To accomplish this, we used immunobot analysis to measure phosphorylated and total eIF2α levels in brain homogenates of Tg2576 mice treated with 2DG, 3NP, or vehicle (). The eIF2α-P(Ser51):eIF2α-T ratios in the brains of 2DG and 3NP-treated mice were elevated to ~130% (p < 0.05) and ~150% (p < 0.01) of vehicle, respectively (), similar to the increases in eIF2α phosphorylation observed in vitro. Importantly, mean increases in eIF2α phosphorylation correlated with the BACE1 elevations that occurred in 2DG and 3NP-treated mice. These results, together with our in vitro BACE1-293 cell and primary neuron data, suggest that chronic energy deprivation in vivo induces the eIF2α translational control pathway, thereby increasing BACE1 levels.
Previous studies had reported that modest overexpression of BACE1 increased Aβ levels and amyloid deposition in transgenic mice (Bodendorf et al., 2002
; Chiocco et al., 2004
; Chiocco and Lamb, 2007
; Lee et al., 2005
; Mohajeri et al., 2004
; Ozmen et al., 2005
; Willem et al., 2004
). Therefore, the elevated BACE1 levels in 2DG and 3NP-treated Tg2576 mice suggested that levels of Aβ and amyloid plaques might also be increased in these animals. To investigate this, we measured Aβ40
in brain homogenates by Aβ40
-specific ELISA and counted amyloid plaques in hemibrain sections from treated Tg2576 mice. 2DG and 3NP treatment caused clear trends toward increased Aβ40
levels (; VEH = 9.10 ± 1.30 ng Aβ40
/mg protein, 2DG = 13.15 ± 1.94, 3NP = 15.56 ± 3.75) and plaque numbers (; VEH = 20.8 ± 3.7; 2DG = 26.7 ± 5.3; 3NP = 36.0 ± 8.6), with a nearly 2-fold increase occurring in 3NP-treated mice. Consistent with these observations, examination of brain sections stained with anti-Aβ antibody (, S5
) or Thioflavin-S (Fig. S5
) showed that plaques in 2DG and 3NP-treated mice appeared larger and more numerous than in vehicle-treated mice. High inter-animal variability in treated mice caused the increases of Aβ40
level and plaque number to not quite reach statistical significance, although it is likely that statistical significance would have been achieved with longer treatment times.
Importantly, we consistently observed that 3NP treatment always produced the strongest increases of phosphorylated eIF2α, BACE1, Aβ40, and plaque number, while 2DG treatment always had more moderate effects (compare ). 3NP is a strong, irreversible inhibitor of glucose metabolism, in contrast to the weaker reversible inhibitor, 2DG. Thus, the effects of the drugs on phosphorylated eIF2α, BACE1, Aβ40, and plaque number reflected their relative potency for inhibiting glucose metabolism. Taken together, these observations make a compelling case that chronic energy deprivation in vivo is likely to promote amyloidogenesis via a mechanism involving eIF2α phosphorylation and BACE1 translational control.
Finally, we wanted to exclude the possibility that other mechanisms were responsible for elevating BACE1 levels and promoting amyloidogenesis in 2DG and 3NP-treated Tg2576 mice, such as altered micro-RNA (miRNA) expression, increased γ-secretase, or decreased Aβ degrading enzyme levels. Recent studies have shown that miRNAs miR-29a, -29b-1, and -9 are decreased in AD and are capable of regulating BACE1 expression in vitro
(Hebert et al., 2008
; Wang et al., 2008
). Therefore, we measured the levels of miR-29a, -29b-1, and -9 in treated Tg2576 mice by TaqMan quantitative RT-PCR analysis. We found that none of these miRNAs were decreased in brains subjected to chronic energy deprivation (Fig. S6
). In fact, miR-29a and -29b-1 were increased
in 3NP-treated mice, as compared to vehicle treatment, which would be expected to decrease
BACE1 levels (Hebert et al., 2008
; Wang et al., 2008
). Next, we performed immunoblot analyses for the amino-terminal fragment (NTF) of presenilin 1 (PS1), a critical subunit of the γ-secretase, and insulin degrading enzyme (IDE) and neprilysin (NEP), two major Aβ degrading enzymes (Fig. S7
). Overall, the levels of these molecules were not significantly affected by chronic energy deprivation in vivo
(Fig. S7A, B
), although a trend toward elevated NEP levels was observed for 3NP-treated mice, a condition that would be expected to decrease
Aβ levels. In addition, PS1-NTF, IDE, and NEP levels were not altered in Sal-treated Tg2576 primary neuron cultures (Fig. S7C, D
). Taken together, these results make it unlikely that miRNAs, γ-secretase, or Aβ degrading enzymes played a major role in the increased levels of BACE1 and Aβ in response to chronic energy deprivation in Tg2576 mice.
Increased eIF2α Phosphorylation Correlates with Elevated BACE1 Levels in 5XFAD Transgenic Mice and Humans with AD
At this point in our analysis, the in vitro
and in vivo
energy deprivation experiments suggested the possibility that impaired energy metabolism in AD could increase BACE1 levels via a mechanism involving eIF2α phosphorylation and exacerbate amyloidogenesis. Reports had already shown that BACE1 levels and activity were increased in AD brains compared to non-demented controls (Fukumoto et al., 2002
; Holsinger et al., 2002
; Li et al., 2004
; Sennvik et al., 2004
; Tyler et al., 2002
; Yang et al., 2003
). Moreover, we had demonstrated that BACE1 levels were dramatically elevated around amyloid plaques in AD patients, Tg2576 mice, and in our aggressive amyloid deposition model, 5XFAD transgenic mice (Zhao et al., 2007
). However, no study had yet addressed whether chronic energy deprivation or eIF2α phosphorylation was potentially involved with the BACE1 increase in AD. If impaired cerebral energy metabolism played a role in sporadic AD, and if the mechanism entailed increased translation of BACE1 as a result of eIF2α phosphorylation, then we would expect to observe increased levels of eIF2α-P and correlation between eIF2α-P and BACE1 levels in AD brain. Similar conditions might also exist in APP transgenic mice, since we observed a robust BACE1 increase in the brains of 5XFAD transgenic mice (Zhao et al., 2007
In order to initially test these hypotheses, we analyzed levels of BACE1, eIF2α-P, and eIF2α-T by immunoblot in the brains of six-month-old 5XFAD mice that had florid amyloid pathology (Oakley et al., 2006
). In agreement with our recent report (Zhao et al., 2007
), we observed that BACE1 levels in 5XFAD brains were increased to ~170% of non-transgenic (non-Tg) littermate controls (p < 0.01; ). As we had predicted, levels of eIF2α-P(Ser51) were elevated in 5XFAD brain as well (). Unexpectedly, however, total eIF2α levels were also increased in 5XFAD brain compared to non-Tg control. Importantly, in spite of the elevated eIF2α-T levels, the eIF2α-P(Ser51):eIF2α-T ratio was significantly increased to ~120% of non-Tg control in 5XFAD brains (p < 0.05; ). The mechanism and functional significance of the increase in total eIF2α remains unclear; however, we speculate that it may represent an adaptive response to chronic stress caused by the aggressive amyloid deposition in 5XFAD mice. It is noteworthy that increased BACE1 mRNA levels were not responsible for the BACE1 elevation in 5XFAD mice (Zhao et al., 2007
), thus suggesting a post-transcriptional mechanism most likely involving eIF2α phosphrylation. In addition, we measured levels of miR-29a, -29b-1, and -9 and found no evidence that these miRNAs had altered expression in 5XFAD brains (Fig. S6
). Finally, it is significant that 5XFAD mice had elevated cerebral eIF2α-P and BACE1 levels in the absence of pharmacologically induced energy deprivation, indicating that eIF2α phosphorylation could be increased by amyloid pathology as well as energy deprivation. This has important implications for a potential positive feedback mechanism that may drive BACE1 elevation and amyloidogenesis in AD (Fig. 9).
eIF2α-P levels are elevated in 5XFAD transgenic mouse and human AD brains and positively correlate with BACE1 levels and amyloid loads
As the final step in our study, it was necessary to verify that eIF2α phosphorylation and BACE1 were elevated in AD brain. To accomplish this, we performed immunoblot analysis to measure levels of BACE1, eIF2α-P, and eIF2α-T in a series of cortical human brain samples from AD patients and age-matched, non-demented (N) controls (). In agreement with previous reports, BACE1 levels were significantly elevated in AD brains (~140% of non-demented (N) control; p < 0.01). As predicted, AD brains also exhibited increased eIF2α-P(Ser51) levels (), and the AD eIF2α-P(Ser51):eIF2α-T ratio was found to be ~150% of N control (p < 0.05). We next performed linear regression analyses and determined that BACE1 level and amyloid load had a significant positive correlation in human brain, as expected (p < 0.05; ). Importantly, BACE1 levels plotted against eIF2α-P(Ser51):eIF2α-T ratios also showed a significant positive correlation (p = 0.0135; ), suggesting that the increase in eIF2α-P may cause the BACE1 elevation in human brain. Finally, amyloid load also exhibited significant positive correlation with eIF2α-P:eIF2α-T ratio (p < 0.01; ), in further support of the hypothesis that eIF2α-P may promote amyloidosis. Taken as a whole, our 5XFAD mouse and human AD results strongly suggest that increased eIF2α phosphorylation plays a role in the elevation of BACE1 levels in the brain and contributes to the development of amyloid pathology.