Rheb GTPase regulates β-secretase levels and amyloid β generation

Abstract

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (β-secretase, BACE1) initiates amyloidogenic processing of APP to generate amyloid β (Aβ), which is a hallmark of Alzheimer disease (AD) pathology. Cerebral levels of BACE1 are elevated in individuals with AD, but the molecular mechanisms are not completely understood. We demonstrate that Rheb GTPase (Ras homolog enriched in brain), which induces mammalian target of rapamycin (mTOR) activity, is a physiological regulator of BACE1 stability and activity. Rheb overexpression depletes BACE1 protein levels and reduces Aβ generation, whereas the RNAi knockdown of endogenous Rheb promotes BACE1 accumulation, and this effect by Rheb is independent of its mTOR signaling. Moreover, GTP-bound Rheb interacts with BACE1 and degrades it through proteasomal and lysosomal pathways. Finally, we demonstrate that Rheb levels are down-regulated in the AD brain, which is consistent with an increased BACE1 expression. Altogether, our study defines Rheb as a novel physiological regulator of BACE1 levels and Aβ generation, and the Rheb-BACE1 circuitry may have a role in brain biology and disease.

Keywords: Aging; Amyloid; GTPase; Protein Stability; Secretases.

FIGURE 1.

Rheb GTPase regulates BACE1 levels in HEK293 cells. A, Myc (control) or Myc-Rheb cDNAs (2 μg each) transfected in HEK293 cells. After 48 h, the cells were lysed, and the equal proteins were loaded on the Western blot gel and probed for the proteins of interest, as indicated under “Experimental Procedures.” Note that BACE1 levels were significantly reduced in Myc-Rheb transfected cells (n = 6, ***, p < 0.001 versus control, Student's t test). B, Rheb reduces BACE1 protein levels in a dose-dependent manner in HEK293 cells. Myc (1 μg) or Myc-Rheb cDNA (0.1, 0.5, 1 μg) was transfected in HEK293 cells, and, after 48 h, the cells were lysed and probed for BACE1 and PERK (protein kinase RNA-like endoplasmic reticulum kinase). ***, p < 0.001, **, p < 0.01. C, BACE1 antibody, D10E5, is as specific as 3D5 to detect BACE1. BACE1^+/+ and BACE1^−/− mice brain lysates were probed for BACE1 and EIF2α. D10E5 or 3D5 antibody (1:1000) detected ∼62-kDa bands only in BACE1^+/+, but not in BACE1^−/− mice brain lysates. D, 3D5 antibody detects Rheb-mediated BACE1 suppression. Myc or Myc-Rheb were transfected in HEK293 cells, and BACE1 was detected using 3D5 antibody, as in panel A.

FIGURE 2.

Rheb GTPase regulates BACE1 levels in brain cells. A, Rheb overexpression reduces BACE1 levels and its activity in brain. Adeno-associated virus (AAV)-GFP or AAV-Rheb-Flag was injected into the left and right hippocampus of P90 mice, respectively. After 3 weeks, the hippocampus was dissected and processed to detect BACE1 and other proteins, as indicated under “Experimental Procedures.” BACE1 levels, sAPPβ, and Aβ (x-40 and x-42) were selectively down-regulated by AAV-Rheb (n = 6, ***, p < 0.001, *, p < 0.05 versus AAV-GFP, Student's t test). B, Rheb overexpression reduces BACE1 levels and its activity in cultured neurons. Adenovirus null (Ad-Control) or adenovirus Rheb (Ad-Rheb) was infected in the primary cortical neurons at 14 days in vitro, and BACE1 and other proteins were detected in lysates, as in Fig. 1A. sAPPβ was measured in the medium, and Aβ was detected by ELISA, as indicated under “Experimental Procedures.” Note that Ad-Rheb markedly inhibited BACE1 levels and reduced BACE1 activity, as demonstrated by decrease in sAPPβ, APP-CTFβ, and Aβ (x-40 and x-42) levels (n = 8, ***, p < 0.001, **, p < 0.01 versus Ad-control). C, depletion of Rheb enhanced BACE1 in the cultured neurons. Primary cortical neurons (14 days in vitro) were infected with lentiviral particles expressing scrambled shRNA or Rheb shRNA1, and after 3 days, the neurons were lysed to detect BACE1, sAPPβ, APP-CTFβ, APP-CTFα, and Aβ (x-40 and x-42) (n = 6, ***, p < 0.001, *, p < 0.05 versus control shRNA (Con shRNA)).

FIGURE 3.

Rheb GTPase degrades BACE1 protein in an mTOR-independent manner. A, Myc or Myc-Rheb cDNA (2 μg each) was transfected in HEK293 cells, and after 48 h, the cells were lysed and RNA was isolated to detect BACE1 levels and GAPDH by RT-PCR, as indicated under “Experimental Procedures.” B, Myc or Myc-Rheb was transfected in HEK293 cells. At 36 h (0-h chase), the cycloheximide (100 μm) was added into the culture medium, and the cells were chased for 3, 6, and 9 h. The rate of half-life of BACE1 was significantly reduced in the Myc-Rheb cells when compared with vector alone, indicating that Rheb degrades BACE1 (*, p < 0.05 versus Myc alone, analysis of variance). PERK was not altered by Myc-Rheb. n.s., not significant. C, Myc or Myc Rheb cDNAs (WT, D60K, C181S, or Q64L, 2 μg each) were transfected into HEK293 cells, and after 48 h, they were lysed to detect BACE1 and other proteins, as well as mTOR signaling (by phosphorylation of S6K (ps6K) at Thr-389), as indicated under “Experimental Procedures.” Rheb D60K was unable to induce BACE1 degradation, whereas Rheb C181S was as effective as Rheb WT (***, p < 0.001 versus Myc alone, Student's t test). D, Myc or Myc-Rheb cDNA (2 μg each) was transfected in HEK293 cells after 36 h, and DMSO (0.1%) or rapamycin (Rapa, 100 nm) was added for 12 h. Rapamycin blocked mTOR activation (as measured by phosphorylation of S6K at Thr389), but did not appreciably affect the Rheb-mediated degradation of BACE1 (***, p < 0.001 versus DMSO, Student's t test).

FIGURE 4.

Rheb binds with BACE1. A, BACE1^+/+ and BACE1^−/− mice brain lysates were treated with BACE1 IgG or control IgG (Con IgG), precipitated with Sepharose beads, and incubated with recombinant Rheb. Rheb co-precipitated with the BACE1 IgG sample, which effectively pulled down BACE1, indicating Rheb and BACE1 interactions. B, HEK293 cells were transfected with HA-Rheb or Myc-BACE1 for 36 h and fixed for the microscopy work, as indicated under “Experimental Procedures.” Note that Rheb and BACE1 readily co-localized in cells with perinuclear localization. C, GST or GST-Rheb (WT, D60K, or C181S) was transfected in HEK293 cells, and after 48 h, lysed and incubated with glutathione beads to detect the interaction of Rheb with endogenous BACE1. BACE1 interacted with Rheb WT or Rheb C181S, but not with Rheb D60K.

FIGURE 5.

Rheb degrades BACE1 through the lysosomal and proteasomal degradation pathway, and Rheb levels are reduced in AD postmortem tissue. A and B, HEK293 cells were transfected with Myc or Myc-Rheb cDNA (2 μg each) for 36 h and treated with DMSO or with different doses of lysosomal inhibitor (10–100 μm), chloroquine (A), or the proteasomal inhibitor (1–25 μm), MG132 (B), for 9 h, and then lysed to detect BACE1 or PDGFR, as described under “Experimental Procedures.” C, chloroquine (Chloro) and MG132 both blocked Rheb-mediated degradation of BACE1 (***, p < 0.001 versus DMSO-treated Myc-Rheb, analysis of variance).

FIGURE 6.

Rheb levels are decreased in AD prefrontal cortex. A, representative Western blot of BACE1 and Rheb in AD versus control samples. B, quantitative analysis of the Western blots showed that Rheb levels are significantly reduced, whereas BACE1 levels significantly increased in AD groups relative to control group (n = 10/group, ***, p < 0.001, *, p < 0.05 versus control, Student's t test). C, Rheb reduction is correlated to BACE1 increase in AD and not in control group.