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Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

Cell Research volume 17, pages 389–401 (2007)Cite this article

Abstract

Amyloid-β (Aβ) peptide, the primary constituent of senile plaques in Alzheimer's disease (AD), is generated by β-secretase- and γ-secretase-mediated sequential proteolysis of the amyloid precursor protein (APP). The aspartic protease, β -site APP cleavage enzyme (BACE), has been identified as the main β-secretase in brain but the regulation of its activity is largely unclear. Here, we demonstrate that both BACE activity and subsequent Aβ production are enhanced after stimulation of receptor tyrosine kinases (RTKs), such as the receptors for epidermal growth factor (EGF) and nerve growth factor (NGF), in cultured cells as well as in mouse hippocampus. Furthermore, stimulation of RTKs also induces BACE internalization into endosomes and Golgi apparatus. This enhancement of BACE activity and Aβ production upon RTK activation could be specifically inhibited by Src family kinase inhibitors and by depletion of endogenous c-Src with RNAi, and could be mimicked by over-expressed c-Src. Moreover, blockage of BACE internalization by a dominant negative form of Rab5 also abolished the enhancement of BACE activity and Aβ production, indicating the requirement of BACE internalization for the enhanced activity. Taken together, our study presents evidence that BACE activity and Aβ production are under the regulation of RTKs and this is achieved via RTK-stimulated BACE internalization, and suggests that an aberration of such regulation might contribute to pathogenic Aβ production.

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Introduction

Alzheimer's disease (AD), one of the most common neurodegenerative disorders in elderly people, is characterized by the formation of senile plaques in patient brains 1. The primary constituent of the senile plaques is amyloid-β peptides (Aβ) that are generated by sequential proteolysis of APP by β-secretase and γ-secretase 2. Nowadays, β-secretase is considered the initial and rate-limiting enzyme during this process. β-site APP cleavage enzyme (BACE), a type I integral membrane aspartic protease, was identified as the main β-secretase 3, 4, 5, 6, 7. As formation of senile plaques could not be detected in BACE-deficient mice 8, 9, BACE has attracted major attentions in AD research during recent years. For example, small molecule inhibitors of BACE have been widely investigated for their potential use in the treatment of AD 10.

Recent studies revealed that BACE is synthesized as an immature and inactive form in the endoplasmic reticulum 11, and undergoes maturation during its transport to cell surface along the secretory pathway 12, 13, 14, 15 , 16. The mature BACE is then internalized from cell surface to endosomes 3, 12, followed by sorting to the trans golgi network (TGN) for recycling or to lysosomes for degradation 15, 16. Interestingly, endosomal accumulation of Aβ was also found in AD patients at the early disease stage 17. These data suggested that internalization of BACE from cell surface to endosomes might be important for its activity with regard to the generation of Aβ. However, the direct relationship between BACE internalization and its activity as well as how BACE internalization is regulated, especially by extracellular signals, remain to be established.

Receptor tyrosine kinases (RTKs) comprise a large family of cell surface receptors, which transduce various extracellular signals to the interior of cells, mediating distinct cellular functions such as proliferation, differentiation, survival, and protein synthesis 18. Upon activation, RTKs undergo phosporylation at specific tyrosine sites and then bind with SH2 (Src homology region) or PTB (phosph-otyrosine binding) domain-containing proteins to activate downstream effectors such as c-Src, MAPK, and PKC 19. Particularly, c-Src has been demonstrated to regulate the internalization of membrane proteins via different mechanisms 20, 21, 22, 23, 24, implicating a means for RTK-mediated regulation of other membrane proteins. RTK internalization is required for their normal signaling. In response to ligand stimulation, RTK internalizes and recruits a protein complex known as the 'signalsome' which contains a number of intracellular signaling molecules including c-Src 25, 26, 27, 28, 29. Interestingly, PDGF stimulation was shown to result in an increased cleavage of the over-expressed APP 30, suggesting a functional role of the RTK signaling pathway in the amyloidogenic process. Therefore, in this study we investigated whether β- or γ-secretase activity could be regulated by RTKs.

Materials and Methods

Antibodies and materials

The polyclonal antibodies against BACE were purchased from Calbiochem (Cat.195100 and Cat.195111). The monoclonal anti-BACE antibody (Cat. MAB5308) and antibody 6E10 against Aβ were from Chemicon. Antibodies against APP-CTF, HA tag and Golgi 58K protein were from Sigma. Antibodies against early endosome antigen-1 (EEA-1) and lysosomal-associated membrane protein (LAMP-1) were from BD Biosciences. Antibodies against TrkA and p75 were from Upstate, and the antibody against EGFR was from Santa Crutz Biotechnology. The following commercially available reagents were used: EGF, NGF (Sigma); PP2, PP3, Herbimycin A, Uo126, BACE inhibitor and the secretase fluorogenic substrates (Calbiochem); AEBSF (AMESCO); Streptavidin-HRP, protein A-Sepharose beads (Amersham Biosciences); and complete protease inhibitor cocktail (Roche).

Plasmid construction

The RNAi plasmid for human c-Src was constructed as described 31. A 22-mer oligonucleotide (oligo1) corresponding to nucleotide 594-615 of the c-Src coding sequence was first inserted into the pBS/U6 vector (a generous gift from Dr Yang Shi, Dept. Pathology, Harvard Medical School) with XhoI and blunted ApaI sites. The inverted motif containing a six-nucleotide spacer and five Ts (oligo2) was then subcloned into the XhoI and EcoRI sites of the intermediate plasmid to generate pBS/U6/c-Src-RNAi. Oligo1 is 5′-GGC CTC AAC GTG AAG CAC TAC A-3′ (forward) and 5′- AGC TTG TAG TGC TTC ACG TTG AGG CC-3′ (reverse). Oligo2 is 5′-AGC TTG TAG TGC TTC ACG TTG AGG CCC TTT TTG-3′ (forward) and 5′-AAT TCA AAA AGG GCC TCA ACG TGA AGC ACT ACA -3′(reverse). For the c-Src expression plasmid, mouse c-Src cDNA (Genbank, accession number NM_009271) was cloned into HindIII and EcoRI sites of pcDNA3 (Invitrogen, Carlsbad, CA).

Cell culture and transfection

Human embryonic kidney (HEK) 293 cells and rat C6 glioma cells were cultured in Modified Eagle's Medium (MEM) and Dulbecco's modified Eagle's medium (DMEM), respectively, supplemented with 10% fetal bovine serum (Life Technologies, Inc.). For generation of stable cell lines, HEK293 and C6 cells were transfected with 3 μg of pcDNA3 containing Swedish APP695 (swAPP) using LipofectAMINE (Invitrogen) according to the manufacturer's instruction. Stable expression clones were selected and maintained in G418 (300 μg/ml). For transient transfection, HEK293 cells were transfected by the calcium phosphate-DNA co-precipitation method as described previously 31.

Intracerebroventricular (i.c.v.) injection of mouse brains

C57/BJ mice were anaesthetized and subjected to intracerebroventricular injection of NGF (2 ml at 100 ng/μl) or NS (normal saline) with the following coordinates: AP (anterior-posterior): −0.6 mm, LR (left-right): −1.2 mm, DV (dorsal-ventral): –1.8 mm for the indicated time (60 min for Aβ production measurement, 30 min for secretase activity assay; n=10 for each assay). Then the hippocampi were isolated and collected for measurement of secretase activities and Aβ production. All animal treatments were processed in Shanghai Laboratory Animal Center in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Measurement of secreted and intracellular Aβ

HEK293 or C6 cells stably expressing swAPP were stimulated for 1 h with EGF (200 ng/ml) or NGF (100 ng/ml), followed by incubation in conditioned medium for 6h. To measure the amount of secreted Aβ, the conditioned medium was directly quantified for Aβ40 and Aβ42 by sandwich ELISA kits (Biosource). To determine intracellular Aβ levels, the stimulated cells were lysed in RIPA buffer (containing the protease inhibitor cocktail and AEBSF); the cell lysates were pre-cleared with APP-CTF antibody, followed by immunoprecipitation of Aβ with the antibody 6E10. The immunoprecipitates were separated by 15% SDS-PAGE and subjected to western blot using 6E10. Each experiment was repeated at least three times. The optical density (OD) of each band was quantified by using Scion Image software. The amount of secreted Aβ produced in mouse hippocampus was determined by using BNT77/BA27 and BNT77/BC05 sandwich ELISA kits (Wako) according to previous reports 32.

Fluorogenic substrate secretase activity assay

This assay was performed as reported 33. Briefly, cultured cells or mouse hippocampi were stimulated with or without RTK agonists for the indicated time periods and homogenized. The resulting aliquots (containing 15 μg of proteins) were centrifuged at 13 000 × g for 15 min. Then, the membrane pellets were recovered and incubated at 37°C for 30 min in 50 μl of assay reaction buffer (for BACE, 50 mM sodium acetate, pH 4.5; for α-secretase, 10 mM Tris-HCl, pH 7.5; for γ-secretase 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 0.25% CHAPSO) containing 10 μM specific fluorogenic substrates for each secretase. After incubation, fluorescence was measured using a spectrometer at excitation/emission wavelength of 320/420 nm (for BACE), 325/393 nm (for α-secretase), and 355/440 nm (for γ-secretase).

Immunofluorescence microscopy

HEK293 cells grown on coverslips were stimulated with EGF (200 ng/ml) for 30 min and fixed with 4% formaldehyde, followed by double staining with anti-BACE antibody (Calbiochem, Cat. 195100, rabbit) and antibodies against EEA-1, LAMP-1, or Golgi 58K protein (mouse). Then Cy3-conjugated anti-rabbit secondary antibody and Cy5-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) were applied. Images were acquired using a laser confocal fluorescence microscope (Leica TCS SP2). The image of the Cy5-stained sample was converted to green pseudocolor. The positive colocalization rate of BACE-organelle marker was calculated from 100 randomly chosen cells in different observed fields.

Fluorescence activated cell sorting (FACS) for internalization studies

HEK293 cells were stimulated with or without EGF (200 ng/ml) and stained sequentially with anti-BACE and FITC-conjugated anti-rabbit secondary antibody. The background fluorescence was assessed by staining the cells with the secondary antibody alone. The stained cells were then analyzed with FACS Calibur (BD Biosciences).

Cell surface biotinylation for internalization studies

Cell surface biotinylation assay was performed as reported 12, and all procedures were carried on ice unless otherwise stated. Briefly, HEK293 cells were labeled with EZ-Link™ Sulfo-NHS-SS-Biotin (Pierce) for 30 min followed by washing with 100 mM glycine to remove the free biotin. Cells were then moved to 37 °C in absence or presence of EGF (200 ng/ml) for the indicated time periods. After washing with the striping buffer (20 mM Tris-HCl, pH 8.6, 50 mM glutathione, 150 mM NaCl, 1 mM EDTA, 0.2% bovine serum albumin) to remove all the biotin that had remained on the cell surface, cells were lysed in RIPA buffer (containing the protease inhibitor cocktail); and the lysates were immunoprecipitated with anti-BACE antibody (Calbiochem, Cat.195111). The resulting immunoprecipitates were subjected to western blot and detected by Streptavidin-HRP.

Statistical analysis

All measurements were performed in at least three independent experiments and the means ± S.E. were calculated. Data were analyzed by Student's t-test for comparison of independent means, with pooled estimates of common variances. For all tests, P<0.05 was considered to be significant.

Results

Stimulation of RTK increases BACE activity and Aβ production

Because the performance of different anti-BACE antibodies may vary for a specific application, we first examined these antibodies in our preliminary experiments. Our preliminary results showed that the anti-BACE from Calbiochem (Cat.195100) was good for immunofluorescence (Supplement 1, Figure A, left and middle panels), the anti-BACE from Calbiochem (Cat.195111) was good for immunoprecipitation (data not shown), and the anti-BACE from Chemicon (Cat. MAB5308) was suitable for western blot (Supplement 1, Figure A, right panel).

Among RTKs, receptors for EGF and NGF are widely distributed and well characterized. Here, the potential regulation of amyloidgenic secretase activities by RTKs was investigated. Activation of endogenous receptors for EGF or NGF through EGF or NGF stimulation was confirmed by Western blot analysis of ERK phosphorylation (Supplement 1, Figure B) 34, and the activities of three secretases were determined simultaneously. The results showed that only BACE activity was increased by RTK stimulation in a time-dependent manner (Figure 1A, 1C) while BACE expression was not affected (Supplement 1, Figure B); and α- and γ- secretase activities were not significantly affected. This enhancement of BACE activity was completely blocked by antibodies against EGFR 35 or NGFR 36 respectively (Figure 1A, 1C). These data indicated that the observed enhancement in BACE activity was dependent on the activation of the corresponding RTK. To verify that RTK stimulation specifically enhances the activity of BACE, corresponding inhibitors for the three secretases were applied respectively: EDTA (2.5 mM) as the inhibitor of α-secretase, DFK167 (100 mM) as the inhibitor for γ-secretase, as well as the BACE inhibitor (125 mM). And the results showed that only the BACE inhibitor could suppress RTK-enhanced BACE activity in HEK293 and C6 cells (Figure 1B and 1D).

Figure 1
figure 1

Activation of RTKs specifically enhances BACE activity. Secretase activities were measured by the fluorogenic substrate assay as described. (A), HEK293 cells were stimulated with EGF (200 ng/ml) for indicated time. In the last panel, cells were pretreated with anti-EGFR (1 μg/ml) for 15 min. HEK293 cells (B) or rat C6 glioma cells (D) were stimulated with EGF (200 ng/ml) or NGF (100 ng/ml) for 30 min, in presence or absence of BACE inhibitor (125 μM), EDTA (2.5 mM) or DFK-167 (100 μM) respectively. (C), C6 cells were stimulated with NGF (100 ng/ml) for indicated time. In the last three panels, cells were pretreated with anti-NGFR (anti-TrkA and/or anti-P75, 1 μg/ml) for 15 min. (E) the hippocampi of C57BL/6J mice were isolated and homogenized, following the i.c.v. injection of NGF (200 ng) for 30 min, n=10; and the proteins were collected for detection of secretase activities in the presence or absence of BACE inhibitor (125 μM), EDTA (2.5 mM) or DFK-167 (100 μM). Data are means ± S.E. of three independent experiments. *, P<0.05 versus untreated cells.

Full size image

Because endogenous Aβ production is too little to be detected in common cell lines 37, HEK293 or C6 cells stably expressing the Swedish APP mutant 695 (denoted as swHEK293 or swC6) were used to examine whether the enhanced BACE activity led to an increase in Aβ production. Following stimulation with the RTK agonist, the secreted Aβ40 and Aβ42 levels were enhanced to 1.967 ± 0.023 and 2.040 ± 0.140 fold in swHEK293 cells (Figure 2A), and to 2.082 ± 0.219 and 2.145 ± 0.306 fold in swC6 cells (Figure 2B). The intracellular Aβ level was also significantly increased (Figure 2C–2F).

Figure 2
figure 2

Activation of RTKs promotes Aβ production. HEK293 and C6 cells stably expressing the Swedish APP mutant 695 (APPswe), abbreviated as swHEK293 or swC6 cells, were stimulated with EGF (200 ng/ml) or NGF (100 ng/ml) for 1 h. The Aβ secreted from swHEK293 (A) and swC6 (B) cells were measured by ELISA. The intracellular Aβ in swHEK293 cells (C) or swC6 cells (D) was detected by immunoprecipitation and western blot. (E) and (F), quantitative representations of results in (C) and (D) respectively, which were from at least three independent experiments. (G), C57BL/6J mice hippocampi were isolated and homogenized after the i.c.v. injection of NGF (200 ng) for 1 h, n=10. The supernatants were collected by ultracentrifugation at 40 000 g for 1 h, and Aβ was measured by ELISA. The values are presented as means ± S.E. from three independent experiments. *, P<0.05 versus untreated cells.

Full size image

The effects of RTK activation on BACE activity as well as on Aβ production were also determined in vivo. Consistent with the observed data from the cultured cells, though being somewhat less potent, NGF stimulation increased both the activity of BACE (Figure 1E) and the resulting production of Aβ40 and Aβ42 (Figure 2E) in mouse hippocampus. The reason underlying the less potent effect shown by the in vivo study might be due to the limited accessibility of NGF to mouse tissues. In conclusion, our results show that RTK activation enhances BACE activity and Aβ production in vitro and in vivo.

RTK-enhanced BACE activity and Aβ production are mediated by c-Src

RTK is reported to transduce signals through various downstream effectors such as MAPK, PKC, PKA, PI3K and Src tyrosine kinases 38, 39, 40. Therefore, we sought to determine which signal pathway is involved in mediating the RTK-enhanced BACE activity and Aβ production. Our results showed that inhibitors of the Src family such as PP2 and Herbimycin A could block the enhancement of BACE activity induced by RTK activation; in contrast, PP3 (a nonfunctional analogue of PP2) and Uo126 (inhibitor of MEK) failed to do so (Figure 3A, 3B). The inhibitors of PKA (H-89), P38 MAPK (SB203580), PI3K (LY294002) and MAPK (PD98059) were found to have no blocking effect on RTK-mediated BACE activation (Supplement 1, Figure C). Further, specific knockdown of the endogenous c-Src, a member of the Src family, by RNAi (Figure 3C), also blocked RTK-mediated enhancement of BACE activity, while the control NS-RNAi had no effect (Figure 3D). Accordingly, inhibition of c-Src activation by its inhibitor (Figure 3E) or by RNAi (Figure 3F) could also block RTK-stimulated Aβ production. Finally, over-expression of c-Src (GSK3β as the control) was found to mimic the effects of RTK activation on both the BACE activity and Aβ production (Figure 4A and 4B) without affecting BACE expression (Figure 4C). Taken together, these results show that c-Src mediates the enhanced BACE activity and Aβ production induced by RTK activation.

Figure 3
figure 3

c-Src is required for enhanced BACE activity and Aβ production induced by RTK activation. (A, B, D), the indicated secretase activities were measured by the fluorogenic substrate assay as described in Materials and Methods. HEK293 (A) or C6 glioma cells (B) were pretreated with PP2 (0.4 μM, 4 μM and 20 μM), Herbimycin A (2 μM), PP3 (10 μM), or Uo126 (20 μM), followed by stimulation with EGF (200 ng/ml) (A) or NGF (100 ng/ml) (B) for indicated time. HEK293 cells were transiently transfected with vectors for NS-RNAi or Src-RNAi (D), and 72 h later the cells were exposed to EGF (200 ng/ml) for 30 min. (E, F), the secreted Aβ was detected by ELISA. (E), swHEK293 cells were stimulated with EGF (200 ng/ml) for 1 h following the pretreatment with PP2 (4 μM), PP3 (10 μM) and Herbimycin A (2 μM). (F), swHEK293 cells were transiently transfected with vectors for NS-RNAi or Src-RNAi, and 72 h later the cells were stimulated with EGF (200 ng/ml) for 1 h. (C), endogenous expression of c-Src in cells transfected with vectors for NS-RNAi or Src-RNAi. NS: none specific, ** indicated the non-specific band. Data are means ± S.E. of three independent experiments. *, P<0.05 versus the absence of inhibitors (A, B, and E) or versus NS-RNAi (D and F).

Full size image
Figure 4
figure 4

c-Src over-expression led to enhanced BACE activity and Aβ production, mimicking that induced by RTK activation. HEK293 cells were transiently transfected with vectors expressing c-Src or GSK3β (as a negative control). The indicated secretase activities (A) and secreted Aβ (B) were measured as described. (C), Cell lysates were collected and the expression of BACE (examined by the antibody from Chemicon, Cat. MAB5308) and Actin was detected by Western blot. Data are means ± S.E. of three independent experiments. *, P<0.05 versus mock transfected cells.

Full size image

RTK activation enhances BACE internalization in a c-Src dependent manner

Because BACE is known to be internalized, and sorted to endosomes and the Golgi apparatus to exert its cleavage activity 3, 12, we thus studied whether RTK activation affects the intracellular distribution of BACE. As shown in Figure 5, BACE was localized in endosomes (EEA-1 as the marker) and Golgi apparatus (Golgi 58K protein as the marker), with little in lysosomes (LAMP-1 as the marker), consistent with other reports 3, 15. Following EGF stimulation, the localization of BACE in endosomes (Figure 5A) was significantly increased, and its localization in the Golgi apparatus was also slightly increased (Figure 5B), suggesting that RTK activation could enhance BACE internalization. This conclusion was also supported by the results shown in Figure 6. In response to EGF, the amount of BACE on cell surface decreased significantly as detected by either FACS analysis (Figure 6A and 6B) or by the cell surface biotinylation assay (Figure 6C–6E). Furthermore, BACE internalization was mediated by c-Src, as evidenced by the observation that it could be blocked by PP2 (Figure 5A, A7–A9, Figure 5B, B7–B9) and mimicked by c-Src over-expression (Figure 7B and 7G).

Figure 5
figure 5

Activation of RTKs increases BACE localization in the endosome and Golgi apparatus. HEK293 cells were stimulated with EGF (200 ng/ml) for 30 min in the presence or absence of PP2 (4 μM) pretreatment (5 min), fixed and double-stained with antibodies against BACE (Calbiochem, Cat. 195100) and molecular markers (green) for endosome (EEA-1 as the marker), Golgi apparatus (Golgi 58K protein as the marker), or lysosome (LAMP-1 as the marker). (A1-A9), BACE and EEA-1; (A1-A3), untreated, scale bar=14 μm; (A4-A6), 30 min EGF stimulation, scale bar=14 μm; (A7-A9), 30 min EGF stimulation following PP2 pretreatment, scale bar=21 μm. (A10), quantitative results of BACE/EEA-1 co-localization in 100 cells. (B1-B9), BACE and Golgi 58K protein; (B1-B3), untreated, scale bar=21 μm; (B4-B6), 30 min EGF stimulation, scale bar=21 μm; (B7-B9), 30 min EGF stimulation following PP2 pretreatment, scale bar=21 μm. (B10), quantitative results of BACE/Golgi co-localization in 100 cells. (C1-C9), BACE and LAMP-1; (C1-C3), untreated, scale bar=21 μm; (C4-C6), 30 min EGF stimulation, scale bar=21 μm; (C7-C9), 30 min EGF stimulation following PP2 pretreatment, scale bar=12 μm. (C10), quantitative results of BACE/lysosome co-localization in 100 cells. All the experiments were performed at least three times. *, P<0.05 versus EGF stimulated cells.

Full size image
Figure 6
figure 6

Activation of RTK decreases BACE localization on cell surface. (A, B), HEK293 cells were stimulated with (EGF 30 min) or without (untreated, 0 min) EGF, stained sequentially with anti-BACE (Calbiochem, Cat.195100) and FITC-conjugated secondary antibody, followed by detection with flow cytometry. Cells stained with the secondary antibody alone were used to assess the fluorescence background (control). The quantitative results from three independent experiments are shown in (B). *, P<0.05 versus the untreated cells. (C, D, E), HEK293 cells were labeled with 0.5 mg/ml EZ-Link™ Sulfo-NHS-SS-Biotin (Pierce) at 4 °C for 30 min. The reaction was stopped by treating with 100 mM glycine for 15 min, and cells were then incubated at 37 °C for the indicated time in MEM medium with (D, EGF) or without (C, warm) EGF (200 ng/ml). The internalized BACE was detected as described in “Materials and Methods”. (E), the statistical plot was generated from three independent experiments performed in (C) and (D). Each dot represents the relative optical density of internalized BACE at different time points compared with the first lane (total BACE expression on the cell surface at time 0).

Full size image
Figure 7
figure 7

BACE internalization enhances BACE activity and Aβ production. HEK293 cells transfected with the indicated constructs were fixed and double-stained with antibodies against BACE (Calbiochem, Cat.195100) and molecular markers (green) for endosome (EEA-1) or Golgi apparatus (Golgi 58K protein). The co-localization of BACE/EEA-1 (A-D) or BACE/Golgi 58K protein (F-I) was detected. (A'-D') and (F'-I') show the success of different transfections, scale bar=30 μm. The positive co-localization rate of BACE/EEA-1 (E) and BACE/Golgi 58K protein (J) in 100 cells is shown; and the experiments were repeated independently three times. (K), HEK293 cells were transfected as above, with GSK3β as a negative control. Forty-eight hours later, membranes were subjected to the analysis of BACE and β-secretase activities. (L). swHEK293 cells were co-transfected as in (K), 48 hours later, the supernatants were collected for analysis of secreted Aβ. Data are means ± S.E. of at least three independent experiments. *, P<0.05 versus cells cotransfected with c-Src and GFP in (E, J, K) and (L).

Full size image

BACE internalization enhances its activity and Aβ production

Since c-Src could enhance both the internalization and the activity of BACE, the relationships between BACE internalization and BACE activity as well as Aβ production were further investigated. For this purpose, we co-expressed c-Src with GFP-Rab5S34N, a dominant negative form of Rab5 capable of disrupting protein internalization to endosomes 41, to block BACE internalization. As expected, c-Src-enhanced BACE internalization was completely blocked by GFP-Rab5S34N, but not by GFP-Rab7T22N (the dominant negative mutant for lysosome formation) 42 (Figure 7C–7E and 7H-7J). Accordingly, the presence of GFP-Rab5S34N, but not GFP-Rab7T22N, also blocked the enhanced BACE activity and Aβ production normally induced by c-Src over-expression (Figure 7K and 7L). In conclusion, BACE internalization was required for enhanced BACE activity and subsequent Aβ production upon c-Src activation.

Discussion

As the only β-secretase known to generate Aβ in brain, BACE has proven to be a key molecule in the formation of senile plaques 8, 9, 43, 44. Up to now, studies of BACE have concentrated on its structure 4, 45, trafficking 11, 16, 46, 47, 48, 49, 50, and specific inhibition 10, but how its activity is regulated remains largely unclear. In this study, we demonstrated that BACE activity both in vitro and in vivo could be efficiently regulated by RTKs, and the regulation of BACE activity led to alterations of subsequent Aβ production. RTKs, a large family of cell surface receptors mediating various extracellular signals, have been reported to regulate APP expression and secretion 51, 52 and to be aberrantly activated at the early disease stages in AD patients 53, 54, 55. In light of the critical roles of BACE activity in Aβ production, our results suggest that RTKs may contribute to the development of AD.

Upon stimulation, RTKs function by activating downstream signaling molecules such as c-Src, MAPK, and PKC 38, 39, 40 or by inducing the internalization of other membrane proteins such as E-cadherin 56. This study demonstrated that stimulation of RTKs induced BACE internalization to enhance BACE activity and subsequent Aβ production, which were independent of MAPK and PKC activation (data not shown). RTK-induced BACE internalization results in accumulation of BACE in endosomes where the acidic environment could provide optimum pH BACE activity. In addition, it has been reported that APP and BACE are co-internalized into endosomes, leading to enhanced APP cleavage there 57, 58, 59, 60. Thus, the abnormally enlarged endosomes observed in the early stages of AD 17 might be due to abnormal internalization of BACE and APP, leading to enhanced Aβ production. It is tempting to speculate that regulation of BACE and APP internalization by RTK activation is critical for Aβ production, and that an aberration of such regulation might contribute to the senile plaque formation.

c-Src is an integral component of the signal transduction apparatus employed by RTKs 61 and plays pivotal roles in the internalization of various membrane proteins such as β2AR, ROMK-1, AT1R, and EGFR. Consistent with these reports, we demonstrated that RTK-induced BACE internalization and subsequent Aβ production also depended on the activity of c-Src. In addition, over-expression of c-Src itself could mimic the effects of RTK activation, indicating that c-Src is not only necessary but also sufficient for BACE internalization. It is known that c-Src mediates internalization of membrane proteins by phosphorylating the internalized protein or components of the internalization machinery 20, 21, 22, 24. Our results suggest that in this case c-Src may phosphorylate components of the internalization machinery because no tyrosine residue was found in the cytosolic tail of BACE 16, 21, 23, 24, 62, 63. Although the precise mechanism of enhanced BACE internalization mediated by c-Src still remains unclear, the results from this study indicate that c-Src may serve as a potential therapeutic target for AD.

AD pathogenesis was recently found to be modulated by environmental factors such as education, mental/leisure activity, depression, and stress, some of which affect signaling events in the brain 64, 65, 66. Remarkably, we found in our recent study that β2-adrenoceptor signaling, which mediates the stress response in animals, regulates γ-secretase activity and senile plaque formation in vivo 67. Our present study shows that extracellular signals, at least those mediated by RTKs, could increase Aβ production by enhancing BACE activity. The physiological significance of this RTK activation-enhanced BACE activity is supported by the results of the mouse hippocampus study, which showed that this enhancement of BACE activity led to increased Aβ production in the major brain region affected by Aβ accumulation. Hence, our study opens a new window for understanding the regulation of BACE activity and Aβ production in AD in response to environmental factors.

(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Accession codes

Accessions

GenBank/EMBL/DDBJ

Abbreviations

AD:

(Alzheimer's disease)

Aβ:

(Amyloid-β peptide)

APP:

(amyloid precursor protein)

BACE:

(β-site APP cleavage enzyme)

RTKs:

(receptor tyrosine kinases)

EGF:

(epidermal growth factor)

NGF:

(nerve growth factor)

EEA-1:

(early endosome antigen-1)

LAMP-1:

(lysosomal-associated membrane protein)

HEK 293:

(human embryonic kidney 293)

PP2:

(4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine)

PP3:

(4-Amino-7-phenylpyrazol [3,4-d] pyrimidine)

MAPK:

(MAP Kinase)

PI3K:

(Phosphatidyl 3-Kinase)

AEBSF:

(4-(2-Aminoethyl) benzenesulfonylfluoride)

ELISA:

(Enzyme-linked Immunosorbent Assay)

NS-RNAi:

(non-specific RNA interference)

GSK3β:

(glycogen synthase kinase 3β)

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Acknowledgements

We thank Dr Z Chen for construction of pcDNA3-c-Src and pBS-U6-Src RNAi plasmids, S Xiang, W Zhang and Angie L Bookout for manuscript revisions, and S Xin and Y Wu for technical assistance. This research was supported by grants from the Ministry of Science and Technology (2003CB515405, 2005CB522406), the National Natural Science Foundation of China (30021003, 30400230, 30625014), the Chinese Academy of Sciences (KSCX1-SW, KSCX2-SW), the Ministry of Education, Shanghai Municipal Commission for Science and Technology (06ZR14098), China Post Doctoral Science Foundation, and Shanghai Postdoctoral Science Foundation.

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  1. Lin Zou and Zhu Wang: These authors contributed equally to this work

Affiliations

  1. Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Lin Zou, Zhu Wang, Li Shen, Guo Bin Bao, Tian Wang, Jiu Hong Kang & Gang Pei

  2. Clinical Molecular Medical Center, Children's Hospital, Chongqing University of Medical Sicences, Chongqing, 400011, China

    Lin Zou

  3. Graduate School of the Chinese Academy of Sciences, Shanghai, 200031, China

    Li Shen

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  1. Lin Zou
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  2. Zhu Wang
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  5. Tian Wang
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Correspondence to Gang Pei.

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Zou, L., Wang, Z., Shen, L. et al. Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization. Cell Res 17, 389–401 (2007). https://doi.org/10.1038/cr.2007.5

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  • DOI: https://doi.org/10.1038/cr.2007.5

Keywords

  • β-site APP cleavage enzyme
  • RTK
  • Amyloid-β
  • Src

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