Correspondence to Virginia M.-Y. Lee: vmylee{at}mail.med.upenn.edu
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Introduction |
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An aspartyl protease, BACE, is the major ß-secretase (Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). BACE cleaves either at Asp1 or Glu11 (numbering relative to the first amino acid in Aß) to release NH2-terminal ectodomains referred to as sAPPß and sAPPß', respectively (Fig. 2 A). The remaining membrane-bound COOH-terminal APP fragments are then cleaved by -secretase to produce full-length Aß1-40/42 or the NH2-terminally truncated Aß11-40/42. BACE overexpression in neuronal (E.B. Lee et al., 2003) and nonneuronal cells (Vassar et al., 1999; Huse et al., 2002; Liu et al., 2002) increases Aß generation, whereas genetic ablation of BACE eliminates Aß production (Cai et al., 2001; Luo et al., 2001).
Less is known about the significance of the subcellular localization of APP processing with respect to amyloid plaque formation. In addition to the ER and the Golgi apparatus, APP is enriched in axons and presynaptic terminals (Schubert et al., 1991; Ferreira et al., 1993). Although Aß is generated in several different organelles in vitro (Wilson et al., 1999), the subcellular site of Aß generation in vivo is more difficult to assess. However, studies indicate that APP undergoes kinesin Idependent vesicular fast axonal transport (Koo et al., 1990; Ferreira et al., 1993) and that APP proteolysis may occur within axonal or presynaptic vesicles (Buxbaum et al., 1998). Furthermore, synaptic activity increases Aß secretion, indicating that the presynaptic terminal is an important regulatory site for Aß generation (Kamenetz et al., 2003). Finally, ablation of the perforant pathway in APP transgenic (Tg) mice decreases amyloid plaque deposition in the hippocampus, suggesting that synaptic Aß contributes to plaque formation (Lazarov et al., 2002; Sheng et al., 2002).
A priori, increased BACE activity is expected to increase Aß pathology. Indeed, modest BACE overexpression in mice increases steady-state Aß levels (Bodendorf et al., 2002). However, we discovered that high BACE overexpression paradoxically decreased Aß deposition despite enhanced ß-cleavage of APP. Furthermore, we found that BACE overexpression altered the subcellular localization of BACE cleavage by increasing ß-cleavage early in the secretory pathway, thereby depleting APP destined for axonal transport. These unexpected findings underscore the importance of the subcellular site of Aß generation in the pathogenesis of AD.
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Results |
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Increased BACE activity also altered steady-state levels of COOH-terminal APP fragments (Fig. 2 C). In monogenic BACE mice, BACE-derived COOH-terminal APP fragments, predominantly C89, were increased relative to wild-type mice, which is consistent with the preferential cleavage of rodent APP at position 11 within Aß (Wang et al., 1996; Gouras et al., 1998; Cai et al., 2001). In APP overexpressing mice, five distinct bands corresponding to APP COOH-terminal fragments were apparent (Fig. 2 C). Several of these bands correspond to phosphorylated COOH-terminal APP fragments as dephosphorylation with alkaline phosphatase collapsed the five bands into three bands corresponding to C99, C89, and C83 (Fig. 2 C, left vs. right). As expected, BACE overexpression increased steady-state levels of C99 and C89, whereas -secretasederived C83 was decreased, which is consistent with a subcellular competition between
- and ß-cleavage (Skovronsky et al., 2000). Interestingly, despite an overall increase in C99, BACE overexpression decreased the amount of phospho-C99 by
5060% in APPxBACE-L and APPxBACE-M mice and over 80% in APPxBACE-H mice.
Decreased steady-state Aß due to BACE overexpression
We hypothesized that the increase in C99 would be paralleled by an increase in Aß. Surprisingly, ELISA quantification of steady-state Aß from young mice (57 mo) indicated that high BACE expression paradoxically decreased full-length Aß production (Fig. 3 A). Although very high BACE overexpression in cell culture models results in cleavage at positions 11 and 34 within Aß, immunoprecipitation of brain lysates with 4G8, albeit less quantitative than ELISA methods, failed to detect any peptides other than full-length Aß and p3 (Fig. 3 B). Thus, truncated Aß peptides such as Aß1-34 and Aß11-40/42 are produced at very low levels relative to full-length Aß, or are rapidly degraded in vivo. To confirm the lack of NH2-terminally truncated Aß peptides, additional ELISA's were performed to detect either full-length Aß1-40/42 or total Aßx-40/42 (Fig. 3, C and D). Equivalent amounts of Aß1-40/42 and Aßx-40/42 were detected suggesting that NH2-terminally truncated Aß species were present at very low levels. These results were corroborated by the near absence of truncated Aß in amyloid deposits of older Tg mice (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1).
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To demonstrate that phospho-APP is selectively transported by fast anterograde axonal transport and that this pool of APP is diminished upon BACE overexpression, we radiolabeled retinal ganglion cells by intravitreal injections of [32P]PO4 (Fig. 6 C) followed by immunoprecipitation of optic nerve lysates for full-length APP. Radiolabeled phospho-APP was present within optic nerves of APP Tg mice, whereas no radiolabeled APP was detected in optic nerves of APPxBACE-H mice (Fig. 6 D). Immunoblotting of the same nitrocellulose membrane showed that only fully glycosylated APP is phosphorylated, and that BACE overexpression selectively reduced mature APP within the optic nerve. Furthermore, the reduction in phospho-APP was specific because 32P-labeled middle molecular weight neurofilament subunit (NFM) levels were not changed by BACE overexpression.
Decreased APP transport in APPxBACE mice
To further demonstrate that BACE overexpression resulted in a shift in the subcellular site of ß-cleavage, axonal transport within sciatic nerves of APP or APPxBACE-H mice was interrupted by ligation for 6 h (Fig. 7 A), after which segments of the sciatic nerve proximal and distal to the ligature were assessed by immunoblotting for full-length APP, phospho-APP, and sAPPßswe. In monogenic APP mice, mature fully glycosylated APP isoforms proximal to the ligature were 50% higher than in unligated sciatic nerves, whereas mature APP distal to the ligature was reduced by
60%, confirming that mature APP is subject to fast axonal transport (Fig. 7 B). Immature APP levels were unchanged after nerve ligation, suggesting that this signal is not derived from axonally transported APP, which is consistent with reports showing that the PrP promoter drives expression within Schwann cells of the sciatic nerve (Follet et al., 2002). In APPxBACE-H mice, mature APP levels at steady-state in unligated sciatic nerves were over fourfold lower relative to monogenic APP mice. Furthermore, sciatic nerve ligation in APPxBACE-H mice resulted in only a slight accumulation of mature APP. Similar results were obtained when phospho-APP was immunoprecipitated by the PhAT antibody. Thus, BACE overexpression selectively decreases the anterograde axonal transport of mature phosphorylated isoforms of APP. Finally, to assess the amount of ß-cleavage within the axonal compartment, we measured sAPPßswe levels proximal to the ligature in sciatic nerves of APP mice. The decrease in sAPPßswe produced within the proximal segment of the sciatic nerve in APPxBACE-H mice relative to APP mice (Fig. 7 B), coupled with the increase in sAPPßswe in total cortical lysates upon BACE overexpression (Fig. 2 A) indicates that APP cleavage had shifted from an axonal/synaptic compartment to the cell body.
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APP turnover and transport in Tg mice
To directly measure APP turnover in neuronal perikarya of BACE mice, pulse-labeling studies were performed in spinal cords and sciatic nerves. L5 spinal cord segments were injected with [35S]-methionine to label newly synthesized APP within cell bodies of the spinal cord. After 30 min, no differences were found in radiolabeled full-length APP in spinal cords of non-Tg and BACE Tg mice indicating that APP synthesis is unaltered by BACE overexpression (Fig. 8, A and B). However, by 8 h, APP levels were significantly reduced in APPxBACE mice indicating that increased BACE activity hastens APP turnover. Significantly, N- and O-glycosylated APP was conspicuously reduced in BACE mice when compared with non-Tg mice at this time point. Furthermore, virtually no radiolabeled APP was recovered from sciatic nerves of BACE Tg mice, indicating once again that the rapid turnover of APP within neuronal cell bodies of the spinal cord results in a diminution in axonal transport of APP (Fig. 8, A and C).
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Discussion |
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To further explore the relationships between the quantitative, qualitative and spatial factors that influence Aß production and deposition, we produced Tg mice that expressed increased levels of BACE. Surprisingly, an inverse relationship between BACE expression and Aß production/deposition was found. Our efforts to understand this paradoxical result led us to discover that BACE overexpression shifted the sites of APP processing such that APP proteolysis occurred predominantly in neuronal perikarya rather than in axons and axon terminals (Fig. 9). This alteration of APP processing upon BACE overexpression, together with the reduction of Aß accumulation, indicates that Aß generated proximally in neuronal perikarya has a different fate than Aß that is generated at or near the synapse.
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Despite increased ß-cleavage, as evidenced by increased sAPPß, sAPPß', sAPPßswe, and C99 levels in APPxBACE mice, Aß levels were reduced by high BACE expression. This finding is contrary to cell culture models in which BACE overexpression leads to increased secretion of Aß (Liu et al., 2002; E.B. Lee et al., 2003). One potential explanation of our results is that the fate of Aß produced in neuronal perikarya and axonal terminals in the in vivo brain are different. For example, the microenvironment wherein Aß is secreted may influence Aß deposition. Synaptic zinc has been shown to play a role in Aß aggregation and deposition, and depletion of synaptic zinc inhibits amyloid formation in vivo (Lee et al., 2002) suggesting that the environment within synaptic vesicles or the microenvironment at synaptic terminals is crucial to Aß amyloidogenesis.
Alternatively, the differential fates of Aß may be related to the localization of Aß degrading activities in brain. Neprilysin is found predominantly in synapses and axons of smaller interneurons (Fukami et al., 2002). The localization and enzymatic properties of neprilysin are consistent with Aß degrading activity within secretory vesicles and the plasma membrane (Iwata et al., 2000). In contrast, endothelin-converting enzyme is likely to degrade intracellular Aß within acidic organelles such as the TGN (Schweizer et al., 1997; Eckman et al., 2001). Finally, cell surface and secreted forms of insulin-degrading enzyme (IDE) have been implicated in Aß catabolism (Qiu et al., 1998; Vekrellis et al., 2000). Genetic ablation of IDE results in accumulation of unphosphorylated APP fragments without altering phosphorylated fragments, suggesting that IDE activity is localized predominantly near the cell soma (Farris et al., 2003). All three of these enzymes have been shown to influence steady-state levels of Aß in vivo, and may serve complimentary roles in Aß catabolism (Iwata et al., 2001; Eckman et al., 2003; Farris et al., 2003; Miller et al., 2003). Thus, altering the subcellular localization of ß-cleavage may disrupt the normal catabolic pathways of Aß, thereby accounting for the different fates of somatic and synaptic Aß.
In support of the view that the ability of certain Aß peptides to deposit into amyloid plaques is related to their susceptibility to degradation, Aß peptides of different lengths were differentially affected by BACE overexpression. The rate-limiting step of Aß degradation in vivo is the production of Aß10-37 (Iwata et al., 2000), suggesting that NH2-terminal truncations may render Aß peptides more prone to degradation. We found that minimal amounts of the NH2-terminally truncated Aß11-40/42 peptide could be detected upon BACE overexpression despite the increase in C89 levels, supporting the hypothesis that this NH2-terminal truncated variant is easily degraded in vivo.
In APPxBACE bigenic mice overexpressing the lowest levels of BACE, we observed an increase in Aß deposition in neocortex, supporting the idea that slight elevations in BACE expression and activity may facilitate the development of AD (Fukumoto et al., 2002; Holsinger et al., 2002). Furthermore, lower levels of BACE overexpression than those reported here increase steady-state Aß levels in Tg mice (Bodendorf et al., 2002). Nonetheless, the effects of BACE overexpression were not specific to APP harboring the Swedish mutation as BACE overexpression resulted in similar reductions of endogenous and exogenous mature APP in non-Tg mice, APPI Tg mice, and human NTera2 cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1).
Regardless of the expression level, Aß deposition was inhibited in the hippocampus. Even at 20 mo old when APPxBACE-M mice accumulate a large amount of brain Aß deposits, hippocampal Aß deposits are markedly reduced (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1). We postulate that the region-specific effects of BACE overexpression on Aß pathology are related to qualitative or quantitative differences in metabolic pathways for APP intrinsic to specific subsets of neurons. For example, mild increases in BACE activity may increase synaptic Aß in smaller cortical interneurons with relatively short axonal processes. However, due to the length of both the perforant pathway and the mossy fiber pathway, slight elevations in perikaryal BACE activity may preclude synaptic processing of APP in the hippocampus.
Similar region-specific amyloid plaque formation has been observed in the brain of AD patients. For example, whereas association cortices and the limbic system are prone to Aß amyloid, other regions such as primary sensory/motor neocortices, striatum, brainstem, and spinal cord are relatively unaffected (Braak and Braak, 1991) despite the widespread expression of APP (Tanzi et al., 1987). Interestingly, we found that layer IV neurons within regions of the mouse somatosensory cortex were spared from Aß pathology in both APP and APPxBACE-M mice (Fig. S3). These layer IV neurons receive a large proportion of their synaptic input from spatially distant thalamic neurons. Although speculative at this point, our results suggest that distinct subsets of neurons and/or the length and number of their efferent inputs may be significant factors that in part determine the regional differences in amyloid pathology found in AD.
In conclusion, although the reduction of Aß deposition upon BACE overexpression was unexpected, our finding that synaptic Aß is crucial to the development of amyloid plaques offers several new avenues of research that may improve our understanding of the pathogenesis of amyloid plaques. Thus, further progress toward understanding APP transport, Aß aggregation within axonal or synaptic vesicles, and the distribution of Aß degrading enzymes may yield insights which may prove to be clinically relevant.
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Materials and methods |
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Immunoprecipitation and Western blot analysis
Proteins were extracted by homogenization of tissue in RIPA buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 5 mM EDTA in TBS, pH 8.0) containing protease inhibitors (1 µg/ml of pepstatin A, leupeptin, L-1-tosylamido-2-phenylethyl chloromethyl ketone, 1-chloro-3-tosylamido-7-amino-2-heptanone, soybean trypsin inhibitor, and 0.5 mM PMSF) followed by centrifugation at 100,000 g for 20 min at 4°C. When indicated, immunoprecipitations were performed before electrophoresis on either Tris-glycine or 10/16% step-gradient Tris-tricine gels, followed by immunoblotting with the antibodies listed in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1. For quantification of BACE, serial dilutions of brain lysates were performed to obtain results within the linear range of quantification. For dephosphorylation experiments, COOH-terminal fragments immunoprecipitated with 5685 were treated with E. coli alkaline phosphatase at 37°C before electrophoresis.
Sandwich ELISA analysis
Brain regions were sonicated in RIPA buffer (1 ml/150 mg tissue) containing protease inhibitors and centrifuged at 100,000 g for 20 min at 4°C. The pellet was sonicated in 70% FA (2 µl/mg tissue) followed by a second of centrifugation. Both RIPA and FA lysates were assayed by sandwich ELISA as previously described (E.B. Lee et al., 2003). Ban50 (antiAß1-10) was used to capture full-length Aß, whereas BC05 and BA27 were used to detect Aß40 and Aß42, respectively. To determine the relative content of NH2-terminally truncated Aß, ELISA plates were coated with either JRF/c40 or JRF/c42 to capture Aß40 and Aß42, respectively. The concentrations of full-length Aß versus total Aß were determined by using JRF/AßN (anti-Aß1-7) or m266 (anti-Aß13-28) as reporting antibodies.
Histology and immunohistochemistry
Mouse brains were fixed in either ethanol or 4% neutral-buffered formalin for 24 h. Samples were dehydrated through a series of graded ethanol solutions to xylene and infiltrated with paraffin as described previously (Trojanowski et al., 1989). Tissue sections (6 µm) were stained using standard avidin-biotin-peroxidase methods using 33' DAB. BaceN1 (rabbit antiNH2 terminus of BACE; Huse et al., 2000), NAB228 (mouse anti-Aß1-11), 4G8 (mouse anti-Aß17-24), m11 (mouse anti-Aßfree11-x) and 82 (rabbit anti-Aßpyro11-x) were used as primary antibodies. Thioflavin S staining was used to detect fibrillar Aß deposits, using Vectashield mounting medium (Vector Laboratories). Images were obtained using 1.25-40x/0.16-0.85 objectives on a microscope (model BX51; Olympus) with a ProgRes C14 Jenoptik camera and software (Laser Optik Systeme). For quantitative image analysis, sections of the somatosensory cortex and hippocampus were stained with NAB228 and analyzed using Image Pro-plus (Media Cybernetics, Inc.).
32P-labeling of phospho-APP
Intravitreal injections of 32P-labeled orthophosphate (250 µCi/2 µl per eye; PerkinElmer) were performed on three pairs of anesthetized APP and APPxBACE-H mice. 6 h after injection, optic nerves were homogenized in RIPA buffer containing protease inhibitors and phosphatase inhibitors (50 mM sodium fluoride and 0.2 mM sodium vanadate), immunoprecipitated with 5685 for full-length APP or RMO26 for NFM (Black and Lee, 1988), separated on a 7.5% Tris-glycine gel, transferred to a nitrocellulose membrane, and exposed to a phosphorimager screen. The same membrane was then immunoblotted with an NH2-terminal APP antibody (Karen).
Sciatic nerve ligation
Mice were anesthetized and one sciatic nerve from each mouse was ligated approximately in the middle. The other sciatic nerve was used as an unligated control. 6 h after ligation, 5-mm sciatic nerve segments proximal and distal to the ligature were sonicated in RIPA buffer containing protease inhibitors and centrifuged at 100,000 g for 20 min at 4°C. Proteins were analyzed by immunoblotting using the antibodies listed in Table S1. Full-length APP, phospho-APP and sAPPßswe were immunoprecipitated with 5685 before immunoblotting.
Spinal cord pulse-labeling
L5 segments of spinal cords from non-Tg and BACE Tg mice were injected with 250 µCi/0.71 µl of [35S]-methionine (PerkinElmer) bilaterally at an infusion rate of 0.1 µl/min. Mice were killed at 0.5, 8, and 16 h after injection when sciatic nerves and spinal cord segments 2-mm rostral and caudal to the injection site were harvested. Samples were homogenized with RIPA buffer, immunoprecipitated with 5685 for full-length APP, and electrophoresed on 7.5% Tris-glycine gels. Radiolabeled APP was quantified by ImageQuant phosphorimager analysis (Molecular Dynamics).
Online supplemental material
Table S1 lists the antibodies used for biochemical analysis. Fig. S1 shows immunohistochemical and biochemical analysis of full-length and truncated Aß in aged Tg mice. Fig. S2 shows the reduction of mature APP in APPI Tg mice and human neuronal cultures upon BACE overexpression. Fig. S3 shows the regional distribution of amyloid pathology in 20-mo-old Tg mice. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200407070/DC1.
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Acknowledgments |
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This work was supported in part by National Institutes of Health training grant T32 AG00255 (to E.B. Lee) and NIA AG11542. R.W. Doms was also supported by a Paul Beeson Faculty Scholar Award, J.Q. Trojanowski is the Measey-Schnabel Professor of Geriatric Medicine and Gerontology and V.M.-Y. Lee is the John H. Ware III Professor of Alzheimer's research.
Submitted: 12 July 2004
Accepted: 22 November 2004
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