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Published as doi: 10.1096/fj.07-8114com.
 (The FASEB Journal. 2007;21:2742-2752.)
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Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway

Il-Sang Yoon, Eunice Chen, Tracy Busse, Emanuela Repetto, Madepalli K. Lakshmana, Edward H. Koo and David E. Kang1

Department of Neurosciences, University of California, San Diego, La Jolla, California, USA

1Correspondence: Department of Neurosciences, MC0691, UC San Diego, Leichtag Biomedical Research 380, 9500 Gilman Dr., La Jolla, CA 92093, USA. E-mail: dekang{at}ucsd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid ß protein (Aß), a small peptide derived from ß- and {gamma}-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that ß- and {gamma}-secretase activities of BACE1 and presenilin, respectively, are concentrated in intracellular lipid raft microdomains. However, the manner in which APP normally traffics to lipid rafts is unknown. In this study, using transient transfection and immuno-precipitation assays, we show that the cytoplasmic domain of low-density lipoprotein receptor-related protein (LRP) interacts with APP and increases Aß secretion and APP ß-CTF (C-terminal fragment) generation by promoting BACE1-APP interaction. We also employed discontinuous sucrose density gradient ultracentrifugation to show that the LRP cytoplasmic domain-mediated effect was accompanied by greatly increased localization of APP and BACE1 to lipid raft membranes, where ß- and {gamma}-secretase activities are highly enriched. Moreover, we provide evidence that endogenous LRP is required for the normal delivery of APP to lipid rafts and Aß generation primarily in the endocytic but not secretory pathway. These results may provide novel insights to block Aß generation by targeting LRP-mediated delivery of APP to raft microdomains. —Yoon, I.-S., Chen, E., Busse, T., Repetto, E., Lakshmana, M. K., Koo, E. H., Kang, D. E. Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway.


Key Words: Alzheimer's disease • BACE1 • secretase • Swedish • siRNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALZHEIMER'S DISEASE (AD), THE MOST COMMON form of dementia, is pathologically characterized by the extracellular accumulation of the amyloid ß (Aß) peptide in senile plaques as well as intracellular accumulation of tau in neurofibrillary tangles. Although the causal relationship between Aß and AD still remains to be firmly established, increasing genetic and biochemical evidence strongly suggests that Aß, especially the longer Aß42 isoform, plays a crucial and early role in AD pathogenesis (1) . The vast majority of amyloid precursor protein (APP) is constitutively cleaved in the middle of the Aß sequence by {alpha}-secretase (TACE/ADAM17 and/or ADAM10) in the nonamyloidogenic pathway, thereby abrogating the generation of an intact Aß peptide. Alternatively, a small proportion of APP is cleaved in the amyloidogenic pathway, leading to the secretion of Aß peptides (37–42 amino acids) via two proteolytic enzyme activities, ß- and {gamma}-secretase, now known as BACE and presenilin, respectively (2) . Endocytic processing of APP from the cell surface is apparently required for Aß generation by delivering APP to intracellular sites enriched in ß- and {gamma}-secretase activities in non-neuronal cells (3 , 4) . It is now well recognized that BACE and presenilins are highly enriched in lipid raft microdomains (5 6 7 8 9) , a subpopulation of cellular membranes that are insoluble in nonionic detergents at 4°C and heavily enriched in cholesterol, sphingomyelin, and glycolipids (10 , 11) . As such, disruption of lipid rafts by cholesterol-depleting agents completely abrogates Aß formation (12) . Moreover, targeting BACE1 exclusively to lipid rafts by glycophosphatidylinositol (GPI) anchor to recombinant BACE1 markedly increases ß-secretase activity (13) . Although only a small fraction of APP associates with rafts, it is clear that the majority of Aß generation occurs in these cholesterol-rich membrane subpopulations. At present, the molecular mechanisms underlying the trafficking of APP to rafts are unknown.

The low-density lipoprotein receptor-related protein (LRP) is a multifunctional endocytosis receptor that mediates the internalization and degradation of ligands involved in diverse metabolic pathways (14) . LRP, a type I transmembrane protein, is synthesized as a 600 kDa precursor protein that is subsequently cleaved in the trans-Golgi compartment by furin to generate a large 515 kDa {alpha} chain and a smaller 85 kDa transmembrane ß chain that remain noncovalently linked (15) . The short cytoplasmic tail of LRP contains two NPxY motifs and two dileucine-based motifs and interacts with a number of cytoplasmic adaptor and scaffold proteins, such as FE65, Shc, PSD-95, Mint2, Disabled-1 (Dab1), and JIP-1 and -2 (16 17 18) . Recent studies (19 20 21) also found that LRP interacts with APP, BACE1, and presenilin 1 presumably through the short cytoplasmic as well as the extracellular domains. The latter studies (20 21 22) also showed that LRP itself undergoes a presenilin-dependent {gamma}-secretase intramembrane proteolysis as well as ß-secretase-like cleavage by BACE1.

Several lines of evidence have implicated the involvement of LRP in AD pathophysiology. First, we and others (23 24 25) have shown that LRP is genetically associated with late-onset AD. Furthermore, we (23 , 26) have shown that LRP genotypes are associated with the age of disease onset and severity of amyloid pathology in AD subjects, much like that shown for apolipoprotein E (apoE) genotypes (27) . Second, three of its key ligands, apoE, {alpha}2-macroglobulin, and APP are also genetically associated with AD and are found in senile plaques in the brains of AD patients along with LRP (27 28 29) , further implicating the LRP pathway in AD pathophysiology. Third, LRP plays a role in the removal of Aß complexed to {alpha}2-macroglobulin and apoE in cultured cells (26 , 30 , 31) , as well as in brain efflux of Aß isoforms at the blood-brain barrier, supporting a model in which LRP plays an important role in extracellular Aß uptake and removal (32) .

By an opposing modality, it has also been shown that LRP promotes Aß generation by altering the processing and/or trafficking of APP, possibly by APP/LRP interactions via the Kunitz protease inhibitor (KPI) domain as well as through its cytoplasmic tail (33 , 34) . Indeed, the loss of LRP or treatment of receptor-associated protein (RAP), an antagonist of all known LRP ligands, greatly reduced Aß release (35) , a phenotype that was reversed when full-length (LRP-FL) or an artificially N-terminal truncated LRP construct was transfected in LRP-deficient cells. Specifically, the C-terminal 370 amino acids of LRP (LRP-CT) containing the transmembrane (TM) domain and the cytoplasmic tail as well as LRP soluble tail (LRP-ST) lacking the TM domain but containing the cytoplasmic tail were sufficient to rescue amyloidogenic processing of APP and Aß release in LRP-deficient cells (34 , 36) . In particular, we found that LRP-ST stimulated Aß secretion at least in part by increasing ß-secretase cleavage of APP without significantly changing the rate of APP endocytosis (36) . Interestingly, a recent study using fluroescence resonance energy transfer (FRET)-based and pull-down assays demonstrated that the LRP cytoplasmic domain interacts with BACE1 (21) . Since APP can also interact with LRP cytoplasmic tail, this led us to hypothesize that the LRP cytoplasmic domain might facilitate amyloidogenic processing of APP by enhancing the association APP with BACE1, the latter that is enriched in lipid raft membranes. In this study, we have confirmed this very prediction. Moreover, we demonstrate that endogenous LRP is critical not only for vast majority of Aß generation but also the delivery of APP to lipid raft microdomains through the endocytic pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
All cell lines were cultured in Dulbecco's modified Eagle's medium (Mediatech/CellGro, Herndon, VA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). Stably transfected Chinese hamster ovary (CHO) cells were generated with wild-type APP751 or Swedish mutant APP751 as described previously (3 , 37) .

cDNA Constructs
LRP constructs used in this study are illustrated in Fig. 1 . LRP minireceptor construct (LRP-D1/2) was generated by PCR and subcloned into pLPCX retroviral vector (Clontech, Palo Alto, CA, USA). The LRP-CT construct was subcloned into the pLHCX retroviral vector (34) . The last 97 amino acids of the cytoplasmic domain (or soluble tail) of LRP ß-subunit (LRP-ST) were generated by PCR as described previously (36) and subcloned into the pEGFP-C3 vector (Invitrogen, Carlsbad, CA, USA) to generate GFP fusion protein. The cDNAs of human APP751 Swedish mutant (Swe-APP751) and human BACE1 were subcloned into the pLHCX and pLPCX retroviral expression vector, respectively (Clontech). Wild-type APP751 was subcloned in pcDNA3 vector (Invitrogen).


Figure 1
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  		Figure 1. Schematic of LRP constructs. Endogenous full-length LRP is proteolytically cleaved by Furin to generate a 515 kDa {alpha} chain containing ligand binding domains 1 to 4 and an 85 kDa ß chain. The LRP minireceptor (LRP-D1/2) contains the ligand binding domains 1 and 2 (amino acids 1–954; numbering from the N terminus of mouse LRP1, NCBI accession number NP_032538) and the C-terminal 703 amino acids of LRP including the ß chain. LRP-CT encodes the last 370 amino acids of the ß chain of LRP and its corresponding signal peptide (SP). LRP-ST represents the last 97 amino acids of the cytoplasmic domain (or soluble tail) of LRP ß chain lacking a signal peptide or TM segment and was fused to EGFP (EGFP-LRP-ST).

Transient transfection
The small interfering RNA (siRNA) targeting human LRP1 (5'-ccgcuccugcaaggccaaguu-3') was custom synthesized by Dharmacon. Transfections of either the single-stranded sense oligo of LRP or the LRP siRNA duplex (final concentration; 50 or 150 nM) were performed using Lipofectamime 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After 24–36 h of the first transfection into the APP751-CHO or SwAPP751-CHO cells, a second transfection was performed to enhance the knock-down effect of the LRP siRNA. Twenty four hours after the second siRNA transfection, conditioned media were collected for Aß detection, and the cells were lysed with 1% CHAPS lysis buffer for the lipid raft fractionation as described below. Transient transfections of cDNA constructs were performed using Lipofectamine 2000 (Invitrogen) as instructed by the manufacturer.

Antibodies
The polyclonal antiserum 1704, which recognizes cytoplasmic domain of LRP1, and the polyclonal antiserum CT15, which reacts with the cytoplasmic region of APP, have been described previously (34) . For the detection of the full-length APP and APP-CTFs, CT15 antibody was used. The 1704 antibody recognizes endogenous LRP of hamster, mouse, and human. Monoclonal antibodies 1G7/5A3 and 26D6, which recognize the ectodomain of APP, were used for the detection of secreted APP and Aß, respectively (38) . Polyclonal antibody B279 that recognize human BACE1 was kindly provided by Dr. R. Yan (Lerner Research Institute, Cleveland, OH, USA). The GFP antibody was purchased from Stressgen (Victoria, Canada), and antibodies against flotillin-1 and caveolin-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunoprecipitation and immunoblotting
For coimmunoprecipitation and Western blotting, cell extracts were prepared with 1% 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS) lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.002% sodium azide, 400 nM Microcystin-LR, 0.5 mM sodium vanadate, and 1x Protease Complete Inhibitor mix (Sigma). To detect Aß, 1 ml of conditioned media was collected after 24 or 48 h and subjected to the immunoprecipitation. The 26D6 antibody was used for both immunoprecipitation and Western blot analysis. The prepared samples were denatured by SDS sample buffer and fractionated by SDS-PAGE in 4–12% NuPAGE Bis-Tris gels (Invitrogen) or 10% Tris-glycine gels. Gel loading was normalized to total protein concentration as measured by the micro BCA method (Pierce, Rockford, IL, USA). Western blotting was carried out with the indicated antibodies and detected by enhanced chemiluminescence (Pierce). Quantitation of the chemiluminescence signal was done using a CCD camera imaging system (GeneGnome, Syngene, Frederick, MD, USA). All experiments were performed at least three times, and each experiment was done in duplicate or in triplicate. Data are mean ± SE.

Lipid raft isolation
CHAPS detergent (1%, w/v)-treated cell extracts were fractionated by discontinuous sucrose density gradient ultracentrifugation according to the previous study (5) . Briefly, confluent cells in 10 cm plates were washed with PBS on ice, scraped in TNE buffer (25 mM Tris, pH 7.4, 150 mM NaCl, and 2 mM EDTA) containing protease inhibitor mix (Sigma), and pelleted by low speed centrifugation. The cell pellets were then disrupted by 10 strokes through a 25-G needle in TNE buffer, and an equal volume of TNE buffer containing 2% CHAPS was mixed together and incubated on ice for 30 min. The CHAPS cell extracts were then mixed with TNE buffer containing sucrose to yield a final concentration of 45% (w/v) sucrose and laid at the bottom of the ultracentrifuge tube. TNE buffer containing 35% and 5% sucrose was successively and carefully laid over the CHAPS cell extracts. The samples were spun at 4°C for 14–16 h at 44,000 rpm in the SW55 rotor. Fractions were then collected in 0.5 ml volumes from top to bottom to yield a total of 10 fractions. The buoyant lipid rafts settled at the interface between the 35 and 5% sucrose as amorphous white material visible to the naked eye that were collected in fractions 2 and 3. Equal volumes of each fraction were then subjected to SDS-PAGE and immunoblotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LRP cytoplasmic tail promotes the physical association between BACE1 and APP
It has been shown by a FRET-based approach that LRP can associate with intracellular APP through both extracellular and cytoplasmic domains (19) , consistent with the finding of APP:LRP complexes. Furthermore, it has also been proposed that cytoplasmic adaptor proteins such as Fe65 might constitute a functional and physical link between LRP cytoplasmic tail and APP (39) . However, APP:LRP complexes that associate via the respective APP and LRP cytoplasmic domains have not yet been demonstrated by coimmunoprecipitation studies in intact cells. Thus, we transiently cotransfected APP751 with an LRP minireceptor containing ligand-binding domains 1 and 2 (LRP-D1/2), LRP-CT, or EGFP-fused LRP-ST (EGFP-LRP-ST) in HEK293T cells to ascertain the interactions (Fig. 1) . LRP-ST was fused to EGFP since LRP-ST alone was relatively unstable (data not shown). Indeed, we could detect specific APP pull-down in LRP immune complexes when cotransfected with an LRP-D1/2 or LRP-CT, while only traces amounts of APP, probably attributable to endogenous LRP binding, were detected when transfected with APP alone (Fig. 2 A). Similarly, APP also specifically coimmunoprecipitated with EGFP-tagged LRP-ST, demonstrating that the cytoplasmic tail of LRP alone can form complexes with APP (Fig. 2B ).


Figure 2
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  		Figure 2. LRP cytoplasmic domain interacts with APP. A) HEK293T cells were transiently transfected with different combinations of LRP-D1/2, LRP-CT, and APP751. Nonidet P-40 lysates were subjected to immunoprecipitation for LRP (11H4) and immunoblotting for APP (CT15, third panel) or direct immunoblotting for APP (CT15, first panel) and LRP (1704). (B) HEK293T cells were transiently transfected with Swe-APP751 and EGFP-LRP-ST or EGFP. Nonidet P-40 lysates were subjected to immunoprecipitation for LRP (11H4) and immunoblotting for APP (CT15, third panel) or direct immunoblotting for APP (CT15, first panel) and LRP (1704). N-glycosylated (N') and N+O-glycosylated (N'/O') forms of APP751 are illustrated.

Recently, it was reported that the cytoplasmic tail of LRP but not the LDL receptor associates with BACE1 and that LRP itself undergoes BACE1-mediated cleavage (21) . As the LRP cytoplasmic domain interacted with both APP and BACE1 and increased APP CTF levels and Aß generation (21 , 36) , we hypothesized that LRP-ST might promote Aß secretion by facilitating APP/BACE1 interaction. To test this, we transiently expressed different combinations of BACE1, "Swedish" mutant APP751 (Swe-APP751), EGFP-LRP-ST, and EGFP alone in HEK293T cells. We initially chose to study Swe-APP751 since it is an excellent substrate for BACE1, anticipating that Swe-APP751 forms more stable complex with BACE1 than wild-type APP. As previously reported (40 , 41) , BACE1 overexpression reduced the level of full-length APP but increased the slower migrating 12 kDa APP ß-CTF at the expense of the 10 kDa APP {alpha}-CTF (Fig. 3 ). Because LRP-ST behaved similar to LRP-D1/2 and LRP-CT, we chose to continue the studies hereon with the EGFP-stabilized LRP-ST construct because it unequivocally demonstrates that the effects are due to the LRP cytoplasmic domain. Coexpression of BACE1 and Swe-APP with EGFP-LRP-ST increased the level of APP ß-CTF by ~5-fold (Fig. 3) , indicating that LRP-ST stimulates BACE1-mediated cleavage of APP. This was accompanied by increased Aß secretion by EGFP-LRP-ST compared to EGFP alone (Fig. 3) . Consistent with our hypothesis that the LRP cytoplasmic domain promotes Aß generation by facilitating APP/BACE1 interaction, the amount of BACE1:APP immune complexes was similarly increased by ~5-fold in the presence of EGFP-LRP-ST in CHAPS solubilized lysates, even though the level of BACE1 or APP expression was virtually identical between transfected samples (Fig. 3) . These results demonstrate that the LRP cytoplasmic domain can promote the amyloidogenic processing of APP via facilitating the physical association of APP with BACE1 and enhancing APP ß-CTF generation, either directly or indirectly.


Figure 3
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  		Figure 3. LRP-ST promotes APP-BACE1 interaction, BACE1 mediated cleavage of APP, and Aß generation. HEK293T cells were transiently transfected with combinations of BACE1, Swe-APP751, EGFP-LRP-ST, and EGFP. First, second panels: CHAPS lysates were directly immunoblotted for full-length APP (APP-FL) and APP CTFs with antibody CT15. Third panel: Lysates were directly immunoblotted for LRP (1704). Fifth, sixth panels: CHAPS lysates were subjected to immunoprecipitation for APP (IG7/5A3) or BACE1 (B279) and immunoblotted for BACE1 (B279) or APP (26D6), respectively. Seventh panel: conditioned medium was subjected to immunoprecipitation-immunoblotting for Aß (26D6).

LRP-ST associates with lipid rafts and enhances the localization of APP and BACE1 to lipid raft microdomains
BACE1 is known to localize to cholesterol-rich lipid rafts, and its activity is highly concentrated in these membrane microdomains (5) . Furthermore, {gamma}-secretase activity and components (presenilins, nicastrin, pen-2, and aph-1) are also highly enriched in raft fractions, whereby the majority of Aß is apparently produced (5 , 8 , 9 , 42) . Because the LRP cytoplasmic domain could promote APP/BACE1 association and enhance Aß generation, we hypothesized that LRP-ST promotes the trafficking of APP and/or BACE1 to lipid raft microdomains. To test this hypothesis, we transiently transfected Swe-APP751 and BACE1 with or without EGFP-LRP-ST, and 1% CHAPS cell extracts were analyzed by discontinuous sucrose density gradient fractionation as described previously (5) . When the fractions were immunoblotted using an antibody against the raft marker flotillin-1, fractions 2 to 4 were enriched for flotillin-1 in both EGFP and EGFP-LRP-ST transfected HEK293T cells (Fig. 4 ). Similar data were obtained with another lipid raft marker caveolin-1 (not shown). Remarkably, EGFP-LRP-ST was strongly localized to more buoyant fraction 2 to 6 compared to EGFP alone, indicating that LRP-ST can associate with lipid rafts membranes (Fig. 4) . This distribution of EGFP-LRP-ST was mirrored by a strong redistribution of full-length APP and APP CTFs from heavier fractions 7–10 toward more buoyant fractions 2–6 compared to that of EGFP control transfection (Fig. 2 , first and second panels), indicating that LRP-ST redistributes a fraction of APP to lipid raft membranes. In particular, APP CTFs ({alpha}- and ß-CTF) were redistributed to raft fractions 2 and 3 more than full-length APP (APP-FL). The presence of the fast migrating {alpha}-CTF in the raft fractions was also reported in previous studies (5 , 43 , 44) . As expected, BACE1 was present in both raft and nonraft fractions in both EGFP and EGFP-ST transfected cells. However, EGFP-LRP-ST transfection markedly further enhanced BACE1 localization in raft fractions 2 and 3 from heavier fractions 7–10, indicating that the LRP cytoplasmic tail not only promotes APP but also BACE1 delivery to lipid rafts. These results were consistent in multiple experiments, and similar results were obtained when wild-type APP751 was cotransfected with EGFP-LRP-ST in HEK293T cells (not shown). Moreover, EGFP-LRP-ST similarly enhanced wild-type APP localization in raft fractions in LRP-deficient 13–5-1 cells (Supplemental Fig. S1), indicating that endogenous LRP is not required for this activity of LRP-ST. These results taken together support our hypothesis that the cytoplasmic domain of LRP promotes APP/BACE1 association by acting as a scaffold and/or facilitating the trafficking of APP and perhaps BACE1 to lipid raft microdomains.


Figure 4
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  		Figure 4. LRP-ST promotes localization of APP and BACE1 to lipid raft microdomains. HEK293T cells were transiently cotransfected with Swe-APP751 and BACE1 with EGFP or EGFP-LRP-ST. Cells were lysed using 1% CHAPS buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. All fractions (1–10) were collected in equal volumes and subjected to immunoblotting for full-length APP and CTFs (CT15, first to fourth panels), BACE1 (B279, fifth, sixth panels), flotillin-1 (seventh, eighth panels), and EGFP (ninth, tenth panels).

LRP is required for the vast majority of Aß secretion and delivery of APP to lipid rafts
We and others previously demonstrated that cells lacking LRP produce lower levels of Aß compared to their wild-type counterparts. This Aß phenotype was reversed with reconstitution of full-length LRP, LRP-CT, or LRP-ST (34 35 36) . To ascertain that endogenous LRP normally contains the activity in the amyloidogenic processing of APP using a different approach, we used the RNAi strategy to reduce endogenous LRP expression in CHO cells stably transfected with wild-type APP751 (CHO-APP751). As shown in Fig. 5 A, transfection of siRNA targeted toward LRP greatly decreased endogenous LRP-ß chain in a dose-dependent manner compared to a single-stranded sense control oligo. This was accompanied by a marked dose-dependent decrease in Aß release into the conditioned medium without altering the levels of full-length APP (Fig. 5A ). In multiple experiments, LRP siRNA reduced Aß secretion by an average of 82.2% (±6.0 SE; n=6) compared to control sense strand oligo transfection. These results are not only consistent with the data from LRP-deficient cells but also clearly illustrate that endogenous LRP is required for the vast majority of Aß secretion from wild-type APP (Fig. 5A, B ). To test whether endogenous LRP is similarly required for lipid raft delivery of APP, control and LRP siRNA transfected CHO-APP751 cells were subjected to 1% CHAPS extraction and sucrose density gradient ultracentrifugation to isolate lipid rafts in the buoyant fractions. In CHO-APP751 cells (unlike HEK293T cells), a significant fraction of full-length APP was present in raft fractions 2 and 3, whereas the vast majority of APP CTFs ({alpha}- and ß-CTFs) was found in these fractions (Fig. 5C ). In heavier fractions 6–10, small amounts of APP CTFs, primarily {alpha}-CTFs were present (Fig. 5C ). Interestingly, >90% of LRP ß chain was concentrated in lipid raft fractions 2 and 3 in these cells (Fig. 5C ). Knockdown of endogenous LRP by siRNA selectively and markedly reduced full-length APP (APP-FL) in lipid raft fractions without significantly altering heavier fractions 5 to 10 (Fig. 5C ). Relative to fraction 10, the amount of APP-FL in raft fractions 2 and 3 was reduced by ~3-fold secondary to LRP siRNA transfection (Fig. 5C, D ). This was accompanied by even more robust reduction in APP CTFs in lipid raft fractions relative to fraction 10 by a factor of >10 on LRP siRNA treatment (Fig. 5C, E ). As expected, LRP siRNA markedly reduced the level of the LRP ß chain (Fig. 5C ). This treatment had no effect on the localization of raft markers, such as caveolin-1 (Fig. 5C ). These results demonstrate that LRP contributes to normal trafficking or localization of APP to lipid rafts and Aß generation. As LRP-ST mimicked endogenous LRP in lipid raft targeting of APP, Aß secretion, and APP ß-CTF generation, we propose that the cytoplasmic domain of LRP significantly contributes to the proamyloidogenic activity of endogenous LRP.


Figure 5
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  		Figure 5. Endogenous LRP is required for the normal trafficking of APP to lipid rafts and Aß generation. A) CHO cells stably overexpressing wild-type APP751 (CHO-751) were transfected twice with control single stranded sense oligo or LRP siRNA duplex (50 and 150 nM final concentration) and conditioned medium was collected for 24 h thereafter as described in Materials and Methods. Lysates were subjected to immunoblotting for LRP (1704) and APP (CT15) and conditioned medium was immunoprecipitated and immunoblotted for Aß (26D6). B) Graph represents quantitations of Aß secretion from LRP siRNA (150 nM) transfected normalized to control oligo-transfected cells. Error bar represents SE (n=6). Note that there is an average of 82.2% reduction in Aß secretion in LRP siRNA compared to control transfected cells. C) After LRP siRNA (150 nM) or control transfections, cells were lysed using 1% CHAPS buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. All fractions (1–10) were collected in equal volumes and subjected to immunoblotting for full-length APP and CTFs (CT15, first to fourth panels), LRP (1704, fifth, sixth panels), and caveolin-1 (seventh, eight panels). D, E) Graphs represent quantitations of APP-FL and APP-CTFs in each fraction relative to fraction 10. Experiments were performed 3–4 times, and representative figures are shown.

Differential effects of LRP on Aß secretion and lipid raft localization in the Swedish APP mutant
The familial AD Swedish APP (Swe-APP) mutation increases Aß secretion by 3–5-fold in various cell culture models through increased ß-secretase cleavage. It is well documented that the vast majority of Aß derived from Swe-APP is generated along the secretory pathway and does not depend on endocytic processing (37 , 45 46 47) , unlike wild-type APP in which ~80% of Aß is generated after endocytosis from the cell surface (3 , 4) . This may be explained by the fact that Swe-APP is a highly preferred substrate for BACE1 compared to wild-type APP, thereby enabling ß-secretase cleavage by BACE1 on the way to the cell surface. In vitro, purified BACE1 exhibits up to 100-fold increased ß-secretase activity on Swe-APP compared to its wild-type counterpart (48) . Given these profound differences, we next asked whether Aß secretion and lipid raft localization of Swe-APP are also altered by endogenous LRP. LRP siRNA transfection led to ~80% reduction in LRP ß chain levels in CHO cells stably transfected with Swe-APP751, comparable to that achieved in wild-type APP751 expressing CHO cells (Fig. 6 A). Moreover, steady-state endogenous LRP levels in Swe-APP751 cells were similar to those in CHO-APP751 cells (not shown). However, in contrast to wild-type APP, the LRP knockdown had little to no effect on Aß secretion from Swe-APP (Fig. 6A, B ). Similar to wild-type APP751, Swe-APP751-derived {alpha}- and ß-CTFs were heavily concentrated in lipid raft fractions 2 and 3, although both {alpha}- and ß-CTFs were detected at similar ratios in heavier nonraft fractions 7–10 (Fig. 6C ). Consistent with the results of Aß secretion, the distribution of APP CTFs in lipid rafts was not altered by LRP siRNA from Swe-APP (Fig. 6C, E ), demonstrating that LRP does not affect Aß production or generation of ß-secretase-derived CTFs in Swe-APP. Interestingly, the relative amounts of full-length Swe-APP in lipid raft fractions 2 and 3 were substantially lower compared to wild-type APP (Figs. 5C , D vs. 6C, D ). However, LRP siRNA further reduced full-length Swe-APP in lipid raft fractions (Fig. 6C, D ). This indicated that LRP does alter Swe-APP localization to lipid raft fractions but not within a pool that that appreciably contributes to the generation of APP CTFs and Aß. As Swe-APP dramatically increases Aß production along the secretory route, this LRP-sensitive pool of full-length Swe-APP likely represents the minor fraction in the endocytic route, which does not significantly contribute to overall Aß secretion. Thus, we interpret these results to indicate that endogenous LRP is critical for lipid raft delivery of APP and Aß generation primarily in the endocytic but not secretory pathway.


Figure 6
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  		Figure 6. Endogenous LRP does not alter Aß generation associated with the secretory pathway of the Swedish APP mutant. A) CHO cells stably overexpressing Swe-APP751 were transfected with control single stranded sense oligo or LRP siRNA duplex (150 nM final concentration) twice over a 56 h period and conditioned medium was collected for 24 h thereafter. Lysates were subjected to immunoblotting for LRP (1704) and APP (CT15) and conditioned medium was immunoprecipitated and immunoblotted for Aß (26D6). B) Graph represents quantitations of Aß secretion from LRP siRNA (150 nM) transfected normalized to control oligo-transfected cells. Error bar represents SE (n=4). C) After LRP siRNA (150 nM) or control transfections, cells were lysed using 1% CHAPS buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. All fractions (1–10) were collected in equal volumes and subjected to immunoblotting for full-length APP and CTFs (CT15, first to fourth panels). D, E) Graphs represent quantitations of APP-FL and APP-CTFs in each fraction relative to fraction 10. Experiments were performed 3 times, and representative figures are shown.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Accumulating evidence has implicated lipid rafts as the major intracellular sites for amyloidogenic processing of APP (5 , 6 , 42) , as ß-secretase (BACE1) and {gamma}-secretase components (PS1, APH-1, PEN-2, and Nicastrin) and their corresponding activities are highly enriched in rafts (7 , 9 , 42) . However, the manner in which APP is normally delivered to lipid rafts is unknown. In this study, we presented several lines of new evidence demonstrating the critical involvement of LRP and its cytoplasmic domain in amyloidogenic trafficking of APP to lipid raft microdomains. First, we showed by coimmunoprecipitation studies that an LRP minireceptor (LRP-D1/2), LRP-CT, and LRP-ST interact with APP, indicating that the cytoplasmic domain of LRP is sufficient for this interaction. Second, we showed that the LRP-ST construct is sufficient to enhance Aß secretion, APP CTF generation, and the physical association of APP with BACE1. Third, we demonstrated that both LRP-ST and endogenous LRP robustly promoted APP trafficking to lipid rafts, where ß- and {gamma}-secretase activities are highly enriched. Fourth, we provided convincing evidence that endogenous LRP is critical for localization of wild-type APP to lipid rafts and Aß generation primarily in the endocytic but not secretory pathway. These results taken together illustrate that LRP, at least in part via its cytoplasmic domain, promotes the amyloidogenic processing of APP by facilitating APP and perhaps BACE1 trafficking to lipid rafts and strengthening their interactions.

Our results indicated that the LRP-ST increased APP CTFs and Aß generation by promoting the physical association between APP and BACE1. Although the manner in which LRP promotes this process is not known with certainty, we propose two mutually inclusive models that can explain this phenotype. First, as the LRP cytoplasmic domain associates with both APP and BACE1 (19 , 21) , LRP-ST may act as a scaffold on which APP and BACE1 are assembled. Indeed, it has been shown that LRP itself can also undergo cleavage by BACE1 much like that of APP (21) . As BACE1 is normally sorted to lipid rafts (5) , the tripartite interactions between APP-LRP-BACE1 may, in part, explain the increased delivery of APP to lipid rafts secondary to LRP-ST overexpression. However, this model by itself does not sufficiently account for the observation that LRP-ST promoted both APP and BACE1 to lipid raft fractions. At present, we do not know whether the interactions between LRP cytoplasmic domain and BACE1 or APP are direct or indirect, since these observations were made by FRET-based or coimmunoprecipitation assays (19 , 21) . In our coimmunoprecipitation experiments, we used 1% CHAPS as detergent for cell lysis. Under these conditions, lipid rafts are not solubilized. Thus, in a second scenario, it is possible that the increased APP/BACE1 pull down secondary to LRP-ST overexpression may not only reflect direct interaction between APP and BACE1 but also the enrichment of both proteins in clusters of CHAPS insoluble lipid raft membranes. If so, the increased lipid raft localization of APP and/or BACE1 by LRP-ST may account for the increased APP/BACE1 association observed in coimmunoprecipitation experiments. The observation that LRP-ST itself strongly associated with lipid rafts and promoted both APP and BACE1 delivery to these membranes suggests that in addition to the interactions among LRP-ST, APP, and BACE1, other proteins enriched in lipid rafts may also physically interact with LRP-ST. For example, LRP cytoplasmic domain is known to bind to PSD-95, a lipid raft resident protein that recruits voltage-gated potassium channel (Kv1.4) and ErbB4 receptor tyrosine kinase to lipid rafts (49 , 50) . It is unlikely that PSD-95, which is expressed mainly in brain, recruited LRP to lipid rafts in the non-neuronal cells (HEK293 or CHO cells) used in this study. However, it is possible that other LRP binding scaffold proteins (e.g., FE65, Shc, Mint2, Dab1, and JIP-1 and -2) or yet unidentified lipid raft targeting proteins may interact with LRP cytoplasmic domain and recruit LRP-APP and/or LRP-BACE1 complexes to the lipid rafts where the frequency of APP/BACE1 interaction would be enhanced. If so, discovering the identity of such LRP-interacting lipid raft targeting molecules could provide further insights to designing strategies to block Aß generation by targeting LRP-mediated delivery of APP and/or BACE to lipid rafts.

Consistent with previous reports, we found that endogenous LRP is required for >80% of Aß secretion from wild-type APP751 in CHO cells. The reduction in Aß was similarly accompanied by strong reductions in the localization of APP and APP-CTFs in lipid raft fractions, demonstrating that LRP normally facilitates the entry of APP to lipid rafts and generation of APP-CTFs in these subcellular microdomains. Indeed, these results showed that LRP-ST without a membrane tether mimics endogenous LRP in promoting lipid raft targeting of APP and Aß generation. Like LRP-ST, endogenous LRP cytoplasmic tail is expected to associate with APP and BACE1. In addition, the extracellular ligand-binding domains II and IV of LRP also interact with the extracellular KPI domain of APP (33 , 51 , 52) . Thus, we cannot rule out the possibility that endogenous LRP alters the lipid raft trafficking of APP via both the extracellular and cytoplasmic domains. Indeed, the loss of LRP cytoplasmic domain in an LRP minireceptor containing ligand-binding domain IV did not abrogate the interaction with APP770 (52) .

Lipid rafts are thought to be assembled in the Golgi apparatus, where sphingolipids are synthesized (53) , and these microdomains are concentrated in the early Golgi cisternae, plasma membrane, and endosomes, although they are apparently excluded from COPI vesicles (10) . We hypothesize that LRP promotes the entry of APP to lipid raft microdomains in the endocytic route of APP trafficking on the plasma membrane and/or endosomes. In both wild-type- and Swe-APP-expressing CHO cells, >90% of APP CTFs were found in lipid raft fractions, consistent with their generation in these microdomains. However, LRP knockdown dramatically reduced APP-CTFs in lipid rafts and Aß secretion in wild-type APP but not in Swe-APP. The critical difference between wild-type APP and Swe-APP is that Swe-APP is a highly preferred substrate for ß-secretase cleavage (48) . As such, the vast majority of Aß from Swe-APP is generated along the secretory pathway and does not require endocytosis (37 , 45 46 47) , whereas endocytic processing is apparently required for >80% of Aß secretion in wild-type APP (3 , 4) . Thus, we propose a model in which CTFs derived from Swe-APP are generated within lipid rafts in the Golgi complex in the secretory pathway independent of LRP, whereas CTFs derived from wild-type APP are generated within lipid rafts on the plasma membrane and/or endosomes in the endocytic pathway in an LRP-dependent manner. This notion is consistent with the function of LRP as an endocytic receptor that delivers cargo from the cell surface to endosomes/lysosomes and recycles back to the cell surface. Both LRP and APP endocytosis are mediated via a clathrin-coated pit mechanism (54 , 55) , although the involvement of lipid rafts in this process has not been investigated. It has been proposed that APP and BACE1 are present in distinct rafts on the plasma membrane and that ß-cleavage occurs after clustering of BACE1 and APP into the same raft platform on endocytosis (6) . This model is consistent with LRP in facilitating APP endocytosis and raft localization. As wild-type APP is a poor substrate for BACE1, LRP-mediated trafficking of APP to BACE1-containing lipid raft microdomains, particularly in endosomes where ß-secretase activity is favored by the slightly acidic pH, would be expected to enhance ß-CTF generation (56) . In contrast to APP-CTFs in lipid raft fractions, LRP knockdown reduced lipid raft localization of full-length APP in both wild-type and Swe-APP, albeit more robustly in wild-type APP. This LRP-sensitive pool in Swe-APP, unlike in wild-type APP, likely represents the minor proportion in the endocytic route of Aß generation. This fraction of Swe-APP, however, would be insufficient to contribute to detectable changes in Aß secretion or APP-CTF generation in lipid rafts as seen in our experiments. Indeed, the relative lipid raft localization of full-length APP in Swe-APP was markedly lower compared to wild-type APP, consistent with the notion that this LRP-sensitive pool of Swe-APP may be those that have escaped ß-secretase cleavage in the secretory pathway. Taken together, we interpret these results to indicate that endogenous LRP primarily promotes the entry of APP to lipid raft membranes and Aß generation in the endocytic pathway on the cell surface and/or endosomes.

Pietrzik and colleagues (34) previously demonstrated that the rates of APP endocytosis and Aß secretion are reduced in LRP-deficient fibroblasts, both of which were corrected by stable transfection of LRP-CT. Although the magnitude of Aß reduction (>80%) was more robust than the slower endocytosis (~50%) in LRP deficient cells, these data are consistent with the notion that LRP-CT restored Aß secretion at least in part by stimulating APP endocytosis. However, the current data also demonstrated that the delivery of APP to lipid raft membranes via an LRP-dependent pathway is also a critical determinant of Aß generation. We propose that LRP-mediated endocytosis and lipid raft delivery of APP are related but separable phenomena. In comparing endogenous full-length LRP and transfected LRP-ST, several similarities and differences can be made. First, both transfected LRP-ST and endogenous LRP promoted Aß generation and localization of APP to lipid rafts, indicating that the cytoplasmic domain of endogenous LRP at least in part contributes to this process. Second, LRP-ST does not have a transmembrane domain and does not alter the endocytosis of APP (36) , whereas endogenous LRP promotes APP endocytosis (34) . Third, overexpression of LRP-ST promoted Aß production from both wild-type and Swedish APP (36 ; Fig. 3 ), whereas endogenous LRP promoted Aß production only from wild-type APP in the endocytic pathway. Thus, these results indicate that without the constraint of the transmembrane domain, the soluble LRP-ST promotes Aß generation in both endocytic and secretory routes of APP trafficking. This appears to be achieved by facilitating the physical association of APP and BACE1 in lipid raft membranes without altering APP endocytosis. Thus, the results from LRP-ST suggest that the effects of endogenous LRP on APP endocytosis and lipid raft delivery of APP are related but separable activities. This raises the intriguing possibility that sequences in the LRP cytoplasmic domain other than the major NPxY or YxxL endocytosis motifs (57) contribute to lipid raft targeting of LRP and APP. Further studies will be required to prove this hypothesis.

To our knowledge, LRP is the first molecule identified so far that is critical for the normal localization of APP to lipid raft membranes. Thus, we propose that the manner in which LRP cytoplasmic domain alters APP trafficking, particularly related to APP/BACE1 interaction and delivery to lipid rafts, can have important implications for a novel approach to inhibit Aß generation. Conceivably, small peptide sequences derived from the LRP cytoplasmic domain or their nonpeptide peptidomimetics that can potentially block LRP-dependent delivery of APP to lipid rafts may be effective in inhibiting Aß generation. This way, unintended side effects associated with inhibiting ß- and {gamma}-secretase activities may be avoided.


   ACKNOWLEDGMENTS
 
This work was supported in part by the Alzheimer's Association (IIRG-05–14752, D. E. Kang), American Health Assistance Foundation (A2005–039, D. E. Kang), and the National Institutes of Health (AG-12376, E. H. Koo). We thank Dr. Markus Kummer for technical and intellectual support. Parts of this paper were presented at the 10th International Conference on Alzheimer's Disease and Related Disorders, July 15–20, 2006, Madrid, Spain.

Received for publication January 24, 2007. Accepted for publication March 26, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hardy, J., Selkoe, D. J. (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297,353-356[Abstract/Free Full Text]
  2. De Strooper, B., Annaert, W. (2000) Proteolytic processing and cell biological functions of the amyloid precursor protein. J. Cell Sci. 113,1857-1870[Abstract]
  3. Koo, E. H., Squazzo, S. L. (1994) Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269,17386-17389[Abstract/Free Full Text]
  4. Perez, R. G., Soriano, S., Hayes, J. D., Ostaszewski, B., Xia, W., Selkoe, D. J., Chen, X., Stokin, G. B., Koo, E. H. (1999) Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J. Biol. Chem. 274,18851-18856[Abstract/Free Full Text]
  5. Riddell, D. R., Christie, G., Hussain, I., Dingwall, C. (2001) Compartmentalization of beta-secretase (Asp2) into low-buoyant density, noncaveolar lipid rafts. Curr. Biol. 11,1288-1293[CrossRef][Medline]
  6. Ehehalt, R., Keller, P., Haass, C., Thiele, C., Simons, K. (2003) Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J. Cell Biol. 160,113-123[Abstract/Free Full Text]
  7. Kawarabayashi, T., Shoji, M., Younkin, L. H., Wen-Lang, L., Dickson, D. W., Murakami, T., Matsubara, E., Abe, K., Ashe, K. H., Younkin, S. G. (2004) Dimeric amyloid beta protein rapidly accumulates in lipid rafts followed by apolipoprotein E and phosphorylated tau accumulation in the Tg2576 mouse model of Alzheimer's disease. J. Neurosci. 24,3801-3809[Abstract/Free Full Text]
  8. Wahrle, S., Das, P., Nyborg, A. C., McLendon, C., Shoji, M., Kawarabayashi, T., Younkin, L. H., Younkin, S. G., Golde, T. E. (2002) Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains. Neurobiol. Dis. 9,11-23[CrossRef][Medline]
  9. Vetrivel, K. S., Cheng, H., Lin, W., Sakurai, T., Li, T., Nukina, N., Wong, P. C., Xu, H., Thinakaran, G. (2004) Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes. J. Biol. Chem. 279,44945-44954[Abstract/Free Full Text]
  10. Golub, T., Wacha, S., Caroni, P. (2004) Spatial and temporal control of signaling through lipid rafts. Curr. Opin. Neurobiol. 14,542-550[CrossRef][Medline]
  11. Lucero, H. A., Robbins, P. W. (2004) Lipid rafts-protein association and the regulation of protein activity. Arch. Biochem. Biophys. 426,208-224[CrossRef][Medline]
  12. Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C. G., Simons, K. (1998) Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proc. Natl. Acad. Sci. U. S. A. 95,6460-6464[Abstract/Free Full Text]
  13. Cordy, J. M., Hussain, I., Dingwall, C., Hooper, N. M., Turner, A. J. (2003) Exclusively targeting beta-secretase to lipid rafts by GPI-anchor addition up-regulates beta-site processing of the amyloid precursor protein. Proc. Natl. Acad. Sci. U. S. A. 100,11735-11740[Abstract/Free Full Text]
  14. Herz, J., Strickland, D. K. (2001) LRP: a multifunctional scavenger and signaling receptor. J. Clin. Invest. 108,779-784[CrossRef][Medline]
  15. Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., Stanley, K. K. (1988) Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J. 7,4119-4127[Medline]
  16. Trommsdorff, M., Borg, J. P., Margolis, B., Herz, J. (1998) Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein. J. Biol. Chem. 273,33556-33560[Abstract/Free Full Text]
  17. Gotthardt, M., Trommsdorff, M., Nevitt, M. F., Shelton, J., Richardson, J. A., Stockinger, W., Nimpf, J., Herz, J. (2000) Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction. J. Biol. Chem. 275,25616-25624[Abstract/Free Full Text]
  18. Barnes, H., Ackermann, E. J., van der, G. P. (2003) v-Src induces Shc binding to tyrosine 63 in the cytoplasmic domain of the LDL receptor-related protein 1. Oncogene 22,3589-3597[CrossRef][Medline]
  19. Kinoshita, A., Whelan, C. M., Smith, C. J., Mikhailenko, I., Rebeck, G. W., Strickland, D. K., Hyman, B. T. (2001) Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: role of the intracellular adapter protein Fe65. J. Neurosci. 21,8354-8361[Abstract/Free Full Text]
  20. Lleo, A., Waldron, E., von Arnim, C. A., Herl, L., Tangredi, M. M., Peltan, I. D., Strickland, D. K., Koo, E. H., Hyman, B. T., Pietrzik, C. U., Berezovska, O. (2005) Low density lipoprotein receptor-related protein (LRP) interacts with presenilin 1 and is a competitive substrate of the amyloid precursor protein (APP) for gamma-secretase. J. Biol. Chem. 280,27303-27309[Abstract/Free Full Text]
  21. von Arnim, C. A., Kinoshita, A., Peltan, I. D., Tangredi, M. M., Herl, L., Lee, B. M., Spoelgen, R., Hshieh, T. T., Ranganathan, S., Battey, F. D., et al (2005) The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate. J. Biol. Chem. 280,17777-17785[Abstract/Free Full Text]
  22. May, P., Reddy, Y. K., Herz, J. (2002) Proteolytic processing of low density lipoprotein receptor-related protein mediates regulated release of its intracellular domain. J. Biol. Chem. 277,18736-18743[Abstract/Free Full Text]
  23. Kang, D. E., Saitoh, T., Chen, X., Xia, Y., Masliah, E., Hansen, L. A., Thomas, R. G., Thal, L. J., Katzman, R. (1997) Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer's disease. Neurology 49,56-61[Abstract/Free Full Text]
  24. Hollenbach, E., Ackermann, S., Hyman, B. T., Rebeck, G. W. (1998) Confirmation of an association between a polymorphism in exon 3 of the low-density lipoprotein receptor-related protein gene and Alzheimer's disease. Neurology 50,1905-1907[Abstract/Free Full Text]
  25. Lambert, J. C., Wavrant-De Vrieze, F., Amouyel, P., Chartier-Harlin, M. C. (1998) Association at LRP gene locus with sporadic late-onset Alzheimer's disease. Lancet 351,1787-1788[CrossRef][Medline]
  26. Kang, D. E., Pietrzik, C. U., Baum, L., Chevallier, N., Merriam, D. E., Kounnas, M. Z., Wagner, S. L., Troncoso, J. C., Kawas, C. H., Katzman, R., Koo, E. H. (2000) Modulation of amyloid beta-protein clearance and Alzheimer's disease susceptibility by the LDL receptor-related protein pathway. J. Clin. Invest. 106,1159-1166[Medline]
  27. Schmechel, D. E., Saunders, A. M., Strittmatter, W. J., Crain, B. J., Hulette, C. M., Joo, S. H., Pericak-Vance, M. A., Goldgaber, D., Roses, A. D. (1993) Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 90,9649-9653[Abstract/Free Full Text]
  28. Rebeck, G. W., Harr, S. D., Strickland, D. K., Hyman, B. T. (1995) Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein. Ann. Neurol. 37,211-217[CrossRef][Medline]
  29. Blacker, D., Wilcox, M. A., Laird, N. M., Rodes, L., Horvath, S. M., Go, R. C., Perry, R., Watson, B., Jr, Bassett, S. S., McInnis, M. G., et al (1998) Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nat. Genet. 19,357-360[CrossRef][Medline]
  30. Narita, M., Holtzman, D. M., Schwartz, A. L., Bu, G. (1997) Alpha2-macroglobulin complexes with and mediates the endocytosis of beta-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. J. Neurochem. 69,1904-1911[Medline]
  31. Yang, D. S., Small, D. H., Seydel, U., Smith, J. D., Hallmayer, J., Gandy, S. E., Martins, R. N. (1999) Apolipoprotein E promotes the binding and uptake of beta-amyloid into Chinese hamster ovary cells in an isoform-specific manner. Neuroscience 90,1217-1226[CrossRef][Medline]
  32. Shibata, M., Yamada, S., Kumar, S. R., Calero, M., Bading, J., Frangione, B., Holtzman, D. M., Miller, C. A., Strickland, D. K., Ghiso, J., Zlokovic, B. V. (2000) Clearance of Alzheimer's amyloid-ss(1–40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J. Clin. Invest. 106,1489-1499[Medline]
  33. Kounnas, M. Z., Moir, R. D., Rebeck, G. W., Bush, A. I., Argraves, W. S., Tanzi, R. E., Hyman, B. T., Strickland, D. K. (1995) LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation. Cell 82,331-340[CrossRef][Medline]
  34. Pietrzik, C. U., Busse, T., Merriam, D. E., Weggen, S., Koo, E. H. (2002) The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing. EMBO J. 21,5691-5700[CrossRef][Medline]
  35. Ulery, P. G., Beers, J., Mikhailenko, I., Tanzi, R. E., Rebeck, G. W., Hyman, B. T., Strickland, D. K. (2000) Modulation of beta-amyloid precursor protein processing by the low density lipoprotein receptor-related protein (LRP). Evidence that LRP contributes to the pathogenesis of Alzheimer's disease. J. Biol. Chem. 275,7410-7415[Abstract/Free Full Text]
  36. Yoon, I. S., Pietrzik, C. U., Kang, D. E., Koo, E. H. (2005) Sequences from the low density lipoprotein receptor-related protein (LRP) cytoplasmic domain enhance amyloid beta protein production via the beta-secretase pathway without altering amyloid precursor protein/LRP nuclear signaling. J. Biol. Chem. 280,20140-20147[Abstract/Free Full Text]
  37. Perez, R. G., Squazzo, S. L., Koo, E. H. (1996) Enhanced release of amyloid beta-protein from codon 670/671 "Swedish" mutant beta-amyloid precursor protein occurs in both secretory and endocytic pathways. J. Biol. Chem. 271,9100-9107[Abstract/Free Full Text]
  38. Koo, E. H., Squazzo, S. L., Selkoe, D. J., Koo, C. H. (1996) Trafficking of cell-surface amyloid beta-protein precursor. I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody. J. Cell Sci. 109,991-998[Abstract]
  39. Pietrzik, C. U., Yoon, I. S., Jaeger, S., Busse, T., Weggen, S., Koo, E. H. (2004) FE65 constitutes the functional link between the low-density lipoprotein receptor-related protein and the amyloid precursor protein. J. Neurosci. 24,4259-4265[Abstract/Free Full Text]
  40. Yan, R., Munzner, J. B., Shuck, M. E., Bienkowski, M. J. (2001) BACE2 functions as an alternative alpha-secretase in cells. J. Biol. Chem. 276,34019-34027[Abstract/Free Full Text]
  41. He, W., Lu, Y., Qahwash, I., Hu, X. Y., Chang, A., Yan, R. (2004) Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nat. Med. 10,959-965[CrossRef][Medline]
  42. Lee, S. J., Liyanage, U., Bickel, P. E., Xia, W., Lansbury, P. T., Jr, Kosik, K. S. (1998) A detergent-insoluble membrane compartment contains A beta in vivo. Nat. Med. 4,730-734[CrossRef][Medline]
  43. Ikezu, T., Trapp, B. D., Song, K. S., Schlegel, A., Lisanti, M. P., Okamoto, T. (1998) Caveolae, plasma membrane microdomains for {alpha}-secretase-mediated processing of the amyloid precursor protein. J. Biol. Chem. 273,10485-10495[Abstract/Free Full Text]
  44. Vetrivel, K. S., Cheng, H., Kim, S.-H., Chen, Y., Barnes, N. Y., Parent, A. T., Sisodia, S. S., Thinakaran, G. (2005) Spatial segregation of {gamma}-secretase and substrates in distinct membrane domains. J. Biol. Chem. 280,25892-25900[Abstract/Free Full Text]
  45. Haass, C., Lemere, C. A., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., Selkoe, D. J. (1995) The Swedish mutation causes early-onset Alzheimer's disease by beta-secretase cleavage within the secretory pathway. Nat. Med. 1,1291-1296[CrossRef][Medline]
  46. Thinakaran, G., Teplow, D. B., Siman, R., Greenberg, B., Sisodia, S. S. (1996) Metabolism of the "Swedish" amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the "beta-secretase" site occurs in the golgi apparatus. J. Biol. Chem. 271,9390-9397[Abstract/Free Full Text]
  47. Steinhilb, M. L., Turner, R. S., Gaut, J. R. (2002) ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways. J. Neurochem. 80,1019-1028[CrossRef][Medline]
  48. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al (1999) Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286,735-741[Abstract/Free Full Text]
  49. Ma, L., Huang, Y. Z., Pitcher, G. M., Valtschanoff, J. G., Ma, Y. H., Feng, L. Y., Lu, B., Xiong, W. C., Salter, M. W., Weinberg, R. J., Mei, L. (2003) Ligand-dependent recruitment of the ErbB4 signaling complex into neuronal lipid rafts. J. Neurosci. 23,3164-3175[Abstract/Free Full Text]
  50. Wong, W., Schlichter, L. C. (2004) Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD-95. J. Biol. Chem. 279,444-452[Abstract/Free Full Text]
  51. Knauer, M. F., Orlando, R. A., Glabe, C. G. (1996) Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP). Brain Res. 740,6-14[CrossRef][Medline]
  52. Cam, J. A., Zerbinatti, C. V., Li, Y., Bu, G. (2005) Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein. J. Biol. Chem. 280,15464-15470[Abstract/Free Full Text]
  53. Simons, K., Ikonen, E. (1997) Functional rafts in cell membranes. Nature 387,569-572[CrossRef][Medline]
  54. Chen, W. J., Goldstein, J. L., Brown, M. S. (1990) NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J. Biol. Chem. 265,3116-3123[Abstract/Free Full Text]
  55. Nordstedt, C., Caporaso, G. L., Thyberg, J., Gandy, S. E., Greengard, P. (1993) Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells. J. Biol. Chem. 268,608-612[Abstract/Free Full Text]
  56. Vassar, R., Citron, M. (2000) Abeta-generating enzymes: recent advances in beta- and gamma-secretase research. Neuron 27,419-422[CrossRef][Medline]
  57. Li, Y., Marzolo, M. P., Van Kerkhof, P., Strous, G. J., Bu, G. (2000) The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein. J. Biol. Chem. 275,17187-17194[Abstract/Free Full Text]



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A. Schneider, L. Rajendran, M. Honsho, M. Gralle, G. Donnert, F. Wouters, S. W. Hell, and M. Simons
Flotillin-Dependent Clustering of the Amyloid Precursor Protein Regulates Its Endocytosis and Amyloidogenic Processing in Neurons
J. Neurosci., March 12, 2008; 28(11): 2874 - 2882.
[Abstract] [Full Text] [PDF]

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