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The Journal of Neuroscience, January 11, 2006, 26(2):418-428; doi:10.1523/JNEUROSCI.3882-05.2006
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Neurobiology of Disease
Interaction of the Cytosolic Domains of sorLA/LR11 with the Amyloid Precursor Protein (APP) and-Secretase
Robert Spoelgen,1 Christine A. F. von Arnim,1 Anne V. Thomas,1 Ithan D. Peltan,1 Mirjam Koker,1 Amy Deng,1 Michael C. Irizarry,1 Olav M. Andersen,2 Thomas E. Willnow,2 andBradley T. Hyman1 1Alzheimer's Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, and 2Max Delbrück Center for Molecular Medicine, D-13125 Berlin, Germany
Abstract
Top
Abstract
Introduction
sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-peptide (A
Key words: sorLA; Alzheimer's disease; amyloid-
Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder with neurofibrillary tangles and amyloid plaques as its pathological hallmarks (Selkoe, 2001). Amyloid plaques are mainly composed of aggregated amyloid-
sorLA is a member of a novel family of vacuolar protein sorting 10 protein (Vps10p) receptors, which contain a domain with high homology to the yeast-sorting protein Vps10p (Yamazaki et al., 1996
-ear-containing ARF-binding (GGA) proteins (Jacobsen et al., 2002
, sAPP
sorLA could act as an endocytic receptor for APP, enhancing APP turnover or directing APP to non-A
Materials and Methods
Generation of expression constructs. The generation of sorLA (human cDNA in pcDNA3.1), C-terminal myc-tagged APP695 (APP-myc), C99 (C99-myc), secreted alkaline phosphatase (SEAP) fused to the N terminus of APP695 (SEAP–APP) and C-terminal V5-tagged BACE (BACE–V5) constructs was performed as described previously (Kinoshita et al., 2001The PCR product was inserted into the XhoI/XbaI restriction sites of modified pSecTag.
To generate the C-terminal green fluorescent protein (GFP)-tagged or flag-tagged construct for sorLA, the sorLA pcDNA3.1 construct was modified using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) to insert an AgeI restriction site. GFP was amplified by PCR and inserted into the new C-terminal AgeI site using the primer pair: 5'-CTAACCGGTATGGTGAGCAAGGGCGAGG-3' and 5'-CTAACCGGTTTACTTGTACAGCTCGTCCATGC-3'.
C-terminal flag-tagged sorLA was generated with a linker sequence using the primer pair: 5'-CCGGTGATTACAAGGATGACGACGATAAGTGAA-3' and 5'-CCGGTTCACTTATCGTCGTCATCCTTGTAATCA-3'.
The GYENPTY site mutant of C99 was generated by substitution of the tyrosine residues 86 and 91 (of C99) to alanine of C99-myc using the QuickChange site-directed mutagenesis kit (Stratagene). Authenticity of all PCR products and the PCR-generated constructs was confirmed by DNA sequencing.
Cell culture conditions and transient transfection. For this study, we used Chinese hamster ovary (CHO) 7W (Wolfe et al., 1999
Primary neuronal cultures were generated from CD1 mice at embryonic day 15–16, as described previously (Berezovska et al., 2003
Immunocytochemistry. Immunostaining was performed 24 h after transient transfection. Cells were fixed in 4% paraformaldehyde for 10 min, washed in Tris-buffered saline (TBS), pH 7.3, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 1.5% normal goat serum for 1 h. To detect the localization of APP695-myc or C99-myc and sorLA, transfected cells were immunostained using monoclonal antibody 8E5 (raised against 444–592 residues of APP770; a gift from Elan Pharmaceuticals, San Francisco, CA) and rabbit N-terminal anti-SOL-sorLA antibody (1:2000) or N-terminal rab LA sorLA antibody (provided by Anders Nykjaer, University of Aarhus, Aarhus, Denmark) or monoclonal 13G8 antibody (raised against 676–695 residues of APP770; 1:500; gift from Elan Pharmaceuticals), monoclonal 9E10 anti-c-myc antibody (Abcam, Cambridge, UK) and rab C-terminal anti-sorLA (raised against 20 C-terminal residues of sorLA; provided by Claus Munck Petersen, University of Aarhus, Aarhus, Denmark). To detect the subcellular localization of BACE, cells transfected with BACE–V5 were immunostained with anti-V5 monoclonal antibody (Sigma, St. Louis, MO). Endogenous BACE was stained or immunporecipitated by rabbit anti-BACE N-terminal antibody (Calbiochem, La Jolla, CA). After three washes in TBS, Alexa 488- and cyanine 3 (Cy3)-conjugated secondary antibodies [from Invitrogen and Jackson ImmunoResearch (West Grove, PA)] were applied for 1 h at room temperature. Immunostained cells were cover-slipped using GVA (glycerol vinyl alcohol) mounting solution (Zymed, South San Francisco, CA) before fluorescence lifetime imaging microscopy (FLIM) measurements or confocal imaging with appropriate filters using a Bio-Rad (Hercules, CA) 1024 confocal microscope.
Fluorescence resonance energy transfer measurements using FLIM. We applied a validated FLIM technique to quantitate protein–protein interactions using multiphoton microscopy (Bacskai et al., 2003
Western blotting. N2a or HEK cells were transfected with APP695-myc, either empty vector or BACE-V5 and sorLA. For cell lysis, cells were treated with 1% Triton X-100 using Complete Protease Inhibitor (Roche Applied Science) and then loaded onto 4–20% Tris-glycine polyacrylamide gels for separation under reducing and denaturating conditions. After gel separation, proteins were transferred to polyvinylidene difluoride (PVDF) membranes and labeled using IR700 and IR800 secondary antibodies (Rockland, Gilbertsville, PA). Proteins were visualized on a LI-COR (Lincoln, NE) Odyssey infrared detection system according to the instructions of the manufacturer.
Immunoprecipitation. Immunoprecipitation of either myc-tagged C99 or GFP-tagged sorLA were performed with µMACS epitope isolation kit (Miltenyi Biotec, Auburn, CA) as recommended by the manufacturers. The magnetic beads were precoupled to either anti-myc or anti-GFP antibodies and were incubated with lysates for 30 min on ice. The complex consisting of beads and proteins was captured on a flow-through magnetic column and washed five times with cold lysis buffer. The proteins then were eluted with hot elution buffer. For negative controls, lysates from cells without the myc- or GFP-tagged protein were used. Lysates and immunoprecipitates were loaded onto 4–20% Tris–glycine polyacrylamide gels (Novex, San Diego, CA) under denaturating and reducing conditions. The proteins were transferred to PVDF membranes (Millipore, Bedford, MA) and blocked in 5% nonfat dried milk. sorLA was detected by rabbit anti-SOL-sorLA antibody, and C99 was detected by mouse 6E10 antibody (Signet, Dedham, MA). Secondary antibodies conjugated to infrared dyes were applied and detected using the LI-COR system.
Immunoprecipitation of BACE–V5 and sorLA were performed with BioMag goat anti-mouse or goat anti-rabbit IgG magnetic beads (PerSeptive Biosystems, Framingham, MA). N2a cells were cotransfected with BACE–V5 and sorLA and lysed in 1% Triton X-100 buffer. Cell lysates were incubated with anti-V5 or anti-SOL–sorLA antibody-loaded beads for 3 h at 4°C. For negative controls, beads were incubated with lysate or antibody alone. After the incubation, beads were washed and boiled in 2x Tris–glycine SDS sample buffer (Invitrogen). Supernatants and immunoprecipitates were loaded onto 4–20% Tris–glycine polyacrylamide gels (Novex) under denaturing conditions. Western blots were performed as described above using anti-V5 and rabbit SOL–sorLA antibody for detection.
Immunoprecipitation of endogenous sorLA by BACE was done with Sepharose beads. Beads and mouse brain lysates were incubated with anti-BACE Ab or PBS at 4°C. After the supernatants were collected, beads were washed with 1% CHAPSO lysis buffer, boiled with 2x Tris–glycine SDS sample buffer and loaded onto 4–20% Tris–glycine polyacrylamide gels (Novex) under denaturing and reducing conditions. The proteins were transferred to PVDF membranes (Millipore), and sorLA was detected by rabbit SOL–sorLA antibody or C-terminal anti-sorLA antibody. Secondary antibodies conjugated to horseradish peroxidase were applied and visualized by chemiluminescence.
Internalizaion assay. CHO 7W (APP751 stable overexpressing cells, provided by D. Selkoe, Brigham and Women's Hospital, Boston, MA) were cultured on six-well plates and transfected with sorLA or empty vector pcDNA3.1. On the next day, cells were washed with cold PBS containing 1 mM CaCl2 and 1 mM MgCl2, 0.2% BSA, and 5 mM glucose and incubated for 30 min on ice with biotinylated 6E10 (Signet). Cells were then incubated at 37°C for different times to allow for endocytosis of 6E10. After endocytosis, surface biotin was masked using streptavidin (Roche Applied Science) for 1 h on ice and quenched with 0.5 mg/ml biocytin (Sigma). Cells were harvested in blocking buffer containing 1% Triton X-100, 0.1% SDS, 0.2% BSA, 50 mM NaCl, 1 mM Tris, pH 7.4, and incubated on IgG-coated 96-well plates at 4°C overnight. Plates were washed with PBS and streptavidin–peroxidase (Roche Applied Science) was applied for 1 h in blocking buffer. Finally, plates were washed and then incubated with 10 mg of o-phenyldiamine HCl (Sigma), 10 µl of 30% H2O2 in 25 ml of 50 mM Na2HPO4, 27 mM citrate, pH 5.0. The readout was done at 490 nm to determine internalized APP against surface APP.
APP ectodomain shedding assay. HEK293 cells cultured in 12-well plates were transfected in triplicate with a
A
Results
Endocytosis of APP by sorLA
In our previous study, we analyzed several aspects of the role of the putative sorting receptor sorLA in APP metabolism. We showed that overexpression of sorLA coincides with sequestration of APP in the Golgi and reduced cell surface exposure of APP (Andersen et al., 200580% in both conditions. These data indicate that sorLA activity does not influence APP uptake from the cell surface but rather affects the kinetics of APP trafficking through the Golgi.
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Figure 1. APP internalization assay shows that APP internalization is not dependent on sorLA. 7W CHO cells were transfected with either empty vector or sorLA. The uptake of cell-surface-biotinylated APP was assessed by determining internalized versus cell surface APP. The internalization of APP is indicated in percentage of cell surface APP over 40 min and revealed no sorLA-dependent uptake of APP. The results are representative of data of three independent assays. The means and SD of one assay performed in triplicate are shown.
APP shedding assay
Recently, we demonstrated that the sorLA overexpression causes a significant reduction of sAPP
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Figure 2. A, APP ectodomain shedding assay shows that APP cleavage is altered by sorLA. HEK293 cells were transfected with SEAP–APP,
sorLA reduces secreted A
The finding that sorLA and sorLA tail both reduce APP cleavage in the presence of BACE suggests that sorLA impactsColocalization of sorLA and C99
Based on the finding that sorLA tail alone was sufficient to reduce APP cleavage and AInteraction of sorLA and C99 in pull-down experiments
To analyze whether sorLA and C99 bind to each other, extracts from C99-myc and sorLA-transfected N2a cells were pulled down with myc-binding beads and probed with rabbit anti-sorLA and mouse 9E10 anti-myc antibody. C99 constructs were used to investigate a specific C-terminal tail interaction, because immunoprecipitation of full-length sorLA with APP might reflect the previously described N-terminal domain interaction. Immunoreactive bands of250 kDa for sorLA and 20 kDa for C99 were detectable in the immunoprecipitated sample, demonstrating that sorLA was pulled down by C99-myc (Fig. 4, lane 1). To check for nonspecific interactions, individually transfected lysates were probed, resulting in the absence of sorLA pull-down by C99-myc only (Fig. 4, lane 2) or in cells only expressing sorLA (Fig. 4, lane 3). Coimmunoprecipitation of sorLA by C99 was confirmed when using lysates from sorLA–GFP- and C99-myc-transfected cells and using GFP-binding beads. Cell extracts from cells expressing either C99-myc or sorLA constructs show no specific pull-down bands (data not shown).
Previous studies have shown that the GYENPTY motif in the cytoplasmatic tail of APP is responsible for the binding of adaptor proteins and seems to mediate endocytosis of APP via the adaptor protein Fe65 and low-density lipoprotein receptor (LDLR)-related protein (LRP) (Lai et al., 1995
GYENPTY-myc mutant construct was also sufficient to pull down sorLA (Fig. 4, lane 4), indicating a GYENPTY-motif-independent interaction of both proteins. For both C99 wild-type and mutant form, the C99 pull-down produced strong immunoreactive bands at 250 kDa, the expected size of sorLA.
Interaction of sorLA and C99 by FLIM analysis in N2a cells
To further study the interaction of C99 and sorLA in intact cells and to determine in which subcellular compartments both proteins interact, we performed FLIM in mouse neuronal N2a cells. FLIM is a new fluorescence resonance energy transfer (FRET)-based technique for detecting intermolecular or intramolecular interactions that take place within a distance of 10 nm or less. The fluorescence lifetime is influenced by the surrounding environment and shortened in the immediate vicinity of a FRET acceptor fluorophore in proportion to the distance between the fluorophores (Berezovska et al., 2003
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Figure 3. Localization of sorLA and C99 in transfected cells. A, B, sorLA and C99 were cotransfected into N2a cells (A) or HEK293 cells (B) and immunostained with anti-tail sorLA antibody visualized by Alexa 488 (green) and mouse 13G8 antibody labeled by Cy3 (red). The panels on the right show colocalization of sorLA and C99 in yellow.
First, we analyzed the proximity of the intracellular domains of APP and sorLA by FLIM. N2A cells were transfected with APP695-myc and sorLA constructs and were stained for the intracellular domain of sorLA by the rabbit C-terminal sorLA antibody labeled by Alexa 488 and for APP by mouse 13G8 labeled by Cy3. The double immunostaining showed colocalization of APP and sorLA in perinuclear and distal compartments. For the FLIM analysis, the change in the lifetime of the donor fluorophore Alexa 488 under different experimental conditions was determined. As a negative control, the lifetime of Alexa 488 attached to the C terminus of sorLA was measured in the absence of acceptor (Cy3), which showed lifetimes of 1985 ± 20 ps, equivalent to Alexa 488–IgG alone (Fig. 5A). The lifetime was significantly shortened when sorLA labeled by Alexa 488 was costained with Cy3-labeled APP695 to 1844 ± 123 ps, indicating FRET attributable to close proximity between sorLA and APP695 (Fig. 5B). The degree of lifetime shortening is displayed in a pseudo-color-coded image, indicating shortening of lifetime in yellow and red, particularly in the perinuclear region and some endosomal-like structures. Equivalent results were obtained when the acceptor and donor fluorophores were exchanged. In cells transfected with only APP, in the absence of acceptor fluorophore (Cy3), lifetimes of 1962 ± 33 ps were detected. The lifetime was shortened to 1881 ± 62 ps when cells were coexpressed with sorLA that was labeled with Cy3 (n = 12; p < 0.001).Although the C-terminal domains of APP695 and sorLA are in close proximity, their interaction reflected by a shortening in lifetime could be attributable to the interaction of the N-terminal domains, moving the C-terminal domains closer together. To test whether there is an N-terminal-independent interaction of the C-terminal domains of APP and sorLA, FLIM analysis of the proximity between sorLA and C99-myc, which lacks most of the APP extracellular site, was performed. C99-myc and sorLA-transfected N2a cells were stained by rabbit C-terminal sorLA antibody labeled by Alexa 488 and for C99-myc by mouse anti-myc 9E10 antibody, which was labeled by Cy3. The lifetime of Alexa 488 attached to the C terminus of sorLA (1985 ± 20 ps) was dramatically shortened when cells coexpressed C99-myc, which was labeled with Cy3, to 1712 ± 131 ps (Fig. 5C, Table 1). To verify the observation of the pull-down experiments, indicating that sorLA binds C99 independent from the GYENPTY motif, we analyzed the proximity of the C99
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Table 1. FLIM assay for sorLA–APP and sorLA–C99 interaction in transfected N2a cells
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Figure 4. Coimmunoprecipitation of C99 and sorLA. C99-myc was pulled down by myc-beads and probed for sorLA by LA–sorLA antibody (lane 1). N2a cells transfected with C99-myc (lane 2) or transfected with sorLA only (lane 3), showed no nonspecific pull-down. Specific coimmunoprecipitation of sorLA was also observed when immunoprecipitating transfected C99
Together, our FLIM results confirm the existence of a GYENPTY-independent C-terminal binding site on sorLA that allows for the interaction with the APP C terminus in addition to the interaction domains located in the extracellular region of the proteins (Andersen et al., 2005The sensitivity of the lifetime measurements to proximity is illustrated by an additional negative control. Fluorophores located in proteins on opposite sides of a membrane are usually considered to be too far apart to show FRET. The lifetime of sorLA labeled at the extracellular N terminus (rabbit anti-sorLA) in the presence of APP labeled at the intracellular C terminus with mouse anti-APP antibody 13G8 labeled by Cy3 was determined (Fig. 5E). The fact that in this control, Alexa 488 lifetime (1938 ± 45; n = 9) is close to the negative control, demonstrates that although sorLA and APP colocalize at the light level, the labeled epitopes are too far apart to support FRET. This proves that a change in the fluorescence lifetime, indicating FRET, is a more sensitive measure of proximity than simple colocalization.
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Figure 5. FLIM analysis of sorLA APP and sorLA C99 interaction within N2a cells. A, For the negative control, cells were transfected with sorLA and stained by C-terminal sorLA antibody labeled by Alexa 488. The left panel shows confocal images of cells stained for sorLA by donor fluorophore Alexa 488. The color-coded FLIM image highlights the lifetime of the donor fluorophore in picoseconds. B, FLIM analysis of sorLA C terminus labeled by Alexa 488 and APP C terminus labeled by Cy3-transfected cells demonstrate shortening of donor lifetime, particularly in the perinuclear region. C, D, N2a cells cotransfected with sorLA, C99-myc (C), or C99
Interaction of sorLA and APP by FLIM analysis in primary neurons
To examine the functional relevance of the interaction between sorLA and APP, we furthermore investigated its presence in primary neurons on the level of endogenous protein by our N- and C-terminal FLIM assay, respectively. Primary neurons were stained for APP by N-terminal 8E5 antibody labeled with Alexa 488 and for sorLA by rabbit SOL–N-terminal antibody labeled with Cy3. In the absence of an acceptor fluorophore, the donor lifetime was 2167 ± 21 (Fig. 6A, Table 2). The lifetime of Alexa 488 attached to the N terminus of APP was significantly shortened when cells were also stained for sorLA labeled with Cy3, to 1919 ± 107 ps (Fig. 6B, Table 2), indicative of close proximity between the two fluorophores. This was confirmed when the acceptor and donor fluorophores were exchanged, because cells stained for sorLA only showed a lifetime of 2059 ± 18 ps, whereas the lifetime was reduced to 1788 ± 99 ps when APP was labeled by Cy3 (data not shown; n = 10; p < 0.001). Because we previously identified the interaction of the C-terminal domains of sorLA and APP in transfected cells, we labeled the sorLA C terminus with Alexa 488 and the APP cytosolic tail with Cy3 on the endogenous proteins. Again, we observed a significant shortening of the donor lifetime to 1985 ± 47 ps (Fig. 6C, Table 2), indicating that the C-terminal interaction between sorLA and APP takes place on an endogenous level as well, consistent with our observation that endogenous sorLA and APP can be coimmunoprecipitated from neurons (Andersen et al., 2005
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Table 2. APP sorLA FLIM analysis on endogenous proteins in primary neurons
Coimmunoprecipitation of sorLA and BACE
When we analyzed APP cleavage in a shedding assay and measured secreted ABACE–APP interaction is reduced by sorLA
Our results demonstrated the interaction of sorLA and BACE by coimmunoprecipitation. In addition, we found that APP cleavage and A
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Table 3. FLIM assay for BACE–APP interaction in N2a cells
Discussion
The potential importance of SorLA in the pathophysiology of Alzheimer's disease is suggested by the observations that sorLA expression is dramatically decreased in the brain of Alzheimer patients and that sorLA null mice have accelerated amyloid production (Scherzer et al., 2004Recent studies have shown that the Vps10p receptor sorLA is directly involved in the intracellular transport and processing of APP (Andersen et al., 2005
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Figure 6. FLIM analysis of APP and sorLA on endogenous proteins. A–C, Primary neurons were immunostained for endogenous APP by N-terminal 8E5 antibody (A, B) or by C-terminal 13G8 antibody (C) labeled by Alexa 488 (A, B) or by Cy3 (C). The lifetime was measured in the presence (B, C) or absence (A) of acceptor fluorophore Cy3 attached to the N terminus of endogenous sorLA (B) or C-terminal of endogenous APP (C). Pseudocolored FLIM images highlight FRET between APP and sorLA in juxtanuclear and distal compartments (B, C).
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Figure 7. Coimmunoprecipitation of BACE and sorLA. sorLA was immunoprecipitated and probed for BACE by BACE antibody from transfected N2a cell extracts. A, Immunoreactive bands of sorLA and BACE in lysates and pull-downs demonstrate BACE–sorLA interaction. B, Interaction of sorLA and BACE could be proven when BACE was immunoprecipitated and sorLA was shown to coimmunoprecipitate. Double bands of BACE show mature and immature form of BACE. C, Coimmunoprecipitation of endogenous BACE and sorLA from mouse brain extract. BACE was immunoprecipitated with rabbit N-terminal BACE antibody and probed for sorLA. D, In the absence of the pull-down antibody, no immunoreactive band was detected for sorLA. IP, Immunopreciptation; CoIP, coimmunopreciptation.
To understand the role of sorLA in APP processing in detail, we investigated the influence of sorLA on APP cleavage and AWe recently demonstrated direct binding of the N-terminal domain of sorLA and APP by surface plasmon resonance analysis, analytical ultracentrifugation, and cell culture experiments (Andersen et al., 2005
A striking result of our current study is the finding that APP cleavage and A
We hypothesized that sorLA impacts a subset of APP–BACE interactions and analyzed whether the sorLA effects could be localized to a specific subcellular compartment. Several lines of data are consistent with a predominant effect of sorLA on APP and BACE in the Golgi. APP and BACE colocalize in the Golgi apparatus and ER (Huse et al., 2000
sorLA is strongly expressed in the Golgi, in which it appears to interact with APP, as suggested by both FLIM studies and the observation that sorLA and APP are mainly colocalized in the same subcellular fraction containing markers of the cis-Golgi (Andersen et al., 2005
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Figure 8. Interaction of APP and BACE dependent on sorLA. For FLIM analysis of APP and BACE proximity, N2a cells were transfected with BACE, BACE–APP, and BACE–APP–sorLA. BACE was labeled on the N-terminal domain by a BACE antibody labeled by Alexa 488 and APP by N-terminal 8E5 antibody labeled by Cy3. A, In the absence of acceptor fluorophore Cy3, lifetime averages at 2051 ± 17 ps. B, C, BACE–APP-transfected cells show BACE–APP interaction mainly in the perinuclear region (B), whereas the FRET interaction in cells that also expressed sorLA was strongly reduced (C). Intensity images of BACE–APP and BACE–APP–sorLA-transfected cells revealed that the immunostaining pattern for BACE or APP is not significantly altered by sorLA.
In conclusion, we envision a model in which sorLA impacts APPMoreover, we also found evidence of an interaction of sorLA with BACE. sorLA and BACE mainly colocalized in the Golgi, suggesting that sorLA also binds BACE in the Golgi and thereby inhibits its access to APP. Although it is also possible that sorLA contributes to retrograde transport of BACE to the Golgi, the observation that sorLA coimmunoprecipitates principally with nonglycosylated immature forms of BACE points to an interaction in the Golgi.
The observations that sorLA is downregulated in AD brain and that, in experimental systems, sorLA reduction and overexpression impact A
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Figure 9. Schematic overview of how sorLA influences APP and BACE interaction. We hypothesize that sorLA regulates trafficking of APP through the Golgi, mainly through slowing release of the precursor to the cell surface and blocking interaction with BACE. Only those APP molecules that are released by sorLA are accessible for proteolytic cleavage either in the Golgi or distal cellular compartments.
Footnotes
Received Sep 13, 2005; revised November 9, 2005; accepted November 14, 2005.This work was supported by National Institutes of Health Grant AG 12406, Deutsche Forschungsgemeinschaft Research Fellowships AR379/1-1 (to C.A.F.v.A.) and TH 1129/1-1 (to A.V.T.), and the J. D. French Alzheimer's Foundation (to M.C.I.). We thank S. F. Lichtenthaler for the SEAP–APP construct.
Correspondence should be addressed to Dr. Bradley T. Hyman, Department of Neurology/Alzheimer Unit, 114 16th Street, Charlestown, MA 02129. E-mail: bhyman{at}partners.org.
DOI:10.1523/JNEUROSCI.3882-05.2006
Copyright © 2006 Society for Neuroscience 0270-6474/06/260418-11$15.00/0
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