Introduction
About 1% of humans aged 60-64 years have AD, increasing steadily to as many as 35%-40% after age 85 [
1]. AD progressively leads to a severely impaired state and complete social dependence. At autopsy, cerebral atrophy, neurofibrillary tangles and amyloid plaques are observed in the hippocampus, entorhinal cortex, amygdala and other areas. Tangles consist of intraneuronal masses of helically wound filaments of the hyperphosphorylated protein, tau. Plaques are extracellular deposits of Aβ, a peptide derived from cleavage of APP, surrounded by dystrophic neurites. Most AD cases present Aβ deposits in cortical and/or meningeal micro vessels. In a minority of cases, this vascular cerebral amyloid angiopathy (CAA) is rather severe [
2].
APP is a type I transmembrane protein that undergoes a series of proteolytic cleavages [
3]. β-secretase cleaves APP into a soluble ectodomain (sAPPβ) and a membrane bound C-terminal fragment of 99 amino acids (C99). C99 is cleaved by the γ-secretase, which consists of a multicomponent complex comprised of Presenilins (PS1 and PS2), Nicastrin (NCT), PEN2 and APH1 [
4]. The γ-cleavage releases two peptides: Aβ peptide, consisting of 2 major species of 40 and 42 amino acids (Aβ40 and Aβ42, respectively) and an intracellular product AID/AICD, Several evidence point to the short AID/AICD as a biologically active intracellular peptide, which may modulate cell death, Notch signaling, gene transcription and Ca
++ homeostasis [
5‐
19]. In an alternative pathway, APP is processed by α-secretase within the Aβ sequence, leading to the production of the soluble sAPPα ectodomain and a membrane bound C-terminal fragment of 83 amino acids (C83). After age, a family history of dementia is major risk factor for AD and 10%-15%, of all AD subjects have a family history consistent with an autosomal dominant trait. These familial cases (FAD) are due to mutations in APP and in Presenilins because they alter the rate of APP processing and Aβ42 generation.
Given the role of APP processing by secretases to AD pathology and APP-mediated functions, identifying the molecules that regulate APP cleavage is physiologically relevant and of therapeutic interest. A genetic screen aimed to the identification of regulators of APP processing led to the identification of BRI2 and BRI3 as APP ligands that inhibit Aβ formation [
20‐
22]. In the same screening, we found CD74 as an APP-interacting protein as well.
Human CD74 is a type II transmembrane protein of 216 amino acids which functions as a molecular chaperone of class II major histocompatibility complex (MIIC) [
23], and at the same time as a receptor for macrophage migration inhibitory factor (MIF), alone [
24] or together with CD44 [
25]. However, little is known about the relationship between CD74 and APP. Because both BRI2 and BRI3 interact with APP and inhibits APP processing [
20‐
22,
26], we investigated the role of CD74 in regulating the processing of APP. Here, we show that CD74 interacts with APP, inhibits Aβ production, and accumulates APP to endocytotic vacuoles produced by CD74.
Materials and methods
Split-ubiquitin screening
The construction of APP as bait, and screening method was described elsewhere [
20].
Cell culture, transfection, plasmids, antibodies and Western blotting
Cell lines, transfection methods, mammalian expression constructs of APP, APP-Ncas, APP Swedish, APP London, APP-YFP, myc-tagged Notch, APLP2 were described [
20,
27]. CD74 is cloned from the two-hybrid screening, and its mouse counterparts, Ii p31 and Ii p41 were generous gifts from Dr. Norbert Koch [
28]. All CD74 constructs, both human and mouse, were cloned into pcDNA3.1 vector (Invitrogen) with a N-terminal FLAG tag. The Dutch and Australian mutations of APP were introduced into APP by QuikChange XL (Stratagene).
The following antibodies and antibody beads were used: αFLAG (M2, Sigma F1804); αmyc (Cell Signaling, 2276); αAPP (22C11, Chemicon MAB348); αsAPPα (IBL 11088); αsAPPβ (IBL 18957); αAPP C-terminal fragments (CTF) (αAPPct, Invitrogen/Zymed 36-6900); αAPLP2 (EMD/Calbiochem 171616); α transferrin receptor; Flag M2 beads (Sigma A2220). Rabbit polyclonal antibody and horse radish peroxidase conjugated secondary antibodies are from Southern Biotechnology.
All Western blot samples were separated by 4-12% Criterion Gels (Bio-Rad 3450125) with NuPAGE MES running buffer (Invitrogen NP0002). The gels were transferred to nitrocellulose membrane (Whatman BA85 Protran) with NuPAGE transfer buffer (Invitrogen NP0006) in a Trans-Blot cell (Bio-Rad). After the transfer, the membrane was blocked in PBS containing 5% skim milk, and incubated with indicated primary antibodies over night in the same buffer. The membrane was washed in PBS containing 0.05% Tween-20, and incubated in corresponding secondary antibodies for 1 hour. The membrane was washed in PBS containing 0.05% Tween-20 again, and the bands were visualized with Super Signal West Pico Chemiluminescent Substrate (Pierce) and Hyblot X-ray films (Denville Scientific).
Immunoprecipitation from HeLa cells
HeLa cells were transfected with indicated combinations of plasmids. The transfected cells were lysed in the Hepes-Triton buffer (20 mM Hepes/NaOH pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Triton-X 100) on ice. The lysates were cleared by spinning at 20,000 g for 10 min. To precipitate the immune complex, the cleared lysates were mixed with FLAG beads, or with indicated antibodies and protein A beads (Pierce). A rabbit polyclonal antibody was used as a negative control of αAPPct precipitation. After being washed three times with the same buffer, the precipitated beads were boiled in 2 × SDS Buffer and analyzed by Westerm blot. The immunoprecipitants analyzed correspond to the four times of the inputs.
Staining of transfected HeLa cells
HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids. The cells on coverslips were fixed, permeabilized with 0.2% Triton X-100, stained as described [
29]. APP-YFP and Notch-GFP were detected with YFP/GFP fluorescence. Anti-FLAG and αAPPct stainings were visualized with Alexa Fluor 594 goat anti-mouse IgG (H+L) (Invitrogen) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen), respectively. Cells were imaged sequentially with the Albert Einstein Analytical Imaging Facility BioRad Radiance 2000 confocal microscope using a 63× NA1.4 oil objective and 488 nm and 568 nm laser lines. The images shown are representative of the phenotypes observed in at least three independent experiments.
Colocalization analysis
Regions of interest were analyzed with ImageJ software (National Institute of Health) and Colocalization Indices plug-in [
30]. Colocalization coefficient (CC) [
31] and intensity correlation quotient (ICQ) [
32] were calculated with the software by setting the threshold of CD74 staining so that the calculation is limited to CD74-positive vacuoles. CC ranges from -1 to 1: 1 indicates complete colocalization; 0, ramdom colocalization, -1, complete negative colocalization. Positive ICQ indicates positive colocalizatioin.
The measurement of APP fragments of HEK293APP cells
HEK293APP cells were transfected with pcDNA3 or with FLAG-CD74. One day after the transfection, the cells were further incubated in fresh media for 24 hours. The conditioned media were collected and cleared by spinning at 20,000 g for 10 min. Cells were lysed in the Hepes-Triton buffer, and protein concentration was determined by Bradford method using bovine serum albumin as a standard. Aβ in the media and the cell lysates were measured using the ELISA kits for Aβ40 and Aβ42 (IBL 17713 and 17711, respectively).
Equal amounts of total lysates and the cleared media were analyzed by Westerm blot.
The measurement of stability of synthetic Aβ added to the media
Synthetic Aβ40 and Aβ42 (American Peptide Company) were dissolved in 50 mM Tris/HCl pH 8.0 at 1 mg/ml and stored at -70°C until use. HeLa cells were transfected with pcDNA3 or FLAG-CD74. One day after transfection, the media was replaced with fresh media containing synthetic Aβ40 and Aβ42 at approximately 100 ng/ml. The media were collected at indicated time points and Aβ was measured as above.
HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. One day after the transfection, the cells were starved for 90 minutes by incubating in DMEM without cysteine and methionine (Invitrogen). The cells were labelled at 500 Ci/ml of 35S cysteine and methionine for 30 minutes and chased in the complete DMEM for indicated times. After the chase, the cells were lysed and immunoprecipitated with αAPPct as above. The precipitants were analyzed by SDS-PAGE and autoradiography.
Discussion
We isolated CD74, together with the familial dementia gene BRI2 and its family member BRI3, during a genetic screening for membrane-bound APP ligands [
20,
21]. CD74 is known to work as a chaperon of class II major histocompatibility complex in B cells, which present antigens to T cells [
34,
38]. Interestingly, the functions of CD74 are not limited to the chaperons in the immune cells. Recently, CD74 has been proposed to serve as a receptor for macrophage migration inhibitory factor [
24].
CD74 associates with Class II MHC in endoplasmic reticulum. After it is transported through post-Golgi compartments, the complex are diverted to endocytic vesicles, and CD74 is proteolytically processed to CLIP region (Figure
1A), which remains bound to Class II MHC [
23,
38]. APP does not associate CD74 in endoplasmic reticulum, since only fully glycosylated APP binds to CD74 (Figure
1B), and CD74 does not change the rate of maturation of APP (Figure
2F). Thus it is unlikely that CD74 functions as ER chaperones as in the case of Class II MHC.
We previously showed that the familial dementia gene BRI2 and its homologue BRI3 are inhibitors of APP processing [
20,
21]. Here, we have established that CD74 binds APP and inhibits the production of Aβ. Like the two members of the
BRI gene family, CD74 is also a type II transmembrane protein, but structurally unrelated to BRIs (Figure
1A). CD74 can immunoprecipitate APP (Figure
1B). Reciprocal immunoprecipitations shows APP can precipitate CD74 (Figure
1C). Both N-terminal 23 amino acids and C-terminal 101 amino acids of CD74 are required for the interaction (Figure
1D). On the APP side, C99 and C83 scarcely bind CD74 (Figure
1A).
This binding of CD74 to APP is similar to that of BRI2 or BRI3, in that only the mature form of APP binds CD74 (Figure
1A). However, there is a clear difference: CD74 does not cause the accumulation of C99 as in the case of BRI2, nor does it bind to C99 (Figure
1B). Or, contrary to BRI2 and BRI3, CD74 does not inhibit secretion of sAPPα or sAPPβ (Figure
2C) [
20‐
22]. Therefore, it is unlikely that CD74 is working on APP by masking the secretase sites as in the case of BRI2 and BRI3. Rather, CD74 might be redirecting APP to intracellular compartments where the production of Aβ in reduced.
As shown in Figure
2D-F, CD74 does reduce the Aβ in the media when APP is co transfected, but it does not change the cytoplasmic Aβ40 (Figure
2D), or the stability of exogenously added Aβ (Figure
2E), or the maturation of APP (Figure
2F). These data further support our notion that CD74 works on APP inside cells after APP is matured, and not by destabilizing Aβ already secreted in the media.
It is noteworthy that the major metabolite of CD74 (CD74NTF14) does not interact with APP, but the minor fragment (NTF33) does (Figure
1C). This observation strongly argues that the CD74-APP interaction is specific because APP distinguishes NTF33 from NTF14; and the interaction occurs before CD74 is irreversibly processed further than NTF33; and at least NTF33 is required for the interaction. We cannot conclude that NTF33 is the minimal region required for the CD74-APP interaction because NTF33 might interact with APP indirectly, in a trimer with full length CD74. The precise dissection of the minimal region responsible for the binding requires further study, and should take into account the fact that CD74 forms trimers [
34,
38].
APP and CD74 displays distinct intracellular distribution. When transfected independently, APP displays reticular and vesicular structures in the cytoplasm. In contrast, CD74 shows larger vacuoles, which are endosomal in nature [
35]. Our observation that APP co localizes with CD74 in vacuolar structure in HeLa cells (Figure
3B, upper panels) supports the notion that CD74 and APP interact. Moreover, the striking APP re-distribution to CD74-positive vacuolar structures suggests that this change is caused by the physical interaction of CD74 and APP. Co expression of CD74 and either Notch, C99 or C83 (all molecules that do not bind CD74) show the specificity of this phenomenon since Notch, C99 and C83 do not accumulate in CD74-positive vacuoles upon co expression (Figure
3B, lower panels, and
3C).
Thus we propose that unlike BRI2 or BRI3, which are working directly on APP by blocking the access of secretases to APP, CD74 interferes with APP processing by regulating the sub-cellular localization of APP in compartments where amyloidogenic APP cleavage is reduced.
Little is known about the relationship between CD74 and AD. In immuno-histochemistry experiments, normal brains do not stain well for CD74 and CD74 is mostly expressed in microglia. However, the brains of AD patients have increased CD74 staining [
39]. Interestingly, a recent study shows that CD74 is accumulated in neurons with paired helical fibers [
40]. Our findings suggest that CD74 may be a player in controlling APP biology and processing, perhaps regulating APP trafficking in neurons. Given the anti-amyloidogenic activity of CD74, it is possible that the increased levels of CD74 in AD brains, potentially caused by increased TNFα in AD patients and in transgenic mouse over expressing APP [
41‐
43], could contribute to a negative feedback mechanism of the organism to fend off noxious APP processing up regulation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SM conceived of the study, carried out yeast and biochemical studies and drafted the manuscript. YM carried out the molecular biological work and immunocytochemical analysis. LD conceived the study, participated in the design and coordination. All authors read and approved the final manuscript.