Sorting nexin-4 regulates β-amyloid production by modulating β-site-activating cleavage enzyme-1
- Na-Young Kim†1, 2, 3, 4,
- Mi-Hyang Cho†1, 2, 3, 4,
- Se-Hoon Won1, 2, 3, 4,
- Hoe-Jin Kang1, 2, 3, 4,
- Seung-Yong Yoon1, 2, 3, 4Email authorView ORCID ID profile and
- Dong-Hou Kim1, 2, 3, 4Email author
© The Author(s). 2017
Received: 4 July 2016
Accepted: 28 December 2016
Published: 21 January 2017
Amyloid precursor protein (APP) is cleaved by β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) to produce β-amyloid (Aβ), a critical pathogenic peptide in Alzheimer’s disease (AD). Aβ generation can be affected by the intracellular trafficking of APP or its related secretases, which is thus important to understanding its pathological alterations. Although sorting nexin (SNX) family proteins regulate this trafficking, the relevance and role of sorting nexin-4 (SNX4) regarding AD has not been studied yet.
In this study, human brain tissue and APP/PS1 mouse brain tissue were used to check the disease relevance of SNX4. To investigate the role of SNX4 in AD pathogenesis, several experiments were done, such as coimmunoprecipitation, Western blotting, immunohistochemistry, and gradient fractionation.
We found that SNX4 protein levels changed in the brains of patients with AD and of AD model mice. Overexpression of SNX4 significantly increased the levels of BACE1 and Aβ. Downregulation of SNX4 had the opposite effect. SNX4 interacts with BACE1 and prevents BACE1 trafficking to the lysosomal degradation system, resulting in an increased half-life of BACE1 and increased production of Aβ.
We show that SNX4 regulates BACE1 trafficking. Our findings suggest novel therapeutic implications of modulating SNX4 to regulate BACE1-mediated β-processing of APP and subsequent Aβ generation.
KeywordsAlzheimer’s disease Sorting nexin BACE1 APP Lysosome
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by senile plaques containing extracellular deposits of the β-amyloid (Aβ) peptide . The Aβ40–42 peptide is derived from the amyloid precursor protein (APP) via the action of two membrane-bound proteolytic enzymes: β- and γ-secretase. β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that mediates the β-secretase cleavage, yielding a soluble ectodomain-secreted APP derivative (sAPPβ), as well as to a membrane-anchored C-terminal fragment (CTF) that subsequently undergoes presenilin-mediated γ-secretase cleavage [2–4]. The γ-secretase cleavage of CTF generates Aβ . Previous reports have shown that APP proteolytic processing occurs at various subcellular sites. Aβ is produced in the trans-Golgi network (TGN), Golgi-associated vesicles, the endosomal system, and the endoplasmic reticulum/intermediate compartment [6–9].
BACE1 has been shown to transit through the secretory pathway and target the endosomal system, cycling between endosomes and the cell surface, probably via TGN [10, 11]. The critical and initial point of Aβ generation is mediated by BACE1; hence, much effort has been made to develop BACE1 inhibitors. It is thus important to understand which molecular machinery regulates the trafficking of BACE1 affecting Aβ generation.
Sorting nexins (SNXs) are a diverse group of cellular trafficking proteins that contain a phospholipid-binding motif (PX). SNXs can form protein-protein complexes and bind specific phospholipids, which suggests a role for these proteins in membrane trafficking and protein sorting [12–14]. The mammalian SNX protein containing a Bin/amphiphysin/Rvs domain (SNX-BAR) retromer is composed of two subcomplexes; a membrane remodeling unit comprising a specific combination of the SNX-BAR domains, including dimers of SNX1/SNX2 and SNX5/SNX6; and a stable trimeric complex of vacuolar protein sorting (VPS) proteins. The trimer of VPS26-VPS29-VSP35 provides cargo selectivity through direct binding of VPS35 to the cytosolic tail of several cargo proteins (e.g., cation-independent mannose-6-phosphate receptor) . The assembly of these two subcomplexes allows the SNX-BAR retromer to coordinate the formation/stabilization of endosomal tubules selectively enriched with the appropriate cargo for endosome-to-TGN retrieval [16, 17].
Recent studies involving sorting nexin-4 (SNX4) regulation of the transferrin receptor have suggested that SNX-BARs play a fundamental, evolutionarily conserved role in tubule-based endosomal sorting [18–20]. Several SNX family members have been found to modulate Aβ generation through different regulatory mechanisms [21–23]. However, the functional roles of over 30 other mammalian SNX proteins remain unknown and deserve further investigation, particularly regarding their potential involvement in AD. In our present study, we first demonstrate that one of the SNX family members, SNX4, can interact with BACE1 and affect its intracellular trafficking, thereby mediating the β-processing of APP in Aβ production.
Human brain tissue and APP/PS1-transgenic mouse brain tissue
Human mediotemporal gyrus samples used in this study
Human SNX4 (GenBank accession number NM_003794) was tagged with green fluorescent protein (GFP) at its N-terminus for fluorescence imaging. These modified SNX4 complementary DNAs were subcloned into a mammalian expression vector, pEGFP-C1 (Invitrogen, Carlsbad, CA, USA). The sequence of all constructs was verified by DNA sequencing. All experiments were performed in SH-SY5Y, HeLa, and HEK293 cells or mouse primary cortical neurons.
Cell culture and isolation of primary mouse cortical neurons
SH-SY5Y, HeLa, and HEK293 cells were maintained in DMEM (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Rockford, IL, USA) and incubated in 5% CO2 at 37 °C. Cultures of primary cortical neurons were prepared from the brains of embryonic day 16 pups as described previously . Briefly, cerebral cortices were dissected in cold calcium- and magnesium-free Hanks’ balanced salt solution and incubated with a 0.125% trypsin solution for 15 minutes at 37 °C. Trypsin was inactivated with DMEM containing 20% FBS, and cortical tissue was dissociated by repeated trituration using a Pasteur pipette. Cell suspensions were diluted in neurobasal medium supplemented with Gibco B-27 components (Life Technologies/Thermo Fisher Scientific, Grand Island, NY, USA) and seeded onto plates coated with poly-d-lysine (catalogue number P7886-100MG; Sigma-Aldrich, St. Louis, MO, USA) and laminin (1 mg/ml; Life Technologies/Thermo Fisher Scientific, Grand Island, NY, USA). Neurons were maintained at 37 °C in a humidified 5% CO2 environment. All animal protocols used in this study were approved by Asan Institute for Life Sciences Animal Care and Use Committee.
Transfection of plasmids and small interfering RNA
The SH-SY5Y, HeLa, and HEK293 cells and primary mouse cortical neurons were transfected with plasmids, scrambled small interfering RNA (siCTL), or a small interfering RNA (siRNA) mixture (siSNX4) of three different siRNAs designed for targeting to SNX4 using Lipofectamine 2000 reagent (catalogue number 11668-019; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guide.
Sense: 5′-CAGAUCAGUUAAAGAGUA-3′, antisense: 5′-UACUCUUUUAACUGAUCUG-3′
Sense: 5′-CAGAAUAAAGGUGCUAGAA-3′, antisense: 5′-UUCUAGCACCUUUAUUCUG-3′
Sense: 5′-GUUUCAAGACCAGCUGUUU-3′, antisense: 5′AAACAGCUGGUCUUGAAAC-3′
Sense: 5′-UGAAUGGAGUGCCAUCGAA-3′, antisense: 5′-UUCGAUGGCACUCCAUUCA-3′
Sense: 5′-GGAAUUCAGGUUUGGACCA-3′, antisense: 5′-UGGUCCAAACCUGAAUUCC-3′
Sense: 5′-GAGUAGCAGAUCGACUCUA-3′, antisense: 5′-UAGAGUCGAUCUGCUACUC-3′
Immunocytochemistry and immunohistochemistry
For immunocytochemistry, SH-SY5Y and HeLa cells were plated onto 18-mm coverslips (Marienfeld, Lauda-Königshofen, Germany) coated with 0.05 mg/ml poly-d-lysine (Sigma-Aldrich, St. Louis, MO, USA). HeLa cells were transfected with pEGFP-C1-SNX4. At 24 h after transfection, cells were fixed with 4% paraformaldehyde. After being washed three times with PBS, cells were permeabilized with PBS containing 0.1% Triton X-100 for 5 minutes at room temperature (RT). Next, the cells were washed three times and blocked with PBS containing 5% bovine serum albumin for 30 minutes at 37 °C. The cells were washed three additional times and incubated with a primary antibody against rat hemagglutinin (HA) (Roche, Basel, Switzerland) for 60 minutes at 37 °C. After cells were washed five times with PBS, a secondary antibody coupled to Texas Red (Invitrogen, Carlsbad, CA, USA) was added for 60 minutes at 37 °C. Finally, the cells were washed five times and mounted for imaging. The cells were examined by confocal microscopy with the LSM 780 microscope (Carl Zeiss, Oberkochen, Germany), and image processing was performed using the ZEN software system (Carl Zeiss, Oberkochen, Germany).
For immunohistochemistry, paraffin-embedded blocks were sectioned and attached to the slide glasses. The paraffin of sectioned tissues was removed with xylene deparaffinizing solution. Next, tissues were dehydrated with various ethanol solutions at 100%, 90%, 80%, 70%, and 50% and washed twice with distilled water. For antigen retrieval, tissues were boiled for 5 minutes in 1 mM ethylenediaminetetraacetic acid (EDTA) solution (pH 8.0). After being washed three times with PBS, tissues were permeabilized with PBS containing 0.1% Triton X-100 for 20 minutes at RT. The cells were washed three times and were blocked with PBS containing 2% bovine serum albumin and 2% horse serum for 30 minutes at 37 °C. Tissues were washed three times and were incubated with a primary antibody against goat anti-SNX4 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-BACE1 (Cell Signaling Technology, Danvers, MA, USA), mouse anti-BACE1 (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Rab11 (Cell Signaling Technology, Danvers, MA, USA). After tissues were washed five times with PBS, a secondary antibody coupled to fluorescein isothiocyanate and Texas Red (Invitrogen, Carlsbad, CA, USA) was added for 60 minutes at 37 °C. After that step, tissues were washed three times with PBS and mounted for imaging. The cells and tissue were examined by confocal microscopy with the LSM 780 microscope, and image processing was performed using the ZEN software system.
Cell surface biotinylation assay
Cells transfected with SNX4 were cooled on ice and washed three times with ice-cold PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 to remove any contaminating proteins. After washing cells twice more with PBS, 0.5 mg of EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Rockford, IL, USA) per milliliter of reaction volume was added and incubated at 4 °C for 60 minutes. After further washing cells twice with PBS, the cells were harvested in PBS and lysed in lysis buffer (1% Nonidet P-40, 40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail [EMD Millipore, Billerica, MA, USA]). Cell lysates were centrifuged at 14,499 × g for 10 minutes at 4 °C to remove any insoluble material. The resulting supernatant was incubated with 50 μl of 50% streptavidin-coated agarose beads (Thermo Fisher Scientific, Rockford, IL, USA) with rotation for 2 h at 4 °C. After the beads were washed three times with lysis buffer, the bound proteins were eluted with SDS sample buffer by boiling for 5 minutes. Total protein and isolated biotinylated proteins were analyzed by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the surface fraction was used as a negative control to confirm fractionation [26, 27].
Coimmunoprecipitation and Western blot analysis
For coimmunoprecipitation and immunoblotting, HEK293 cells or cultured mouse cortical neurons transiently expressing BACE1-HA and GFP (mock) or GFP-SNX4 construct or mouse brain tissues were lysed with lysis buffer for 1 h at 4 °C. Cell lysates were centrifuged at 14,499 × g for 10 minutes at 4 °C to remove any insoluble material. Immunoprecipitation was performed by overnight incubation with anti-BACE1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-GFP (Roche, Basel, Switzerland), or anti-HA (Roche, Basel, Switzerland) antibody. Immune complexes were captured using protein G sepharose (GE Healthcare Life Sciences, Piscataway, NJ, USA), followed by washing with lysis buffer three times. Immunoprecipitated samples or 5% of the input lysates were used for immunoblotting. For Western blot analysis, protein lysates from HEK293 cells or primary mouse cortical neurons or mouse brain tissue were homogenized in 1× IGEPAL (I8896; Sigma-Aldrich, St. Louis, MO, USA), a protein extraction solution, according to the manufacturer’s instructions and incubated at −20 °C with rotation for 30 minutes. The suspension was microcentrifuged at 15,682 × g for 15 minutes at 4 °C, and the supernatant was collected. Protein concentrations were measured by Bradford assay, and proteins were mixed with 5× sample buffer (60 mM Tris-HCl, pH 6.8, 2% wt/vol SDS, 25% vol/vol glycerol, 14.4 mM vol/vol β-mercaptoethanol, and bromophenol blue), boiled at 100 °C for 5 minutes, and stored at −20 °C. Proteins were resolved by SDS-PAGE at a constant voltage (110 V) and transferred at 100 V for 1.5 h to polyvinylidene difluoride membranes (0.2-mm pore size; Bio-Rad Laboratories, Hercules, CA, USA). After 1-h incubation in blocking buffer (PBS containing 0.1% vol/vol Tween-20 [PBST]) containing 3% wt/vol bovine serum albumin and 5% vol/vol skim milk, blots were incubated with primary antibodies overnight at 4 °C. Blots were next washed in PBST buffer, incubated with HRP-conjugated anti-immunoglobulin G (1:5000; Thermo Fisher Scientific, Rockford, IL, USA), and visualized using enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and x-ray film. The primary antibodies and dilutions used in the Western blot analysis were goat anti-SNX4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-BACE1 (1:5000; Abcam, Cambridge, UK), mouse anti-GFP (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-β-actin (1:10,000; Sigma-Aldrich, St. Louis, MO, USA), mouse anti-GAPDH (1:2000; EMD Millipore, Billerica, MA, USA), mouse anti-early endosome antigen 1 (EEA1) (1:2000; BD Biosciences, San Jose, CA, USA), rabbit anti-Rab7 (1:2000; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-Rab11 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-Aβ (6E10, 1:5000; Covance, Princeton, NJ, USA). The band intensities were measured and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Protein synthesis inhibition
HEK293 cells stably expressing BACE1 were transfected with mock or SNX4 constructs for 24 h, followed by treatment with 10 μg/ml cycloheximide, a protein synthesis inhibitor (Sigma-Aldrich, St. Louis, MO, USA) for 0, 1, and 6 h.
To produce a single-cell suspension, SH-SY5Y cells were plated in 10 ml of DMEM (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 5% FBS (Thermo Fisher Scientific, Rockford, IL, USA) and grown to 90% confluency with 5% CO2 at 37 °C. SH-SY5Y cells were cotransfected with BACE1 and SNX4 constructs or mock treatment for 48 h. After transfection, the cells were washed three times with cold PBS and harvested. The suspension was microcentrifuged at 13,362 × g for 5 minutes at 4 °C, and the supernatant was removed. In accordance with the manufacturer’s instructions, the pellet was resuspended in gradient fractionation solution A (catalogue number 89839; Thermo Fisher Scientific, Rockford, IL, USA) and incubated with added protease inhibitor at −4 °C for 2 minutes. The mixed solution was homogenized on ice, added to gradient fractionation solution B, and centrifuged at 500 × g for 10 minutes at 4 °C. The supernatant was transferred to a 1.5-ml tube, and protein concentrations were measured by the Bradford method. Equal amounts of protein were mixed with OptiPrep gradient solution (Sigma-Aldrich, St. Louis, MO, USA). The supernatant containing total protein in 640 μl was loaded onto the top of a step gradient composed of 320 μl of 30% gradient, 320 μl of 27% gradient, 160 μl of 23% gradient, 320 μl of 20% gradient, and 160 μl of 17% gradient (total approximately 2 ml). Gradients were centrifuged at 145,000 × g for 2 h at 4 °C. Fourteen 130-μl fractions were collected from the top of the gradient. The distributions of BACE1, EEA1, Rab7, Rab11, and β-actin along the gradient were assayed by SDS-PAGE followed by Western blot analysis.
Quantitative analysis of fluorescence intensity
The fluorescence intensity of immunostained cells in images were measured using the ZEN 2011 software system (blue edition). Each intensity value was corrected with background fluorescence intensity of nonstained cells and normalized by each control. The intensity value in all figures was analyzed from about 50–100 cells, and then p values were calculated using Student’s t test.
Data are presented as mean ± SEM and were analyzed using Student’s t test. A p value less than 0.05 was considered to be statistically significant.
Altered levels of SNX4 in the brains of patients with AD and APP/PS1 mice
SNX4 increases BACE1 and β-processing of APP
Our observations of increased SNX4 levels in the brains of young APP/PS1 mice and decreased SNX4 levels in the brains of old APP/PS1 mice and patients with late-stage AD prompted us to investigate the role of SNX4 by overexpression and knockdown. To this end, HEK293 cells were cotransfected with BACE1 and SNX4 to examine if SNX4 affected Aβ generation. Levels of BACE1 were increased in SNX4-transfected cells compared with mock-transfected cells (Fig. 3a and b). sAPPβ and Aβ was also increased in the culture media by SNX4 overexpression (Fig. 3a and b). Next, HeLa cells were cotransfected with BACE1-HA and SNX4 and immunostained with HA antibody. In these experiments, BACE1 was increased in SNX4-transfected cells compared with mock-transfected cells (Fig. 3c). Primary mouse cortical neurons were also cotransfected with BACE1 and SNX4 to confirm the effects in neurons, and we found that levels of BACE1 and secreted sAPPβ and Aβ were increased in SNX4-transfected neurons compared with mock-transfected cells (Fig. 3d and e). Immunocytochemistry also showed that BACE1 was increased in SNX4-transfected neurons compared with mock-transfected neurons (Fig. 3f). These results show that SNX4 overexpression increases BACE1 levels and subsequently leads to an increase in the BACE1-mediated, APP-processing product Aβ.
SNX4 knockdown leads to decreased BACE1 and β-processing of APP
SNX4 interacts with BACE1
SNX4 shifts BACE1 from the degradation pathway and increases its half-life
SNX4 regulates cell membrane levels of BACE1
In our present study, we found that SNX4 levels are altered in the brains of patients with AD and in AD model mice. Our data reveal that SNX4 interacts with BACE1 and increases its steady-state levels, leading to increased generation of Aβ. Our results also indicate that SNX4 enhances the recycling of BACE1 from sorting endosomes to the plasma membrane. This recycling of BACE1 by SNX4 prevents the trafficking of BACE1 to late endosomes and lysosomes for degradation, increasing the half-life of BACE1 and β-processing of APP.
SNX4 is a member of the PX domain-containing trafficking molecule family involved in membrane trafficking [19, 20]. One feature of SNXs is their ability to support cargo complex formation by binding specific lipids and aiding donor membrane curvature via their PX and BAR domains [28, 29]. Another role of SNXs is to tightly control the levels of their selected target cargo proteins in a given organelle. SNX4 localizes to Rab11+ recycling endosomes, which have abundant SNX4 but relatively low levels of SNX1 and SNX8 . SNX4 is also involved in the recycling of the transferrin receptor. Sorting tubules are formed by SNX4 from Rab4+/Rab11+ endosomes, indicating that recycling endosomes are extended by SNX4-dependent membrane trafficking, whereas SNX1 or SNX8 associates with TGN-targeted tubules in the early-to-late endosome pathway  Collectively, SNX4 may regulate the recycling of specific cargoes to the plasma membrane. This is supported by our finding that SNX4 recycles BACE1 to the plasma membrane and protects it from the degradation pathway (Figs. 6, and 7).
The critical and initial point of Aβ generation is mediated by BACE1; hence, it is very important to understand which molecular machinery regulates the trafficking of BACE1, affecting Aβ generation. Although we found the direct interaction of BACE1 and SNX4 (Fig. 5) that accounts for changes in BACE1-mediated APP processing (Fig. 3, and 4), APP processing could also rely on the APP level itself. SNX4 also regulated the APP full-length levels (Fig. 3, and 4), which could affect BACE1-mediated APP processing. We think that SNX4 could regulate both APP and BACE1, affecting Aβ levels by the direct interaction of BACE1 and SNX4 and the indirect and unknown mechanism of APP. BACE1 has been shown to transit through the secretory pathway and target the endosomal system, cycling between endosomes and the cell surface, probably via the TGN [10, 11]. In addition to our findings of SNX4’s roles in BACE1 trafficking, several molecular mechanisms of regulating BACE1 have been reported. The endosomal trafficking of BACE1 appears to be partially regulated by an acidic cluster-dileucine motif in its cytoplasmic tail [30–32]. This motif has been shown to interact with the Vps27/Hrs/signal-transducing adapter molecule domain of Golgi-localized γ-adaptin ear-containing ADP-ribosylation factor-binding (GGA) proteins GGA1, GGA2, and GGA3, adapter proteins that mediate sorting between the TGN and endosomes [31, 33]. Recently, GGA3 was shown to bind BACE1 via the ubiquitin-sorting machinery and to regulate BACE1 degradation . Although we showed that BACE1 level is regulated by SNX4, BACE1 was not decreased in the brains of 24-month-old mice or in late-stage AD brains (Fig. 1, and 2). This phenomenon let us analogize that BACE1 escaping from the recycling pathway may be not degraded, despite a decrease of SNX4 due to other factors, including inefficiency of lysosomal degradation or the endocytic pathway, as observed in AD-like pathological conditions [35, 36]. Decreased BACE1 levels were protected by inhibiting lysosomal acidification or endocytosis using bafilomycin A1 or chlorpromazine in siSNX4-transfected cells (Additional file 5: Figure S5), which may explain how BACE1 is not decreased in the brains of 24-month-old mice or in late-stage AD brains. Although it has been reported that BACE1 levels were increased in patients with AD , some studies have shown that BACE1 levels were not elevated in AD temporal cortex , in line with our present results.
Our data indicate that SNX4-mediated regulation of the steady-state levels and trafficking of BACE1, as well as the subsequent increase in BACE1-mediated cleavage, may be relevant to AD progression. Regulating the expression levels of BACE1 to reduce Aβ production remains a promising strategy for therapeutic intervention in AD. Inhibition of BACE1 expression by strategies such as SNX4 modulation may be a critical strategy in developing AD therapeutics.
Amyloid precursor protein
β-Site amyloid precursor protein-cleaving enzyme 1
Early endosome antigen 1
Glyceraldehyde 3-phosphate dehydrogenase
Green fluorescent protein
Golgi-localized γ-adaptin ear-containing ADP-ribosylation factor-binding protein
Major histocompatibility complex class I
PBS containing 0.1% vol/vol Tween-20
Soluble ectodomain-secreted β-amyloid precursor protein derivative
Scrambled small interfering RNA
Small interfering RNA
Small interfering RNA mixture
SNX protein containing a Bin/amphiphysin/Rvs domain
Vacuolar protein sorting
We thank the Netherlands Brain Bank for supplying the human brain material and also thank the brain tissue donors and their relatives for enabling the neuropathological studies described in this paper.
This work was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (2015R1A2A1A10053683) and the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C1630).
Availability of data and material
There is not any supporting data in this study.
NYK carried out the cell experiments, Western blot experiments, immunohistochemistry, and gradient fractionation and drafted the manuscript. MHC carried out the cell experiments, Western blot experiments, immunocytochemistry, and surface biotinylation; analyzed the data; drafted the manuscript; and revised the manuscript. SHW participated in the cell experiments and Western blot experiments. HJK carried out the Western blot experiments. SYY designed the research, drafted the manuscript, supervised the overall experiments, and revised the manuscript. DHK designed the research, drafted the manuscript, and supervised the overall experiments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Written informed consent was obtained from the Netherlands Brain Bank for publication of patients’ individual details. The consent forms are held by the authors and are available for review by the Editor-in-Chief of this journal.
Ethics approval and consent to participate
The Netherlands Brain Bank had all necessary consent from any patients involved in the study, including consent to participate in the study where appropriate. All experimental procedures in the study that involved animals were approved by the institutional animal care and use committee of Asan Institute for Life Sciences.
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