Introduction
Alzheimer’s disease (AD) is one of the most common debilitating neurodegenerative diseases [
1]. The neuropathological hallmarks of AD primarily derive from aggregation and deposition of misfolded proteins, in particular the β-amyloid (Aβ) peptide that oligomerizes to form extracellular senile plaques [
2] and the hyperphosphorylated tau protein that aggregates into neurofibrillary tangles [
3,
4]. Numerous studies have shown that AD symptoms directly correlate with the amount of pathogenic oligomeric Aβ in the brain [
5,
6]. Aβ is generated from the Amyloid Precursor Protein (APP), a type I membrane protein [
5,
7]. APP can undergo either non-amyloidogenic or amyloidogenic processing depending on the secretases that cleave the protein. In the non-amyloidogenic cascade, APP is sequentially cleaved by α-secretase and γ-secretase thus generating p3 and AICD fragments [
8]. In the amyloidogenic processing, BACE1 first generates βCTF that is further processed by the γ-secretase complex to produce pathogenic Aβ peptides and AICD fragments [
9‐
15]. Aβ peptides oligomerize and aggregate in the form of plaques, resulting in inflammation and neuronal cell death [
16,
17].
APP is a ubiquitously expressed ~105 kDa transmembrane glycoprotein [
5]. In the brain, APP is found at both presynaptic and postsynaptic terminals and plays an important role in various neuronal functions such as synapse formation, neuronal migration, neurite outgrowth, synaptic plasticity, synaptic transmission and learning and memory [
18‐
21]. APP is known to traffic axonally by interacting with the kinesin light chain and localize at the presynaptic terminal [
22‐
25]. There, it undergoes proteolytic processing and releases Aβ at the synapse [
25‐
27]. Although numerous APP-interacting proteins have been identified [
28,
29], not much is known about synaptic proteins that interact with the ectodomain of APP and may regulate Aβ generation.
The Synaptotagmin (Syt) family of proteins is known to regulate membrane trafficking and fusion [
30,
31]. Syts are type-I transmembrane proteins with a short amino-terminal domain and a large carboxyl-terminal cytoplasmic region harboring the Ca
2+-binding domains C2A and C2B (Additional file
1: Figure S1B) [
30,
32,
33]. Importantly, Syt-1, −2 and −9 are known to act as calcium sensors on synaptic vesicles and play a major role in synaptic vesicle membrane fusion events [
34]. Recent studies have also shown that APP is present in purified synaptic vesicle preparations along with its secretases and Aβ [
35,
36].
Here, we performed an unbiased MS-coupled affinity chromatography screen to search for synaptic proteins that interact with the extracellular domain of APP. Our study identifies Syt-1, −2 and −9 as novel APP-ectodomain binding proteins and demonstrates that Syt-1 is a physiological interactor of APP. Moreover, we show that both Syt-1 and Syt-9 increase Aβ levels likely via BACE1-mediated APP processing and thus may play an important role in Alzheimer’s disease pathology. Overall, our studies reveal an important novel function for Syt family of synaptic vesicle proteins in regulating Aβ generation.
Materials and methods
Antibodies and reagents
Rabbit anti-APP antibody and anti-sAPPβ antibodies were described previously [
37]. Syt-1 mouse monoclonal and Syt-5/9 rabbit polyclonal antibodies were obtained from Synaptic Systems (Goettingen, Germany). Mouse monoclonal anti-GAPDH antibody was purchased from Cell Signaling (Danvers, MA) while mouse anti-V5 antibody was from Life Technologies (Grand Island, NY). Anti-Syt-1 mouse monoclonal antibody (ASV30) and Isopropyl-beta-D-thiogalactopyranoside (IPTG) were from Thermo Fisher Scientific (Waltham, MA).
Plasmids construction
To generate NH2-terminal GST-tagged APP ectodomain constructs, different ectodomain regions of APP were first amplified with the help of polymerase chain reaction (PCR) using the following forward and reverse primers, E1 domain: forward, ATATATGTCGACTCTGGAGGTACCCACTGATGG; reverse, TATATAGCGGCCGCCTAAGCCAGTGGGCAACACAC; E1+ KPI domain: forward, ATATATGTCGACTCTGGAGGTACCCACTGATGG; reverse, TATATAGCGGCCGCCTAGGCATCAGGGGTACTGGC; E2 domain: forward, ATATATGTCGACTGCAGCCAGTACCCCTGATG; reverse, TATATAGCGGCCGCCTAGGCCAAGACGTCATCTGAATAG; CBD domain: forward, ATATATGTCGACTGCAGCCAGTACCCCTGATG; reverse, TATATAGCGGCCGCCTATTTGTTTGAACCCACATCTTC; APP full-length ectodomain: forward, ATATATGTCGACTCTGGAGGTACCCACTGATGG; reverse, TATATAGCGGCCGCCTATTTGTTTGAACCCACATCTTC. For the GST-tagged Syt-1 ectodomain construct, the entire 57 amino acids long NH2-terminal region of Syt-1 was amplified using FP: ATATATGGATCCATGGTGAGCGAGAGTCACCATG, and RP: TATATAGCGGCCGCCTAGGCCCACGGTGGCAATG. Following amplifications, APP fragments and the Syt-1 ectodomain were subcloned in frame with the GST tag into pGEX6P-2 vector (GE Healthcare Life Sciences, Pittsburgh, PA). Human SCN2B cDNA (BC036793) was obtained from Harvard Plasmid DNA Resource Core (Harvard Medical School, Boston, MA). NH2-terminal GST-tagged SCN2B ectodomain construct was generated by amplifying the ectodomain coding region of SCN2B using forward primer, ATATATGGATCCATGGAGGTCACAGTACCTGCC and reverse primer, TATATAGCGGCCGCCTAGGCCACCGTGGAGTCC and then ligated in frame with GST tag into pGEX6P-2 vector. For generation of COOH-terminal V5-tag Syt-1, Syt-2 and Syt-9 constructs, the entire coding region of Syt-1, Syt-2 and Syt-9 were amplified using the following forward and reverse primers; Syt-1: forward, ATATATGCTAGCATGGTGAGCGAGAGTCACCATG; reverse, TATATAGCGGCCGCCCTTCTTGACGGCCAGCATG; Syt-2: forward, ATATATGCTAGCATGGCAAGGAACATTTTCAAGAGGAACCAG; reverse, TATATAGCGGCCGCCCTTGTTCTTGCCCAGGAGTGC; Syt-9: forward, ATATATGCTAGCATGGCATTCCCGGAGCCCCCAAC; reverse, TATATAGAATTCGGGGCGCAGGCAGCAGC. The amplified fragments were then subcloned into pCDNA6-V5 vector (Life Technologies, Grand Island, NY). All the generated constructs were sequenced and verified at the MGH DNA sequencing core facility (Boston, MA).
Generation and purification of GST-tagged recombinant proteins
GST-tagged ectodomain fragments of APP and SCN2B were generated and purified using similar methodology as described earlier [
38]. Briefly, plasmids were transformed into
E. coli BL21 cells (Life Technologies, Grand Island, NY) and then allowed to grow at 37 °C until the optical density of the culture reached between 0.6-1.0. Cultures were induced by 1 mM IPTG for 3 h at 24 °C. Cells were lysed and the GST-tagged recombinant proteins were purified by incubating the soluble fractions with glutathione resin for 4 h at 4 °C. Samples were washed and stored at 4 °C until further use.
Glutathione S-transferase (GST) pull-down assay and Colloidal Blue staining
GST pull-down assays were performed as described previously [
38]. In brief, adult male ICR (CD-1) mice were purchased from Charles River Laboratory and anaesthetized with the help of Isoflurane (Hospira Inc, IL). Brains were removed quickly, homogenized in the ice-cold lysis buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 % Triton X-100 supplemented with protease and phosphatase inhibitors cocktails (Roche Life Science, Indianapolis, IN). After removal of the insoluble fractions, soluble supernatant was incubated at 4 °C with equal amount of GST-tagged recombinant purified proteins coupled with glutathione resin. Samples were washed, eluted out and separated on one-dimensional gel electrophoresis using 4-12 % Bis-Tris Gel (Life technologies, Grand Island, NY). Gels were then subjected to Colloidal Blue staining and the excised bands were subjected to mass spectrometry-based analysis.
Protein sequence analysis by LC-MS/MS
Excised Colloidal Blue-stained gel bands were cut into approximately 1 mm
3 pieces and then subjected to a modified in-gel trypsin digestion procedure as described previously [
39]. Gel pieces were washed, dehydrated with acetonitrile and then rehydrated with 50 mM NH
4HCO
3 containing 12.5 ng/μl modified sequencing-grade trypsin (Promega, Madison, WI) for 45 min at 4 °C. Peptides were extracted by removing the NH
4HCO
3 solution, followed by one wash with a solution containing 50 % acetonitrile and 1 % formic acid, dried and stored at 4 °C. On the day of analysis, samples were reconstituted in HPLC solvent A (2.5 % acetonitrile, 0.1 % formic acid) and loaded onto a nano-scale reverse-phase HPLC capillary column via a Famos auto sampler (LC Packings, San Francisco, CA). Peptides were eluted with the help of increasing concentrations of solvent B (97.5 % acetonitrile, 0.1 % formic acid), subjected to electrospray ionization and then entered into an LTQ Velos ion-trap mass spectrometer (Thermo Fisher, San Jose, CA). Peptides were detected, isolated, and fragmented to generate a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program Sequest (ThermoFisher, San Jose, CA).
Immunogold electron microscopy
PC12 cells were washed and fixed in a solution containing 4 % paraformaldehyde and 0.2 % glutaraldehyde in 1X PBS. Following 5 washes, cells were pelleted, resuspended in warm 2 % agarose, cut into small blocks and incubated with 2.3 M sucrose at 4 °C for overnight. Ultrathin cryosections were generated on a Leica EM FCS at −80 °C and collected on the formvar-carbon coated nickel grids. For double immunolabeling, grids were first blocked on drops of 1 % BSA and 5 % goat serum and then incubated with mouse anti-Syt-1 antibody for 1 h followed by anti-mouse secondary antibody coupled with 10 nm gold particle for 1 h. After rinsing, grids were incubated again with rabbit anti-APP antibody for 1 h followed by anti-rabbit secondary antibody coupled with 15 nm gold particles. Grids were washed, stained on drops of Tylose and Uranyl acetate and then allowed to dry. The grids were examined at 80 kV in a JEOL JEM 1011 transmission electron microscope and the images were acquired using an AMT digital imaging system (Advanced Microscopy Techniques, Danvers, MA).
In situ proximity ligation assay (PLA)
In situ proximity ligation assay was performed using the In situ PLA kit (OLink Bioscience, Sweden) according to the manufacturer’s protocol. Briefly, PC12 cells were first blocked and then incubated with rabbit anti-APP (C66) and mouse anti-Syt-1 antibody for 2 h. Cells were washed 3 times and then incubated with two different in situ probes for 1 h at 37 °C. After 3 washes, ligation solution was added to the cells for 30 min followed by polymerase solution for 2 h. Later, cells were mounted in the mounting medium and visualized under confocal microscope using 20X objective. Image were captured at identical settings and later processed by Metamorph software.
Generation of Syt-1 and Syt-9 stable cell lines
Syt-1 and Syt-9 stable cell lines were generated on both CHO-APP [
37] and PC12 cells. In brief, CHO-APP and PC12 cells were transfected with 4 μg of V5-tagged Syt-1 or Syt-9 cDNA with the help of Effectene transfection reagent (Qiagen, Valencia). Cells were later trypsinized and then replated in the presence of 10 μg/μl of Blasticidin selection marker (Life Technologies, Grand Island, NY). Cells were grown for 2 weeks before Blasticidin-resistant cells were further plated in a series of serial dilution in a 96 well tissue culture plate. Single cell colonies were picked and analyzed by Western blotting for optimal expression of Syt-1 or Syt-9 with the help of a mouse anti-V5 antibody.
Primary dissociated neuronal culture
Primary dissociated neuronal culture was prepared as described previously [
37].
Lentiviral infection
Syt-1 shRNA and control DsRed lentiviral particles were generated at MGH Vector Core facility (Charlestown, MA). Mouse primary neuronal cultures were infected with 1 × 106 lentiviral particles at DIV5 and half the media was replaced 12 h after infection. Cultures were allowed to grow for 10 additional days after infection before analysis of APP processing and Aβ40/42 release.
Immunofluorescence microscopy
Primary neuronal cultures grown on cover glass in 12 well tissue culture plates were fixed at 12 days in vitro (DIV12) with 4 % paraformaldehyde for 30 min. Cultures were washed and permeabilized with 0.1 % Triton X-100 and 5 % donkey serum for 1 h at room temperature. Cells on coverglass were then incubated with rabbit anti-APP (C66) and mouse anti-Syt-1 antibodies at 4 °C for overnight. Following washing, cells were again incubated with Alexa Fluor488-conjugated anti-rabbit and Alexa568-conjugated anti-mouse secondary antibodies for 2 h at room temperature. Cells on coverglass were then washed and then mounted on glass slides with the help of DAPI containing mounting media (Life Technologies, Grand Island, NY). Images were acquired on Olympus IX-70 microscope at similar exposure settings and later processed by IP lab software.
Western blot analysis
Cells were lysed in 1X GTIP buffer (10 mM Tris–HCl, pH 6.8, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100 and 0.25 % Nonidet P-40) supplemented with protease and phosphatase inhibitors cocktail. The soluble fraction was obtained by centrifugation and the protein concentration was measured with the help of a BCA protein assay kit (Pierce Biotechnology, Rockford, USA). 40–60 μg of protein samples were separated by gel electrophoresis using 4-12 % Bis-Tris gels and later transferred on PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5 % skimmed milk for 1 h at room temperature and then incubated with the indicated primary antibodies overnight at 4 °C at the following dilutions: rabbit anti-APP (C66) 1:1000, mouse anti-V5 1:5000, mouse anti-Syt-1 1:1000, rabbit anti Syt-9 1:1000, rabbit anti-sAPPβ 1:250, mouse anti-sAPPα 1:1000. Following incubation with HRP-conjugated secondary antibodies, blots were developed with the help of ECL chemiluminiscence detection reagent using Biomax film (Kodak).
Aβ ELISA
CHO cells and PC12 cells stably expressing Syt-1 or Syt-9 and PC12 Syt-1 stable KD cells were plated on 60-mm tissue culture plates. Conditioned media was collected after 24 h and subjected to standard sandwich ELISA for measurement of secreted Aβ40 and Aβ42 using an Aβ ELISA Human/Rat kit (Wako Pure chemical). Aβ40 and Aβ42 from mouse primary neuronal cultures were determined by performing sandwich ELISA on conditioned media using 21F12/2G3 as capture antibody for Aβ42/ Aβ40 and 266 (Abeta13-26) as detector. Aβ values were normalized by the cellular protein amount and expressed as pg/ml/mg.
Statistical analyses
All statistical analyses were performed using a 2-tailed Student’s t test. Error bars represented in graphs denote the SE.
Discussion
APP regulates numerous physiological functions in brain, but it is also cleaved to generate Aβ species involved in the pathology of the Alzheimer’s disease [
48‐
50]. To understand how APP functions are regulated in both physiological and pathological conditions, numerous studies have focused on the identification of APP-interacting proteins [
28,
29,
51]. Although these studies identified some mouse brain APP-associated proteins, these mainly bound to the APP-CTF. Only a few proteins such as contactin, F-spondin and Nogo-66 have been reported so far to interact with the APP extracellular domains [
41,
52,
53].
Here, we performed an unbiased proteomic screen using purified GST-tagged APP-ectodomain fragments and have identified members of the Synaptotagmin family as novel APP-ectodomain interacting proteins. Using transgenic mice, previous studies had identified Syt-1 and Syt-11 as pulling down with APP and thus suggested a close association between these Syts and APP [
29,
54]. However, these studies have not characterized the interaction between APP and Syts and its effect on APP processing. Our data not only identify Syt-1 as an APP-interacting protein but also show that Syt-1, −2, and −9 exist in complex with APP both
in vitro and
in vivo, the interaction is mediated by APP’s linker region between the E1 and KPI domains, and it results in altered APP processing.
The 17 different isoforms of Synaptotagmins not only differ in structure but also in subcellular localization [
55,
56]. Syt-1 and Syt-2 were shown to localize to synaptic vesicles while Syt-9 is targeted to both axons and dendrites [
57]. Recently, it was shown that APP is localized to synaptic vesicles and trafficked via neuronal activity-dependent release of synaptic vesicles [
36]. The interaction of Syts with APP could mediate the localization of APP to synaptic vesicles and its activity-dependent localization to presynaptic membranes. Syt-1 is the best-characterized isoform among the Synaptotagmin family of proteins. Syt-1 acts as a dual sensor as it regulates both the fusion of synaptic vesicles to the pre-synaptic membrane as well as their endocytosis [
58]. Endocytosis has been shown to play an important role in the generation of Aβ as APP is endocytosed via a clathrin-mediated process from the plasma membrane into endosome-like structures to generate Aβ peptides [
59,
60]. These studies suggest the possibility that Syt-1 containing vesicles may acquire APP during synaptic vesicle endocytosis from the presynaptic membrane and regulate APP processing and Aβ generation in the endosomes. However, the precise role of Syt-1 in APP trafficking, localization, and metabolism warrants further investigation.
APP is known to localize both at the presynaptic and postsynaptic terminals where it acts as a trans synaptic adhesion molecule and promotes synapse formation [
18]. Presence of APP at the synapse regulates synaptic transmission and neurotransmitter release, best shown in APP/APLP2 double knockout mouse models. Indeed, APP/APLP2 double knockout mice exhibit reduced synaptic vesicle density, active zone size, and number of docked synaptic vesicles per active zone as compared to APLP2 single knockout [
21,
61,
62]. Moreover, overexpression of APP in PC12 cells promoted exocytosis and increased basal and constitutive secretion [
63]. Given the essential role of Syt-1 in synaptic vesicle exocytosis, docking and neurotransmitter release [
30], its interaction with APP may regulate its function. This could partially or fully explain altered synaptic vesicle biology in APP knockout mice. Future studies will be needed to fully explore the role of APP in regulating Syt-1-mediated synaptic vesicle exocytosis and endocytosis.
Evidence suggests that APP undergoes BACE1 and PS1/γ-secretase-dependent proteolytic processing in the presynaptic compartments where it may generate Aβ peptides released from the nerve terminals [
25]. Genetic inactivation of PS1 in the presynaptic compartment significantly increases APP-CTF accumulation, supporting a role for the presynaptic compartment as a major site for APP processing and Aβ generation [
64]. In addition, PS1 and other γ-secretase complex components were found in purified synaptic vesicle preparations from adult rat brain [
35]. Similarly to the γ-secretase complex components, BACE1 was also found in the synaptic vesicle fraction [
36,
65]. Moreover, using a transgenic AD mouse model, Vassar
et al. have observed strong colocalization of BACE1 with APP in the swollen dystrophic presynaptic terminals surrounding Aβ plaques [
66]. This suggests abnormal accumulation of BACE1 at presynaptic sites and enhanced BACE1-mediated processing of APP, potentially contributing to AD pathogenesis. Our results indicate that an APP-interacting protein, Syt-1, regulates APP processing in the presynaptic compartment. Specifically, our data suggest that Syt-1 modulates BACE1-mediated cleavage of APP as overexpression of Syt-1 enhanced sAPPβ and Aβ levels while loss of Syt-1 in PC12 cells resulted in lower endogenous Aβ generation and sAPPβ levels in the conditioned media with decreased APP-CTF levels. Moreover, our results indicate that Syt-1 also regulates α-cleavage of APP and loss of Syt-1 may also impair α-cleavage as we have observed increased sAPPα in Syt-1 overexpressing cells. BACE1-mediated cleavage of APP in presynaptic vesicles was previously reported [
65]. Interestingly, the pH of synaptic vesicles is around 5.0 - 5.7, which would allow for an ideal microenvironment for optimal BACE1 activity and Aβ generation [
67,
68]. Given the essential role of Syt-1 in synaptic vesicle biology and trafficking, it is very likely that BACE1-mediated cleavage of APP and Aβ generation are regulated by Syt-1 particularly in synaptic vesicles. Thus, Syt-1 interaction with APP could represent a novel therapeutic target in the treatment or prevention of AD.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DMK, VG designed the study. DMK, VG and OB contributed to the analysis of data. VG and CD performed the experiments and collected data. VG and DMK wrote and revised the manuscript. All authors read and approved the final manuscript.