PS1 conformation is dynamically regulated by Ca2+ influx in intact neurons

Neuronal activation is reported to modulate Aβ production and elevate the Aβ42/40 ratio [13, 19, 21]. The latter correlates with distinct conformation of the PS1/γ-secretase [22–25]. To probe for possible dynamic changes in PS1/γ-secretase in live cells in response to neuronal stimulation we used our previously developed ratiometric spectral Förster Resonance Energy Transfer (FRET) assay that utilizes GFP-PS1-RFP (G-PS1-R) as a reporter of PS1 conformation (Fig. 1a). As determined previously, the G-PS1-R protein can traffic through the secretory pathway to the plasma membrane, shows similar subcellular distribution to that of endogenous PS1, can be incorporated into the γ-secretase complex, is efficiently endoproteolyzed, and reconstitutes the γ-secretase enzymatic activity in PS1/2 double knockout mouse embryonic fibroblasts (MEF) [25]. The change in the proximity between RFP and GFP fluorophore-tagged PS1 loop- and NT-domains (FRET efficiency) corresponds to the change in the ratio of RFP (598 nm) to GFP (513 nm) fluorescence intensity (R/G ratio). The higher the R/G ratio the closer the two domains are, indicating so-called “closed” PS1 conformation.

Fig. 1 PS1 conformation and Aβ production change upon KCl or glutamate treatment. a Schematic representation of the “open” (left) and “closed” (right) PS1 conformation; green and red circles represent green fluorescent protein (GFP) and red fluorescence protein (RFP) fused to PS1 NT and L6-7, respectively, to generate FRET reporter probe. b Time-lapse recording of PS1 conformational changes in live neurons transfected with GFP-PS1-RFP FRET reporter probe and treated with KCl, glutamate, or water control. The Spectral FRET data are presented as a change in the RFP/GFP ratio, 50 to 100 neurons were analyzed for each condition; the graph shows mean ± SEM; detailed statistical analysis of the Spectral FRET data evaluation is described in the Methods. c Time-lapse recording of Oregon Green 488 BAPTA-1 AM fluorescence intensity changes reflecting intracellular calcium load in primary neurons treated with KCl, glutamate, or water control. Three independent experiments, mean ± SEM. d ELISA measurements of secreted Aβ40 and Aβ42 in conditioned-medium collected from KCl (KCl+) or H 2 O (KCl-) treated mouse cortical neurons. The Aβ levels determined in pmol were normalized to the amount of total protein [g] extracted from the cells in the corresponding well. Data are presented as mean% ± SEM, n = 4; 100 % = 91.11 pmol/g for Aβ40 and 11.04 pmol/g for Aβ42. Statistical significance was determined using the Mann-Whitney U test, *p < 0.05. PS1 presenilin 1, Aβ amyloid β, NT N-terminus, FRET Förster Resonance Energy Transfer Full size image

Primary neurons (12–14 days in vitro [DIV]) were transfected with the G-PS1-R and imaged every ~30 seconds prior to and after KCl bath treatment inducing membrane depolarization. We detected a rapid increase in the R/G ratio within the first minute of the stimulation, which lasted for at least 30 minutes (Fig. 1b and Additional file 1). This suggests that PS1 has a dynamic structure that responds rapidly to KCl treatment by changing the NT-loop proximity. To confirm this finding, we used another stimulus, glutamate (Glu) that was applied transiently by puffing it directly onto the imaged neuron. Again, a rapid increase in the R/G ratio was observed, indicating a change in the PS1 conformation. In this case, however, the R/G ratio returned to the baseline within 2 minutes, indicating that PS1 conformation was able to recover when the stimulant diffused (Fig. 1b). No change in the R/G ratio was observed in H 2 O-treated neurons or in neurons treated with Glu in the Ca2+/Mg2+ free media. Additional file 1 shows the GFP and RFP emission intensities and the R/G ratio in cells expressing GFP-PS1 (negative FRET control), GFP-RFP fusion (positive FRET control) or GFP-PS1-RFP, during 30 minutes recording. No GFP or RFP photobleaching was observed under the settings used. The increased R/G ratio after KCl application reflects increased FRET efficiency and is observed in G-PS1-R expressing cells only.

To verify that both treatments increase intracellular calcium load ([Ca2+] I ), sister cultures were preloaded with Oregon Green 488 BAPTA-1 AM and imaged using time-lapse settings. Changes in the [Ca2+] I strongly correlated with the R/G ratio (Fig. 1c).

These data reveal the dynamic nature of the PS1/γ-secretase and suggest that continuous insult (high intracellular Ca2+) maintains PS1/γ-secretase in a “closed” conformation, while a transient stressor modulates PS1 reversibly.

Furthermore, after 15 minutes of KCl treatment we could already detect increased levels of the secreted Aβ40 (150.5 ± 21.63 %, p = 0.0294) and Aβ42 (168.1 ± 27.24 %, p = 0.0286), resulting in an increased Aβ42/40 ratio (Fig. 1d) and consistent with the PS1 adopting a “closed” conformation at high intracellular Ca2+.

Synaptotagmin 1 is a novel calcium-dependent PS1-binding partner

To search for potential Ca2+-dependent modulators of the PS1/γ-secretase at the synapse, we performed an unbiased mass spectrometry (MS) proteomics screen of wild type (wt) mouse brain lysed in 1 % Triton X-100 in the presence or absence of Ca2+. Using GST-tagged peptides corresponding to PS1 domains or GST alone as a control, synaptotagmin 1 (Syt1) was identified as a strong candidate for novel PS1-interacting presynaptic protein (Table 1, Fig. 2, and Additional file 2). Syt1 interaction with PS1 was the strongest when GST-fused PS1 L6-7 peptide was used as “bait”, although a smaller number of Syt1 peptides was also pulled down with the GST-PS1 NT peptide. Since both L6-7 and PS1 NT sequences used for the pull down are localized within the post-endoproteolytic PS1 N-terminal fragment, the data suggest that Syt1 interacts with the PS1 NTF. Importantly, Ca2+ level affected the interaction between PS1 and Syt1, with robust PS1-Syt1 binding observed in the presence of high Ca2+. Identification of known Ca2+-insensitive PS1 interactors, such as catenin delta1, reaffirmed the specificity of the assay for Ca2+-dependent PS1 binding partners.

Table 1 Mass spectrometry screen identified Syt1 as a novel synaptic PS1-binding protein that shows calcium-dependent profile of the interaction Full size table

Fig. 2 Mass spectrometry analysis of the PS1 interacting proteins. a Coomassie-stained gel of the proteins eluted from the column. The gel slices with bands of different sizes were excised from the Ca2+ (+) and Ca2+ (-) condition from the GST-PS1 pull-downs, and were sent for mass spectrometry analysis (indicated by lines to the right of the gel). The slice selection was based on the differences in the protein profiles/band intensities between GST-control and GST-PS1 pull-down, GST-PS1 pull-down from brain lysates vs. lysis buffer, and between Ca2+ (+) and Ca2+ (-) condition (these bands are indicated with the asterisks). The bands containing Syt1 are indicated with arrowheads. Three independent MS screens were performed. The Table shows number of peptides identified and the sequence coverage for Syt1 in Ca2+ (-) and Ca2+ (+) conditions; catenin1 delta is shown as Ca2+-independent control. b Image of the coomassie stained gel from a different MS experiment shows differences in the band intensities for proteins pulled down with the GST-PS1 L6-7 (the area is overexposed in Fig. 2a). The arrowhead points to the band containing Syt1. c Schematic representation of the PS1 molecule and the GST-fusion peptides used in the MS screen of the Triton X-100-digested mouse brain lysates for PS1-binding partners; gray cylinders correspond to PS1 transmembrane domains. PS1 fragments used in the pull-down are labeled in red, green and blue. PS1 presenilin 1, GST glutathione S-transferase, Syt1 synaptotagmin 1, MS mass spectrometry Full size image

PS1 co-localizes with Syt1 at presynaptic terminals

To establish if PS1 co-localizes with Syt1 in the presynaptic terminals, we triple-immunostained synaptoneurosomes (SNSs) isolated from wt mouse cerebral cortex for: 1) PS1, 2) Syt1, and 3) either synapsin 1 (Syn1) and vesicular glutamate transporter 1 (vGlut1) or microtubule-associated protein 2 (MAP2), as pre- and postsynaptic markers, respectively.

Bright field imaging and immunostaining demonstrate the presence of the snowman-shaped SNSs formed by pre- and post-synaptic terminals (inserts in Fig. 3a) and co-localization of endogenous PS1 with Syt1, Syn1, and vGlut1 at the presynapse, in addition to PS1 presence at the postsynapse (Fig. 3a). Of note, a fraction of the synaptic terminals was PS1 negative in both pre- and post-synaptic compartments. Enrichment of the PS1/γ-secretase in the SNSs was further confirmed by western blotting and PS1-colloidal gold immunoelectron microscopy of mouse brain tissue (Fig. 3b and c). The latter showed that PS1 is present in synaptic vesicles of both the reserve and release-ready pools at the presynaptic membrane and within the postsynaptic terminals.

Fig. 3 PS1/γ-secretase present in the pre- and post-synaptic terminals. a Confocal microscopy imaging shows co-localization of PS1 with Syt1 (left) and with synapsin1 and vGlut1 (right) in presynaptic compartments of isolated synaptoneurosomes (SNSs, arrowheads). Arrows show MAP2-positive post-synaptic buttons. Insert on the left shows a bright field image of isolated SNS. b Western blot analysis of PS1 CTF, Nct, Pen-2, Syt1 and β actin as a control in total homogenate (H) and synaptoneurosome (S) fractions from wild type mouse brain. Enrichment of the γ-secretase components in SNS vs. total brain homogenate is quantified. Data are presented as mean ± SEM, n = 4. Statistical significance was determined using the unpaired student t-test; * p < 0.05, *** p < 0.001. c Electron micrographs of synaptic terminals from mouse cortex immunostained for PS1 (gold particles). Red circles and arrows indicate positive staining in presynaptic and postsynaptic compartments, respectively. PS1 presenilin 1, Syt1 synaptotagmin 1, vGlut1 vesicular glutamate transporter 1, MAP2 microtubule-associated protein 2, CTF C-terminal fragment, Nct nicastrin, Pen-2 presenilin enhancer 2 Full size image

PS1 interacts with Syt1 on endogenous levels

The use of recombinant GST-PS1 peptide pull-down in the mass spectrometry screen has some limitations due to the possibility of improper folding. Therefore, to verify occurrence of the interaction on endogenous level, we employed a different approach: co-immunoprecipitation (co-IP) of PS1/γ-secretase with Syt1 using 1 % CHAPSO-solubilized extracts of mouse hippocampi, primary neurons, and cerebral cortex synaptoneurosomes (SNSs). Indeed, Syt1 was co-immunoprecipitated with PS1 (Fig. 4), suggesting a physiological function of the interactions between PS1 and Syt1 in the brain. Of note, other members of the γ-secretase complex were also pulled down with Syt1 from the 1 % CHAPSO-solubilized extracts, in which interactions among components of the γ-secretase complex are retained (Fig. 4a).

Fig. 4 PS1 interacts with Syt1 on endogenous level. a Syt1 co-immunoprecipitates with PS1 from mouse hippocampi. The co-IP assay was conducted using anti-PS1 CT and anti-PS1 NT antibodies for pull-down; the detection antibodies are indicated to the left of each blot, n = 3. b PS1 co-immunoprecipitates with Syt1 from mouse cortical primary neurons. The co-IP assay was conducted using anti-Syt1 antibody for pull-down; the detection antibodies are indicated to the left of the blot. Other γ-secretase components pulled-down with Syt1 in the 1 % CHAPSO buffer are shown, n = 4. c Syt1 co-immunoprecipitates with PS1 from synaptoneurosome (SNS) fractions in a Ca2+-dependent manner. SNS were solubilized in 1 % CHAPSO buffer in the presence or absence of 2 mM CaCl 2 . N-terminal PS1 antibody (or IgG control) were used for pull-co-immunoprecipitated with PS1 NTF (normalized to the respective PS1 NTF band intensities). All the data are presented as mean ± SEM, n = 4. Statistical significance was determined using the unpaired student t-test, * p < 0.05. d Syt1 co-immunoprecipitates with PS1 from mouse SNSs solubilized in 1 % TritonX-100 buffer in the presence of 2 mM CaCl 2 when anti-PS1 NT, but not anti-PS1 loop or anti-PS1 CT antibody, is used for pull-down. The detection antibodies are indicated on the left side of the blot. Schematic representation of the PS1 molecule; the presumed Syt1 interaction sites are shown. PS1 presenilin 1, Syt1 synaptotagmin 1, IP immunoprecipitation, CT C-terminus, NT N-terminus, NTF N-terminal fragment Full size image

To validate the Ca2+-dependence of the interactions, SNS fractions were subjected to IP in the presence of 2 mM CaCl 2 (Ca2++) or 2 mM EGTA (Ca2+-). Indeed, more Syt1 was co-immunoprecipitated with PS1 in the high Ca2+ condition (Fig. 4c). There was no significant effect of Ca2+ on the immunoreactivity of total lysates (input lanes in Fig. 4c) or the efficiency of PS1 NTF and CTF interaction.

To examine which fragment of PS1 (NTF or CTF) predominantly binds Syt1, SNS fractions were lysed in 1 % Tx-100 buffer, disrupting interactions between the γ-secretase components [26]. The Syt1 band was detected only when anti-PS1 NT antibody was used for the pull-down, suggesting that Ca2+-bound Syt1 selectively interacts with the PS1 N-terminal fragment (Fig. 4d). Since the largest amount of the Syt1 peptides during the MS screen was pulled down with the PS1 peptide corresponding to the amino acids (aa) 263–376 of the L6–7 domain, the region was further narrowed to the aa 263–293, the C-terminal part of the PS1 NTF. Of note, a smaller number of the Syt1 peptides was also detected in the GST-PS1 NT (aa 1–80) pull-down; hence, it is possible that this region provides an additional, lower-affinity interface for the interaction (Fig. 4d).

Next, we tested whether Ca2+ influx into mouse primary neurons due to KCl-induced membrane depolarization or calcium ionophore treatment would enhance endogenous interaction between PS1 and Syt1. For this, 21–28 days in vitro cultured neurons were treated with KCl or Ca2+ ionophore (A23187) and subjected to co-IP/western blotting. An increased level of Syt1 co-immunoprecipitated with PS1 in the KCl-stimulated neurons (134.34 ± 3.14 %, p = 0.0004), compared to those treated with vehicle (Fig. 5a). An even higher increase was detected when a stronger stimulant, Ca2+ ionophore, was used (208.6 ± 22.78 %, p = 0.0089). Collectively, these data provide strong evidence for endogenous, activity- and Ca2+ influx-regulated PS1-Syt1 interactions in neurons.

Fig. 5 PS1 interacts with Syt1 in Ca2+-dependent manner in primary neurons. a Co-immunoprecipitation experiments demonstrate that KCl (n = 3) or calcium ionophore A23187 treatment (n = 3) of primary neurons that trigger Ca2+ influx strengthen the interaction between PS1 and Syt1. The asterisk shows a non-specific, heavy chain IgG band. The graph presents quantitative analysis of the Syt1 band intensity, mean ± SEM. Statistical significance was determined using the unpaired student t-test, ** p < 0.01, *** p < 0.001. b FLIM analysis of the PS1-Syt1 proximity in mouse cortical primary neurons treated for 5 minutes with 50 mM KCl (KCl+) or water control (KCl-). Fluorescence images show PS1 (green) and Syt1 (red) immunoreactivity. Scale bar: 5 μm. Pseudo-colored FLIM images depict lifetime of the Alexa 488 donor fluorophore. Colorimetric scale shows fluorescence lifetime in picoseconds. Zoomed boxed area shows FLIM image superimposed onto a table indicating average lifetimes for each pixel (~0.2 um2) of the image. Shortest lifetimes (yellow-to-red) reflect closest proximity between PS1 and Syt1. Bar graph presents [%] FRET efficiency (PS1-Syt1 proximity) recorded in the outlined regions of interest (ROIs) corresponding to the neuronal cell bodies or the processes. (mean ± SEM; n = 82 for cell bodies KCl(-), n = 63 for cell bodies KCl(+), n = 128 for processes KCl(-) and n = 146 for processes KCl(+); unpaired student t-test, *** p < 0.001). PS1 presenilin 1, Syt1 synaptotagmin 1, FLIM fluorescence lifetime imaging microscopy, FRET Förster Resonance Energy Transfer Full size image

Syt1-PS1 interaction in intact neurons is stimulated by KCl treatment

To further validate and characterize endogenous PS1-Syt1 interactions in intact neurons, we employed an antibody-based fluorescence lifetime imaging microscopy (FLIM) analysis. The FRET efficiency (%E FRET ), reflecting relative proximity between the fluorophore-labeled PS1 L6-7 epitope and Syt1, was increased in neurons stimulated with KCl, compared to vehicle-treated, suggesting more PS1-Syt1 interactions in the former (Fig. 5b).

To determine subcellular localization of the PS1 and Syt1 interactions, donor lifetime was color-coded and mapped on a pixel-by-pixel basis through the entire image. Shortest lifetimes (yellow-to-red pixels) were recorded mainly along the processes and co-localized with the dotted pattern of Syt1 immunoreactivity, presenting synaptic boutons as sites of the KCl-induced PS1-Syt1 interaction (Fig. 5b). Of note, KCl treatment resulted in a significant increase in the %E FRET along the processes, without having a considerable effect on PS1-Syt1 interactions in the cell bodies. These findings reaffirm that PS1 and Syt1 interaction occurs on endogenous level, and that Ca2+-influx induces the interaction primarily at the Syt1-positive loci along the dendrites in primary neurons.

Syt1 3D-A mutations impair Ca2+-dependent interactions between PS1 and Syt1

To gain further insight into the Ca2+-dependency of the Syt1 interaction with PS1, we constructed a 3D-A Syt1 mutant by substituting for alanine three aspartate residues important for Ca2+ binding [27]. PC12 cells lacking Syt1 (Syt1 KD) were transfected with wild type or 3D-A Syt1-V5 plasmids, treated with KCl or vehicle, harvested, and subjected to co-IP/western blotting. As expected, significantly enhanced interaction of wt Syt1-V5 with PS1 was observed in response to KCl. In contrast, 3D-A Syt1-V5 binding to PS1 was significantly reduced compared to that of wt Syt1 in KCl-treated cells (Fig. 6a, c). Analogously, the interaction between mutant 3D-A Syt1-V5 and PS1 in Ca2+ ionophore-treated Chinese hamster ovary (CHO) cells was significantly weaker compared to that of wt Syt1-V5 and PS1 (Fig. 6b, c). These results support the importance of Ca2+ binding to Syt1 in the regulation of the PS1-Syt1 interactions.

Fig. 6 Mutations in Syt1 at Ca2+–binding aspartate residues disrupt Ca2+-dependent Syt1-PS1 interaction. a Western blot analysis of the Syt1 co-immunoprecipitated with PS1 from 1 % CHAPSO lysed Syt1 KD PC12 cells transiently transfected with Syt1.wt or Syt1.3D-A encoding vectors, following 15-minute 50 mM KCl (KCl+) or water control (KCl-) treatment. Inp.- input cell lysate shows a comparable level of Syt1 in cells transfected with wild type and 3D-A mutant Syt1. IgG (control) or PS1 CT antibody was used for pull-down. b PS70 CHO cells were transfected with Syt1.wt or Syt1.3D-A expression plasmids, treated for 15 minutes with 5 μM Ca2+ ionophore (A23187), and lysed in 1 % CHAPSO buffer. PS1-Syt1 complexes were immunoprecipitated using anti-PS1 NT, anti-PS1 CT or anti-PS1 loop antibodies. The immunoprecipitation efficiency was determined by re-probing the membrane with PS1 antibodies used for pull-down. c Quantitative analysis of the Syt1 co-immunoprecipitated with PS1 from PC12 (n = 3) and CHO (n = 6) in high calcium condition after KCl and calcium ionophore treatment, respectively. More Syt1.wt than Syt1.3D-A was co-immunoprecipitated with PS1 in the above conditions. All the data are presented as mean ± SEM. Statistical significance was determined using the unpaired student t-test, *** p < 0.001. Syt1 synaptotagmin 1, PS1 presenilin 1, wt wild type, CT C-terminus, NT N-terminus, CHO Chinese hamster ovary Full size image

Syt1 affects endogenous Aβ production and PS1 conformation

The above data provide strong evidence that Ca2+ influx promotes Syt1-PS1 interactions and dynamically modulates PS1 conformation in neurons. Therefore, we reasoned that Syt1 might be involved in regulation of the Aβ production/secretion and/or PS1 conformation. To gain insight into how Syt1 may modulate Aβ levels, we employed Syt1 knockdown and overexpression approaches using parental and Syt1 KD PC12 cells (Additional file 3). Aβ40 and Aβ42 levels were reduced by 37.69 ± 2.15 % and 25.63 ± 4.31 % (p < 0.0001), respectively, in Syt1 KD cells (Fig. 7a). The deficits were partially rescued by transient Syt1-V5 expression in Syt1 KD cells. Conversely, transient overexpression of Syt1-V5 in parental PC12 cells led to increased levels of both Aβ40 and Aβ42, by 16.30 ± 5.46 % and 22.16 ± 8.66 % (p < 0.05), respectively. Of note, the levels of intracellular Aβ40 and Aβ42 were not significantly different between the parental and Syt1 KD PC12 cells (Fig. 7b), suggesting that Aβ generation may be impaired.

Fig. 7 Aβ production/secretion and PS1 conformation are altered by Syt1 KD. a, b ELISA measurements of the endogenous (A) secreted and (B) intracellular Aβ40 and Aβ42 in parental and Syt1 KD PC12 cells stimulated for 15 minutes with 50 mM KCl. The secreted Aβ levels determined in pmol were normalized to the total protein extracted from the cells in the same well. (A) n = 5 for parental + Syt1-V5 and n = 6 for all other conditions; (B) n = 4. 100 % equals to 212.93 pmol/g and 20.06 pmol/g for secreted Aβ40 and Aβ42, respectively. 100 % for intracellular Aβ40 and Aβ42 equals to 1.92 pmol/g and 0.575 pmol/g, respectively. c FLIM analysis of the FRET efficiency [%] reflecting PS1 conformational changes in parental and Syt1 KD PC12 cells; n = 101 cells for parental Ca2+(-), n = 103 for parental Ca2+(+), n = 84 for Syt1 KD Ca2+(-) and n = 86 for Syt1 KD Ca2+(+). Higher values correspond to “closed” conformational state of PS1. The cells were placed in Ca2+/Mg2+-free (-) or Ca2+/Mg2+-containing (+) medium, prior to treatment with 50 mM KCl. d ELISA measurements of the endogenous Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the conditioned medium from parental and Syt1 KD cells (n = 8). e FLIM analysis of endogenous APP and PS1 proximity (FRET efficiency [%]) in parental and Syt1 KD PC12 cells; n = 83 cells for parental Ca2+(-), n = 89 for parental Ca2+(+), n = 70 for Syt1 KD Ca2+(-) and n = 64 for Syt1 KD Ca2+(+). The cells were treated for 5 minutes with 50 mM KCl in Ca2+/Mg2+-free or normal medium, fixed, and immunostained for PS1 and APP. The data in all panels of the Figure are presented as mean ± SEM. The statistical significance was determined using the unpaired student t-test. *p < 0.05; ** p < 0.01; ***p < 0.001. Aβ amyloid β, PS1 presenilin 1, Syt1 synaptotagmin 1, FLIM fluorescence lifetime imaging microscopy, FRET Förster Resonance Energy Transfer, APP amyloid precursor protein Full size image

To test whether KCl/Ca2+ influx-induced changes in PS1 conformation and Aβ production/secretion observed in neurons (Fig. 1b, d) are mediated by Syt1, we monitored PS1 NT-loop proximity and Aβ40 and Aβ42 levels in parental and Syt1 KD PC12 cells. The cells were transfected with G-PS1-R FRET reporter and treated with KCl in regular or Ca2+/Mg2+ free media. Similarly to neurons, a significant increase in the %E FRET was observed in parental PC12 cells in the regular, Ca2+/Mg2+-containing (but not in the Ca2+/Mg2+-free) media, indicating that KCl-triggered pathological “closed” PS1 conformation requires the presence of Ca2+ ions (Fig. 7c). This corresponded to the KCl-induced increase in the Aβ40 and Aβ42 levels and the Aβ42/40 ratio in parental PC12 cells (Fig. 7d). On the contrary, no changes in PS1 conformation and Aβ in response to Ca2+ influx were detected in Syt1KD cells. Interestingly, whereas %E FRET (Fig. 7c) and the Aβ42/40 ratio (Fig. 7d) were not affected by calcium in Syt1 KD, their baseline values were significantly higher in the absence of Syt1. These data suggest that, although the total level of Aβ is reduced by Syt1 KD, the presence of Syt1 is required for the maintenance of the Aβ42/40 ratio and PS1 in the physiological, “normal” conformation.

Since Aβ levels were significantly reduced in Syt1 KD cells, we tested whether interactions between APP substrate and PS1/γ-secretase may be affected. We detected decreased proximity between the PS1 loop domain and APP CT in Syt1 KD cells, suggesting reduced PS1-APP interaction in the absence of Syt1 (Fig. 7e). In addition, while KCl treatment of parental PC12 cells increased %E FRET (PS1-APP proximity), it had no effect on the %E FRET in Syt1 KD cells. Taken together, these data suggest that Syt1 may modulate Aβ levels by influencing the assembly and/or activity of PS1/γ-secretase and its interaction with the APP substrate.

Syt1 modulates γ-secretase activity

A co-IP/western-blot analysis of the PS1 CTF co-immunoprecipitation with PS1 NTF and with the other γ-secretase components in parental and Syt1 KD PC12 cells showed increased binding between PS1 CTF and NTF, as well as between PS1 and Pen-2 and Aph1a in the absence of Syt1 (Fig. 8a). This indicates that Syt1 deficiency affects γ-secretase architecture and promotes tighter association of its components. The finding is consistent with the more compact “closed” PS1 conformation in Syt1 KD cells, as detected by the FLIM assay. At the same time, decreased levels of the PS1 N-terminal and C-terminal fragments along with the diminished Pen-2 were detected in Syt1 KD cells (Fig. 8b), suggesting reduction of the functional γ-secretase in the absence of Syt1.

Fig. 8 Syt1 influences the architecture of γ-secretase complex and Aβ generation but not AICD production. a Western blot analysis of the γ-secretase complex members co-immunoprecipitated from parental and Syt1 KD PC12 cells lysed in 1 % CHAPSO (n = 4). The bottom gel shows the level of Syt1 in the input lysates and after pull-down with PS1 C-terminal antibody or IgG control. The graph presents quantitative analysis of the PS1 NTF, Pen-2 and Aph1a co-IPed with the PS1 C-terminal fragments. The intensities of the bands were measured by densitometry, and adjusted to the intensity of the input bands and to the PS1 CTF (IP efficiency control). b Quantitative analysis of the PS1 FL, PS1 NTF, PS1 CTF, and Pen-2 in total protein extracts from parental and Syt1 KD PC12 cells (n = 4 for PS1 FL, n = 8 for PS1 NTF and PS1 CTF, n = 7 for Pen-2). The intensities of the bands were normalized to β actin levels. c Cell-free γ-secretase activity assay using parental and Syt1 KD PC12 cell membrane preparations and recombinant C100-FLAG as a substrate. The amounts of Aβ40 and Aβ42 were determined by sandwich ELISA and normalized to the levels of PS1 CTF in the corresponding samples. The intensities of AICD-FLAG bands were measured by densitometry and normalized to PS1 CTF. The quantitative data in all panels are presented as mean ± SEM, n = 4. Statistical significance was determined using the unpaired student t-test, * p < 0.05, ** p < 0.01. Syt1 synaptotagmin, Aβ amyloid β, AICD APP intracellular domain, APP amyloid precursor protein, PS1 presenilin 1, NTF N-terminal fragment, Pen-2 presenilin enhancer 2, IP immunoprecipitation, CTF C-terminal fragment, FL full length Full size image

To test more directly if Syt1 may affect PS1/γ-secretase activity, we performed a cell-free in vitro γ-secretase activity assay using recombinant C100-FLAG substrate (APP β-CTF) and membrane preparations from parental and Syt1 KD PC12 cells containing active γ-secretase. A ~30 % reduction of the Aβ40 production was detected in Syt1 KD cells (Fig. 8c). A similar trend was recorded for Aβ42 but the data did not reach statistical significance. These data confirm partial loss of γ-secretase catalytic activity in the absence of Syt1. Of note, the APP intracellular domain (AICD) level remained unaltered (Fig. 8c), supporting the hypothesis that γ-secretase cleavage of the APP β-CTF occurs in a step-wise manner [28, 29], and loss of γ-secretase activity results in “incomplete digestion” of the APP CTF substrate in Syt1 KD cells. Likewise, the γ-secretase-dependent cleavage of Notch1 at the membrane-cytoplasm interface leading to generation of the Notch1 intracellular domain (NICD) was not affected in the absence of Syt1 in PC12 Syt1 KD cells (Fig. 9).

Fig. 9 Notch1 intracellular domain (NICD) generation is not affected in Syt1 KD PC12 cells. Parental and Syt1 KD PC12 cells were transfected with Myc-tagged, constitutively active Notch1 with deleted extracellular domain (NΔECD), which is an immediate substrate of the γ-secretase. The cells were treated with DMSO or γ-secretase inhibitor — DAPT. Transfection with a plasmid encoding Notch1 intracellular domain (NICD) was used as a control. Western blot analysis shows no difference in the production of the Notch1 intracellular domain, as detected by anti-myc antibody, between parental and Syt1 KD PC12 cells. Data are presented as the NICD/NΔECD ratio; mean ± SEM, n = 4. Statistical significance was determined using unpaired student t-test. Syt1 synaptotagmin 1 Full size image

Syt1 modulates APP processing

The role of Syt1 in APP processing was further investigated by analyzing the levels of endogenous APP C-terminal fragments and secreted APPβ (sAPPβ) in the presence and absence of Syt1 in PC12 cells. About a 22.57 ± 5.21 % (p < 0.001) decrease in the total APP CTF/FL ratio was observed in Syt1 KD cells, suggesting that α- and/or β-secretase cleavage of APP may be affected (Fig. 10a). Indeed, the sAPPβ/APP FL ratio was decreased by about 32.25 ± 9.15 % (p < 0.001) in the absence of Syt1 (Fig. 10a). On the other hand, in CHO cells lacking endogenous Syt1 but stably overexpressing human APP and Syt1-V5 or only APP, the APP CTF/APP FL, sAPPα/APP FL, and sAPPβ/APP FL ratios were significantly increased in cells overexpressing Syt1 (Fig. 10b).

Fig. 10 APP CTF and sAPPβ production are reduced in the absence of Syt1. Western blot analysis of the APP processing in PC12 (a endogenous APP) and CHO (b stably overexpressed human APP) cells. The levels of sAPPβ, sAPPα, and APP CTFs (total) were normalized to FL APP. Data are presented as mean ± SEM, (A) n = 6 for APP CTF and n = 8 for sAPPβ, (B) n = 7 for APP CTF, n = 6 for sAPPα, n = 5 for sAPPβ. Statistical significance was determined using the unpaired student t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. APP amyloid precursor protein, sAPPβ secreted APPβ, Syt1 synaptotagmin 1, CHO Chinese hamster ovary cells, CTF C-terminal fragment, FL full length Full size image

BACE1 maturation and degradation are affected in Syt1 KD cells

The reduced amount of sAPPβ suggests that in addition to the γ-secretase activity, BACE1 activity may also be affected in Syt1 KD cells. Indeed, the level of endogenous total BACE1 was significantly decreased, whereas the level of immature BACE1 detected by prodomain-specific antibody was increased in Syt1 KD cells, relative to that in parental PC12 cells. Thus, the total/immature BACE1 ratio was reduced by 58.19 ± 5.14 % (p < 0.0001) in the absence of Syt1 (Fig. 11a).

Fig. 11 Syt1 modulates BACE1 maturation and stability. a Levels of total and immature BACE1 in protein lysates from parental and Syt1 KD PC12 cells were determined by western blotting using anti-BACE1 antibodies directed against the C-terminus or prodomain of BACE1, respectively (n = 5). The specificity of the antibodies was confirmed using overexpression of human BACE1 in CHO cells (top gel) and brain lysates from BACE1 KO and control littermate mice (bottom gel). The intensities of all BACE1 bands were quantified by densitometry and normalized to the levels of β actin. Data are presented as mean ± SEM. Statistical significance was determined using unpaired student t-test. ** p < 0.01, *** p < 0.001. b Analysis of BACE1 degradation. Parental and Syt1 KD PC12 cells were treated with cycloheximide and harvested 0, 2, 4, 8, 24, and 48 hours after the treatment (n = 5). Western blot presents BACE1 expression over time. Anti-BACE1 C-terminal antibody was used for detection. The quantitative analysis of BACE1 levels revealed a significantly reduced half-life of BACE1 in Syt1 KD PC12 cells compared to the parental PC12 control (marked with black dashed lines). Statistical significance was determined using 2-way ANOVA with Bonferroni post-test, *** p < 0.001 Full size image

The low levels of mature BACE1 were at least partially due to a drastic reduction in the BACE1 protein half-life in Syt1 KD cells, as revealed by the cycloheximide pulse chase assay (Fig. 11b). Of note, no difference in the stability of PS1 CTF and APP between parental and Syt1 KD cells was observed (Additional file 4).

Syt1 affects localization of APP processing enzymes

To determine whether Syt1 may modulate Aβ production by influencing trafficking of BACE1, PS1/γ-secretase, or APP, we analyzed their distribution in the parental and Syt1 KD PC12 cells using subcellular fractionation (Fig. 12a). We found that lack of Syt1 affects trafficking of PS1 CTF and BACE1 but not APP. The PS1 CTF has been retained within the earlier secretory compartments in Syt1 KD cells. On the other hand, the distribution of BACE1 has shifted towards the trans-Golgi network in the absence of Syt1.

Fig. 12 Subcellular compartmentalization of PS1, APP, and BACE1 in the presence and absence of Syt1. a Western blots show distribution of PS1, APP, and BACE1 in 13 subcellular fractions from parental and Syt1 KD cells. The arrows indicate a shift in the subcellular distribution in Syt1 KD cells. The enrichment of the respective subcellular compartments was determined by western blotting with anti-calreticulin, anti-GM130, anti-Tgn46, and anti-Rab11 antibodies. The fractions that correspond to the respective intracellular compartments are indicated above the western blots. b Bar graphs present quantitative analysis of the PS1, APP, and BACE1 levels at the plasma membrane, in total protein lysates, and the ratio of plasma membrane/total protein. Data are presented as mean ± SEM, n = 4 for PS1 in the membrane fraction, n = 8 for PS1 in total lysate, n = 5 for APP, and n = 5 for BACE1. Statistical significance was determined using the unpaired student t-test, * p < 0.05, ** p < 0.01; ***p < 0.001. PS1 presenilin 1, APP amyloid precursor protein, BACE1 β-secretase 1, Syt1 synaptotagmin 1 Full size image

The cell-surface biotinylation approach revealed a significantly reduced amount of BACE1 and PS1 CTF on the surface of Syt1 KD cells as compared to parental PC12 cells (Fig. 12b). However, since the levels of mature, functionally active γ-secretase (PS1 CTF/NTF) and BACE1 were considerably reduced in the absence of Syt1, we calculated the ratio of plasma membrane to total level for each of the proteins. The adjusted values were not significantly different between the cell lines, although there was a trend towards reduced PS1 CTF plasma membrane/total ratio in Syt1 KD cells, suggesting that the impaired intracellular transport of PS1 might cause this slight reduction of the PS1/γ-secretase at the cell surface.

To determine if the observed mislocalization of PS1 and BACE1 in the absence of Syt1 was due to a broader effect of the Syt1 KD on protein trafficking, we analyzed subcellular distribution of another synaptic protein, synaptophysin (Syp), as well as other γ-secretase members/transmembrane proteins, Nct and Pen-2, in Syt1 KD and parental PC12 cell lines (Additional file 5A and B bottom panel). No major differences in the abundance of the target proteins within the respective fractions were observed between the cell lines. Furthermore, the overall transport fidelity was not noticeably altered by the lack of Syt1, as shown by comparable distribution of the compartment specific markers within the respective fractions in PC12 cells with and without Syt1 (Additional file 5B). This further reaffirms that Syt1 selectively affects PS1 and BACE1, and that their misdistribution is not due to impairments in the overall intracellular transport fidelity in the Syt1 KD cells.

Syt1 is unlikely a substrate for PS1/γ-secretase

Since Syt1 has a relatively short N terminus, single-pass transmembrane domain and a larger cytoplasmic region, a structure similar to the previously known PS1/γ-secretase substrates, we tested if Syt1 may be a novel γ-secretase substrate. There was no difference in the levels of Syt1 and its proteolytic fragments between DMSO- and γ-secretase inhibitors-treated primary neurons and mouse embryonic fibroblasts (MEF), or in MEFs lacking PS1/PS2 (Additional file 6). Hence, it is unlikely that Syt1 is a PS1/γ-secretase substrate.