Fig 2: 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 ?-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 CaCl2. 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 CaCl2 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

Fig 3: 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

Fig 4: 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 ß 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

Fig 5: 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 ß-secretase 1, Syt1 synaptotagmin 1