Fig 2: AD-related hallmarks relationship to presynaptic proteins decrease. (A) Representative images of the whole hippocampus and zoom-in pictures of the molecular region of the DG in controls (left) and AD (right) brain sections labelled respectively for NeuN and Aß1-16. The regions of interests are indicated by outer molecular layer (OML) and inner molecular layer (IML) located next to the granule cell layer (GCL). Scale bars are 1 mm (low magnification) and 100 µm (high magnification). (B) Scatter plots with bars of the mean percentage of area covered by amyloid staining in different hippocampal molecular layers (OML, IML, CA3-ML, CA1-ML). The amyloid burden is not significantly different between the regions (one-way ANOVA, P = 0.13). (C) Graph of the mean pixel intensity of the presynaptic protein CPLX1, STX1A, SYT1 and SYNGR1 in OML in function of the amyloid burden. Each dot represents the value of one AD case. There was no correlation between the amyloid burden and the extent of the presynaptic proteins reduction. (D) Representative images of the whole hippocampus and zoom-in pictures of the molecular region of the DG in controls (left) and AD (right) brain sections labelled respectively for NeuN and phosphorylated-Tau (AT8). The regions of interests are indicated by outer (OML) and inner (IML) molecular layers located next to the granule cell layer (GCL). Scale bars are 1 mm (low magnification) and 100 µm (high magnification). (E) Scatter plots with bars of the mean percentage of area covered by neurites positive for phosphorylated-Tau (AT8) in different hippocampal subregions (OML, IML, CA3, CA1). The dystrophic neurites burden is not significantly different between the regions (one-way ANOVA, P = 0.56). (F) Graph of the mean pixel intensity of the presynaptic protein CPLX1, STX1A, SYT1 and SYNGR1 in OML in function of the burden in AT8-positive dystrophic neurites. Each dot represents the value in one AD case. There was no correlation between the burden in AT8-positive dystrophic neurites and the extent of the presynaptic proteins reduction.

Fig 3: OML-specific reduction in presynaptic proteins. (A) Schematic diagram showing the main hippocampal sub-regions in a section labelled for CPLX1 in a healthy hippocampus. (B) Schematic diagram showing the hippocampal trisynaptic circuit. (C) Schematic diagram showing the main afferents projections in OML and IML. (D, G, J, M, P) Representative images of the whole hippocampus and zoom-in pictures of the molecular region of the DG in controls (left) and AD (right) brain sections labelled respectively for CPLX1, STX1A, SYT1, SYNGR1, VAMP2. The regions of interests are indicated by outer molecular layer (OML) and inner molecular layer (IML), which are located right next to the granule cell layer (GCL). Scale bar is 1 mm (low magnification) and 100 µm (high magnification). (E, H, K, N, Q) Scatter plots of the mean pixel intensity of the fluorescent labelling in OML and IML respectively for CPLX1, STX1A, SYT1, SYNGR1, VAMP2. The fluorescence pixel intensities were significantly decreased in AD cases by 44% for CPLX1 (E, ctrl: 18.7 ± 6.4, AD: 10.6 ± 2.4; P-value = 0.0051), 42% for STX1A (H, ctrl: 17.1 ± 5.7, AD: 9.9 ± 4.4; P-value = 0.016), 29% for SYT1 (K, ctrl: 14.3 ± 3.6, AD: 10.1 ± 3.2; P-value = 0.031) and 34% for SYNGR1 (N, ctrl: 16.9 ± 6.8, AD: 11.3 ± 2.2; P-value = 0.042). In contrast, the levels of these proteins were not altered in the IML of AD cases. (Q) The levels of VAMP2 showed a non-significant reduction by 45% (ctrl: 13.7 ± 8.2, AD: 7.5 ± 3.0; P-value = 0.068) and 38% (ctrl: 16.6 ± 8.2, AD: 10.3 ± 4.1; P-value = 0.075) both in the OML and the IML, respectively. Note that in a healthy brain, CPLX1, STX1A and SYT1 displayed a stronger IR in the OML with respect to IML (D, G, J), while SYNGR1- and VAMP2-IR was more prominent in the IML than the OML (M, P). (F, I, L, O, R) Scatter plots of the mean pixel intensity OML/IML ratio of the fluorescent labellings respectively for CPLX1, STX1A, SYT1, SYNGR1, VAMP2. The ratios were significantly decreased in AD cases by 36% for CPLX1 (F, ctrl: 2.0 ± 0.2, AD: 1.3 ± 0.2; P < 0.0001), 35% for STX1A (I, ctrl: 1.2 ± 0.2, AD: 0.8 ± 0.1; P = 0.0003), 28% for SYT1 (L, ctrl: 1.1 ± 0.1, AD: 0.8 ± 0.1; P = 0.0001) and 25% for SYNGR1 (O, ctrl: 0.8 ± 0.1, AD: 0.6 ± 0.1; P < 0.0001). The ratio for VAMP2 was unaltered. Statistical descriptions are described as mean ± S.D. P-values refer to unpaired, two-tailed t-test.

Fig 4: Synaptic vesicle proteins but not postsynaptic proteins concentrate within APP accumulations (A) Human hippocampal sections immuno-labeled against APP-Cter and Shank2 and with methoxy-X04. Shank2 is prominently observed in neuronal soma and in the neuropil in both control and AD condition. (B) Close-up pictures of stainings as in ‘A’ showing the absence of Shank2 in APP accumulations surrounding an amyloid plaque labeled with methoxy-X04. (C) Human hippocampal sections immuno-labeled against APP-Cter and synaptotagmin-1 (Syt1) and with methoxy-X04. In controls, Syt1 is prominently observed in the neuropil, especially in presynaptic terminals in the CA4 region. In AD, Syt1 strikingly accumulates at the level of APP accumulations. (D) Close-up pictures of stainings as in ‘C’ showing the presence of Syt1 in APP accumulations close to an amyloid plaque labeled with methoxy-X04. (E) Close-up pictures of stainings showing the presence of VAMP2 in APP accumulations close to an amyloid plaque labeled with methoxy-X04 (full-hippocampus acquisition related to this condition are shown in FigS4A). (F) Close-up pictures of stainings showing the presence of syntaxin-1a in APP accumulations nearby an amyloid plaque labeled with methoxy-X04 (full-hippocampus acquisition related to this condition are shown in FigS4B). (G) Scatter plot of the MOC between APP-Cter and the synaptic markers labeled in Fig4. The colocalization is high for the presynaptic markers Syt1, VAMP2 and Stx1A but low for Shank2. (H) Scatter plot of the intensity of presynaptic markers labelings within APP accumulations. The data are normalized to the intensity outside the APP accumulations represented here by a dashed line. The analysis revealed that synaptic vesicle proteins Syt1 and VAMP2 are enriched inside the APP accumulations whereas the cell surface protein Stx1A is not