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. Author manuscript; available in PMC: 2021 Feb 21.
Published in final edited form as: Cell Syst. 2020 Dec 15;12(2):141–158.e9. doi: 10.1016/j.cels.2020.11.007

Figure 5: Altered presynaptic proteins colocalize with Aβ puncta in AppNL-G-F/NL-G-F brains and co-aggregate with Aβ in AppNL-F/NL-F and AppNL-G-F/NL-G-F brain extracts.

Figure 5:

(A-C) Representative stitched 10X magnification IHC analysis of six-month-old App KI brains with Syt1 (green), Aβ (red), and Vglut (blue) antibodies. Localization patterns of synaptotagmin 1 (Syt1) are unaffected in (A) AppNL/NL and (B) AppNL-F/NL-F. (C) Syt1 colocalizes with Aβ puncta in AppNL-G-F/NL-G-F hippocampus and cortex. (D) Representative stitched 10X magnification IHC analysis with Syt1 (green), Aβ (red), and Vgat (blue) antibodies in AppNL-G-F/NL-G-F brains. Vgat does not colocalize with Aβ in AppNL-G-F/NL-G-F brain. (E) Quantification of 10X magnification images, quantifying the number of Aβ puncta ≥ 4 pixel radii colocalized with presynaptic proteins in the cortex. Circles represent individual biological replicates. Data represent average number of colocalized puncta ± SEM. (F) Representative 63X magnification IHC analysis of Aβ plaques in AppNL-G-F/NL-G-F brain sections immuno-stained with Snap91, Snca, Calm, Pip5k1c, Snap25, Vamp1, Stx1b, Syt1, and Vamp2 (green), Aβ (red), and Vglut (blue) or Vgat (blue). (G-H) Representative MIMS quantitative images from hippocampal sections of a F1 15N labeled (G) AppNL-G-F/NL-G-F or (H) AppNL/NL mouse that was chased with 14N for 6 months. Hue saturation intensity image represents 12C15N / 12C14N ratio as an intensity for each pixel, indicating areas where 15N remains in the CA1. Arrows indicate puncta with elevated 15N / 14N ratio. N indicates nuclei. (I) AppNL-G-F/NL-G-F hippocampal section that was adjacent to MIMS section (top), immunostained for Vamp1 (green) and Aβ (red). Arrows indicate puncta positive for 15N, Vamp1, and Aβ. (J) Coomassie stain of SDS gel analysis with detergent insoluble fractions from eight-month-old App KI cortices. (K-N) Immunoblots for (K) Aβ, (L) APP, (M) Stx1b, and (N) Syt1, with quantitation of the band intensity between the well and 250 kD normalized to the same area from the Coomassie stain. Data represent average band intensity ± SD relative to AppNL/NL except for Aβ. (F, K-N) N = 3 mice per genotype, analyzed with one-way ANOVA, followed by Fisher’s LSD, * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001. Scale bar = 1 mm (A-D), 100 μm (F, I), 50 μm (G, H).