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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 2: Development of an in vivo screen to identify proteins with impaired turnover in App KI brains.

Figure 2:

(A) Dynamic 15N labeling of App KI mice. Schematic depicting the metabolic labeling procedure of AppNL/NL, AppNL-F/NL-F, and AppNL-G-F/NL-G-F dames (F0) with an 15N diet for six to seven months. Second generation 15N-labeled mice (F1) are further labeled with 15N during nursing and switched to an 14N diet at one month of age. Hippocampal, cortical, and cerebellar extracts from F0 and F1 mice at 7 months of age were analyzed by MS. Proteins remaining fully unlabeled (14N) in the F0 generation and heavy labeled (15N) in the F1 generation were monitored. (B) Global protein turnover [15N protein remaining = 15N / (15N+14N)] in F1 App KI mice was not significantly different between the App KI genotypes after six-month chase. N = 3565 – 4000 proteins from 4 mice per genotype. Bar represents median, analyzed by one-way ANOVA. (C) Protein turnover in AppNL-F/NL-F and AppNL-G-F/NL-G-F relative to AppNL/NL. (Top) F1 AppNL-F/NL-F and (Middle) F1 AppNL-G-F/NL-G-F protein turnover datasets of hippocampal, cortical, or cerebellar extracts relative to AppNL/NL based on 15N protein remaining. N = 3 – 4 mice per genotype. (Bottom) F0 AppNL-G-F/NL-G-F protein turnover datasets of hippocampal, cortical, or cerebellar extracts relative to AppNL/NL based on 14N protein remaining. N = 2 – 3 mice per genotype. (D) Gene ontology (GO) cell component enrichment analysis of proteins with ≥ 33% impaired turnover in AppNL-F/NL-F and AppNL-G-F/NL-G-F relative to AppNL/NL (blue circles from Figure 2C). Plots depict fold enrichment versus FDR (-log10), analyzed by Fisher’s exact test with FDR estimation. Brown represents GO terms related to axon / presynapse, yellow represents GO terms related to dendrite / postsynapse, purple represents GO terms related to entire synapse, dark grey is the GO term myelin, and grey represents all other GO terms.