In Vivo Prenylomic Profiling in the Brain of a Transgenic Mouse Model of Alzheimer's Disease Reveals Increased Prenylation of a Key Set of Proteins

Abstract

Dysregulation of protein prenylation has been implicated in many diseases, including Alzheimer's disease (AD). Prenylomic analysis, the combination of metabolic incorporation of an isoprenoid analogue (C15AlkOPP) into prenylated proteins with a bottom-up proteomic analysis, has allowed the identification of prenylated proteins in various cellular models. Here, transgenic AD mice were administered with C15AlkOPP through intracerebroventricular (ICV) infusion over 13 days. Using prenylomic analysis, 36 prenylated proteins were enriched in the brains of AD mice. Importantly, the prenylated forms of 15 proteins were consistently upregulated in AD mice compared to nontransgenic wild-type controls. These results highlight the power of this in vivo metabolic labeling approach to identify multiple post-translationally modified proteins that may serve as potential therapeutic targets for a disease that has proved refractory to treatment thus far. Moreover, this method should be applicable to many other types of protein modifications, significantly broadening its scope.

Figure 1.. Brain metabolic labeling after a single bolus ICV injection and 13-day ICV infusion of C15AlkOPP

(A) Structures of endogenous substrates for prenylation, alkyne-containing diphosphate analogue (C15AlkOPP) used in this study and the alcohol (C15AlkOH) analogue. (B) Schematic representation of ICV bolus injection and ICV infusion and (C) the relative locations of brain coronal sections (400 μm) from the injection/infusion site that were used for the representative in-gel fluorescence. The brain section (600 μm) located between section #1 and #2 was PFA-fixed for in situ click reaction and imaging. (D) In-gel fluorescence and Coomassie blue gel staining images of brain regions depicted in B. Brains were harvested 48 hours after the left ICV bolus injection of 150 mM C15AlkOPP (10 μL) or 10 mM Simvastatin (SV) (10 μL) + 150 mM C15AlkOPP (10 μL); or 13 days after the initiation of left ICV infusion of followings: 150 mM C15AlkOPP, or 10 mM simvastatin (SV) + 150 mM C15AlkOPP (1:3).

Figure 2:. Prenylomic profiling of WT mice after ICV infusion of C15AlkOPP with or without a statin treatment.

(A) Schematic representation of the prenylomic workflow depicting the enrichment of prenylated proteins, their digestion, and the subsequent division of peptides into three replicates. This scheme shows the process for C15AlkOPP treated samples. The vehicle sample was treated in the same manner with the only change being that vehicle replicates were labeled with TMT 126, TMT127, and TMT128 reagents. (B) Gel fluorescence analysis of the brain samples used in this analysis. After harvesting, the left side of the brain was lysed and subjected to a click reaction with TAMRA-azide to visualize the level of alkyne-probe incorporation in these samples. Lane 1 contains vehicle sample, Lane 2 contains C15AlkOPP sample and Lane 3 contains the sample treated with C15AlkOPP and statin. (C) Volcano plot comparing C15AlkOPP with vehicle ICV infusion (FDR=5%). (D) Volcano plot comparing C15AlkOPP and simvastatin coadministration with vehicle ICV infusion (FDR=5%). (E) Venn diagrams showing the distribution of prenylated proteins obtained from the volcano plots shown in panels C and D. These are further subdivided by type of prenylation shown in color. Proteins that are grouped differently across the pairs are entered as one protein. Example: In C15AlkOPP treatment alone Rab3a,3b,3c are grouped, and in the SV+C15AlkOPP treated mouse Rab 3a,3b, and 3c are not grouped. Therefore, for simplicity, Rab 3a,3b and 3c are one data point in the Venn diagram. Color scheme: prenylated proteins that are known substrates for FTase (red); prenylated proteins that are substrates for GGTase I (blue); prenylated proteins that are known substrates for GGTase II (green).

Figure 3.. Brain metabolic labeling of APP/PS1 and wild type control mice.

(A) Schematic representation of the site of ICV infusion and (B) brain area used for the representative in-gel fluorescence and the prenylome profiling. (C) In-gel fluorescence and Coomassie blue gel staining images of WT and APP/PS1 mice after 13-day left ICV infusion of 100 mM C15AlkOPP. Full-length APP (FL-APP) was measured via immunoblot assay to confirm the genotypes.

Figure 4.. Prenylomic analysis of three pairs of APP/PS1 vs WT mice both subjected to ICV infusion of C15AlkOPP.

(A) Volcano plots for each pair of mice (FDR=5% for all three plots). Data was processed in MaxQuant with the same parameters with non-prenlyated proteins removed for clarity. Color scheme: prenylated proteins that are known substrates for FTase (red); prenylated proteins that are substrates for GGTase I (blue); prenylated proteins that are known substrates for GGTase II (green). (B) Venn diagram showing the distribution of prenylated proteins across the three pairs of mice. This is further subdivided by the type of prenylation shown in color. Proteins that are grouped differently across the pairs are entered as one protein. Example: In pair 2 Hras;Kras;Nras are grouped, and in pair 3 Kras is not grouped and Hras;Nras are. So for simplicity, Hras;Nras and Kras are one data point in the Venn diagram. (C) Graph indicating the fold-change for the 15 proteins observed across all three pairs.

Figure 5.. Gene Ontology (GO) term, KEGG pathway enrichment analyses and protein-protein interaction network of identified prenylated proteins and known AD network proteins.

(A) Top enriched GO terms and KEGG pathways among prenylated proteins enriched in APP/PS1 mice. (B) Protein-protein interaction network showing direct/indirect interactions between AD network proteins and enriched prenylated proteins in APP/PS1 mice. A total of 19 prenylated proteins have known interactions with proteins involved in AD pathogenesis including amyloid precursor protein (App) and microtubule-associated protein tau (Mapt).

Figure 6:. Transcriptomic and native abundance comparisons showed that prenylation is upregulated in the APP/PS1 mice.

A) comparisons of the log₂ fold change for the 15 common enriched proteins found in the prenylomic analysis of APP/PS1 mice (orange) compared to a total bottom-up proteomic analysis of APP/PS1 mice compared to WT from externally reported data from proteome exchange (green). B) comparisons of the log₂ fold change for the 15 common enriched proteins found in the prenylomic analysis of APP/PS1 mice (orange) compared to previously reported RNAseq data comparing the expression of proteins in APP/PS1 mice to WT mice. Numerical values for plot are in SI Table 2.