Exploring temporal and sex-linked dysregulation in Alzheimer disease phosphoproteome

Abstract

This study aims to characterize dysregulation of phosphorylation for the 5XFAD mouse model of Alzheimer disease (AD). Employing global phosphoproteome measurements, we analyze temporal (3, 6, and 9 months) and sex-dependent effects on mouse hippocampus tissue to unveil molecular signatures associated with AD initiation and progression. Our findings reveal consistent phosphorylation of known AD biomarkers APOE and GFAP in 5XFAD mice, alongside candidates BIG3, CLCN6, and STX7, suggesting their potential as biomarkers for AD pathology. In addition, we identify PDK1 as a significantly dysregulated kinase at 9 months in females, and the regulation of gap junction activity as a key pathway associated with Alzheimer disease across all time points. AD-Xplorer, the interactive browser of our dataset, enables exploration of AD-related changes in phosphorylation, protein expression, kinase activities, and pathways. AD-Xplorer aids in biomarker discovery and therapeutic target identification, emphasizing temporal and sex-specific nature of significant phosphoproteomic signatures. Available at: https://yilmazs.shinyapps.io/ADXplorer.

Keywords: Neuroscience; cell biology; molecular biology.

Graphical abstract

Figure 1

Phosphosites identified as differentially phosphorylated for Alzheimer disease 5XFAD model at different time points (A–C) Volcano plots indicating the screened phosphosites with high or low phosphorylation at 3-/6-/9-month time points. The dashed horizontal lines in each plot indicate the statistical significance threshold (p < 0.1 and FDR ≤ 0.1) and the vertical line the threshold on fold changes (>2). The statistically significant phosphosites at FDR ≤ 0.1 level are marked as stars (∗). The top differentially expressed phosphosites are highlighted in each panel. (D) Venn diagram showing the number of phosphosites that pass the screening (p ≤ 0.1 and 0.5 ≥ FC ≥ 2) for 3/6/9 months data. Whereas, the numbers in parentheses indicate the significant ones at FDR ≤ 0.1 level. (E and F) Identified phosphosites that pass the screening in sex-specific analysis. The y axis indicates a lower-bound of the 95% confidence intervals for each phosphosite.

Figure 2

Proteins identified as differentially phosphorylated for Alzheimer disease 5XFAD model across different time points (Left) The bars indicate the mean phosphorylation for each protein identified as consistently phosphorylated, and the error bars indicate 95% confidence intervals. Proteins are marked according to their statistical significance levels: ∗p ≤ 0.1, ∗∗FDR ≤ 0.1, and ∗∗∗FDR ≤ 0.01.

Figure 3

Inferred kinase activities across 3-/6-/9-month time points and different sexes For each subplot, the top seven most significant kinases are shown. The error bars indicate 95% confidence intervals. Kinases are marked according to their statistical significance levels: ∗p ≤ 0.1, ∗∗FDR ≤ 0.1, and ∗∗∗FDR ≤ 0.01.

Figure 4

Dysregulation of PDK1 at protein expression and phosphorylation level for different sexes and time points of Alzheimer disease 5XFAD model (A) Protein expression (log2-FC) of PDK1 for all time points and sexes. (B) The auto-phosphorylation (log2-FC) of PDK1 for all time points and sexes. (C) Inferred activity score of PDK1 for all time points and sexes. (D) Phosphorylation (log2-FC) of known targets of PDK1 for 9 months’ data. For all plots, the coloring is done to reflect the degree of statistical significance based on Z scores.

Figure 5

Validation data results for candidate proteins Proteins are marked according to their statistical significance levels: ∗p ≤ 0.1, ∗∗FDR ≤ 0.1, and ∗∗∗FDR ≤ 0.01.