Fig 1: Differential expression analysis of proteins in brains of 5xFAD mice. a Volcano plots depicting the protein expression logarithmic fold-changes (log10 fold-change, x-axis) and the adjusted p-values (- log10 p-value, y-axis). Differentially Expressed proteins across all time points (Age 2, 5, and 8) in cortex and hippocampus were analyzed (DE.cortex.Age2, DE.cortex.Age5 and DE.cortex.Age8; DE.hippo.Age2, DE.hippo.Age5 and DE.hippo.Age8). All proteins highlighted in blue have significantly altered expression. The candidate AD biomarker Arl8b, marked in red, is significantly upregulated in hippocampus. Gene names for potential proteins of interest with highly significant fold-changes are indicated. Proteins not significantly changed in their abundance are highlighted in gray. Significance was determined using a two-tailed t-test and a Benjamini–Hochberg False Discovery Rate (FDR) set to 5%. b Numbers of differentially expressed proteins obtained by comparing the protein expression measurements of 5xFAD mice at different ages with their corresponding, age-matched, wild-type controls in cortex and hippocampus. The identifiers of the amounts of proteins analyzed were denoted analogously as in panel a. Bars indicate the numbers of significant down- (blue) and upregulated (red) proteins. c Numbers of proteins significantly differentially regulated. A2TC (Age 2, Tissue Cortex), A5TC and A8TC label the numbers of proteins for which the transgene effect is significant in cortex, at 2, 5, and 8 months, respectively. Proteins more abundant in 5xFAD mice are shown in red, while proteins more abundant in wild-type mice are shown in blue. A2TH (Age 2, Tissue Hippocampus), A5TH, and A8TH label the same comparison in hippocampus. A52TC (Age 5 vs 2, Tissue Cortex), A85TC and A82TC refer to the numbers of proteins for which the transgene effect is significantly different at two time points (5 vs 2, 8 vs 5, and 8 vs 2 months) in cortex. The numbers of proteins which increase in abundance with age are shown in red, while the numbers of proteins that decrease with age are shown in blue. The corresponding labelling for the hippocampus samples are A52TH (Age 5 vs 2, Tissue Hippocampus), A85TH and A82TH. Finally, A2THC (Age 2, Tissue Hippocampus vs Cortex), A5THC and A8THC report the numbers of proteins for which the transgene effect is significantly different in cortex and hippocampus at 2, 5, and 8 months, respectively. Higher hippocampus abundance is shown in red on the right, and higher cortex abundance in blue on the left. The underlying data are available as Additional file 3: Supplementary Excel File 1c. d–f Venn diagrams showing the numbers of total (d), downregulated (e), and upregulated (f) proteins in cortex and hippocampus, including only significantly differentially expressed proteins (DEPs). The amounts of DEPs were denoted analogously to panel c but were combined across all time points (months 2, 5, and 8); the combination of DEPs is indicated by the x (e.g., AxTC). g Correlation analysis of DEPs for A2TC, A8TC, A2TH, A5TH, and A8TH. The degree of correlation was assessed by Spearman correlation coefficients (rS) and corresponding FDR-adjusted p-values (**, p < 0.01; ***, p < 0.001). Crosses indicate no correlation. The colors denote the values of the Spearman correlation coefficients. h Example Spearman inverse correlation of A2TC versus A8TC also shown in panel g. Time-dependent changes in the abundance of 8 mitochondrial proteins (i) and transcripts (j) that play a key role in oxidative phosphorylation and ATP production. The temporal changes of the LFCs across all ages (2, 5, and 8 months) in cortex (orange) and hippocampus (blue) are shown. The statistical significance of the differentially expressed proteins and transcripts was measured with a two-tailed t-test, adjusted by the Benjamini–Hochberg multiple testing correction (*, p < 0.05, **, p < 0.01; ***, p < 0.001). All analyses are based on mean values of measured intensities from five biological replicates of tg mice per age and tissue (n = 5)
Fig 2: Identification of Aß-correlated and anticorrelated protein alterations in 5xFAD brains. a Numbers of proteins that correlate (corr, in red) or anticorrelate (acorr, in blue) with Aß aggregates in hippocampus and cortex and are differentially expressed at 2 (DE.cortex.Age2, DE.hippo.Age2), 5 (DE.cortex.Age5, DE.hippo.Age5), or 8 (DE.cortex.Age8, DE.hippo.Age8) months. The identifiers were denoted analogously as in Fig. 2a. The degree of correlation was assessed by Pearson correlation and corresponding FDR-adjusted p-values. b Volcano plots depicting the protein expression logarithmic fold-changes (log10 fold-change, x-axis) and the adjusted p-values (- log10 p-value, y-axis) across all time points (age 2, 5, and 8 months) for DEPs that correlate (corr) or anticorrelate (acorr) with Aß aggregates in hippocampus (h) and cortex (c). All proteins highlighted in blue are expressed significantly differently. The Aß-correlating AD biomarker candidate Arl8b is marked in red. Proteins of interest with highly significant fold-changes are indicated with gene names. Proteins not significantly changed are highlighted in gray. Significance was determined using a two-tailed t-test and a Benjamini–Hochberg False Discovery Rate (FDR) set to 5%. c, d Changes of APOE transcript (c) and protein (d) levels in hippocampus (H) and cortex (C) of 2-, 5-, and 8-month-old 5xFAD mice. LFC, log2 fold-change. e Number of differentially expressed genes (DEGs, light red) that overlap (purple) with Aß aggregate-correlated (corr) and anticorrelated (acorr) DEPs (light blue). f Time-dependent changes in the abundance of 9 correlating or anticorrelating molecules; both protein (top) and transcript (bottom) level changes are shown. The temporal changes of the t-scores across all ages (2, 5, and 8 months) in cortex (orange) and hippocampus (blue) are illustrated. The statistical significance of differentially expressed proteins and transcripts was measured with a two-tailed t-test, adjusted by the Benjamini–Hochberg multiple testing correction (*, p < 0.05, **, p < 0.01; ***, p < 0.001). g Ingenuity pathway analysis (IPA) for the correlating or anticorrelating molecules that are changed both at the protein and transcript level as shown in panels e and f. The statistical significance of the association between the DEPs and the canonical pathways was measured with right-tailed Fisher’s exact test to calculate the p-values, adjusted by the Benjamini–Hochberg multiple testing correction. All analyses are based on mean values of measured intensities from five biological replicates of tg mice per age and tissue (n = 5)
Fig 3: The protein Arl8b is upregulated in hippocampal tissues of 5xFAD mice. a Time-depended change of Arl8b protein abundance in hippocampal and cortical tissues of 5xFAD transgenic animals. LFC, log2 fold-change. b Hippocampal brain homogenates prepared from four 8-month-old 5xFAD (1 to 4) and control (ctrl; 1 to 4) mice were analyzed by SDS-PAGE and immunoblotting using anti-Arl8b. As a control, a tubulin (anti-alpha Tubulin, #T6074) immunoblot was performed. c Quantification of Arl8b expression in relation to tubulin using band intensities of immunoblots in b. Relative intensity values (mean ± SD) are shown for AD (n = 4) and Ctrl (n = 4) mice. Statistical significance was assessed between AD and Ctrl mice using an unpaired, two-tailed t-test (*, p = 0.0137). d Immunofluorescence analysis of 5xFAD mouse (8 months) brain slices using AlexaFluor594-labelled 6E10 antibody (red); an anti-Arl8b antibody combined with an AlexaFluor647-labelled anti-rabbit IgG (turquoise) was applied to detect Arl8b. The scale bar shown in the 6E10 image also applies to the Arl8b and merge image. The picture on the right shows a magnification (magnif.) of an area indicated in the merged picture. e Brain slices of 8-month-old 5xFAD mice were stained with the primary antibodies indicated in the images. For detection with 352 and Lamp1 antibodies, an AlexaFluor594-labelled anti-mouse IgG (red) was used; for Arl8b detection, an AlexaFluor647-labelled anti-rabbit IgG (turquoise) was applied. Antibody 352 specifically recognizes Aß42 fibrillar aggregates [30]. f Pearson correlation between the volumes of Arl8b accumulations and 6E10-stained amyloid-beta plaques in hippocampus of 2- (H2), 5- (H5), and 8- (H8) month-old 5xFAD mice. A total of 60 plaques were analyzed in brain slices derived from 5- and 8-month-old 5xFAD mice. For 2-month-old 5xFAD mice, less than 60 plaques were analyzed, since amyloid burden at this age is low. The statistical significance of the association between the volumes of Arl8b accumulations and 6E10-stained amyloid-ß plaques was measured with a two-tailed t-test (*, p = 0.024; ****, p < 0.0001)
Fig 4: Analysis of Arl8b expression in human brain and CSF samples. a Detection of Arl8b protein aggregates in postmortem brain homogenates of 10 AD patients (1 to 10, black lettering) and 10 age-matched controls (11 to 20, red lettering) using a native MFA. Triplicates per sample were filtered. For immunoblotting, an anti-Arl8a/b antibody was used. b Quantification of protein aggregates retained on filter membrane in a was performed using Aida image analysis software. Data are expressed as mean ± SD. The statistical significance was assessed with an unpaired, two-tailed t-test (****, p < 0.0001). c Determination of Arl8b concentrations in CSF samples of 38 AD patients and 44 control individuals (Ctrl) using an ELISA. Data represent mean ± SD. Statistical significance was determined using an unpaired, two-tailed t-test (***, p = 0.0002). d Arl8b ELISA using CSF samples of 10 Huntington’s disease (HD) patients, 10 controls (Ctrl HD), 3 AD patients, and 3 controls (Ctrl AD). Data are mean ± SD. Statistical significance was evaluated using an unpaired, two-tailed t-test between HD and Ctrl HD, and AD and Ctrl AD groups (**, p = 0.0041). e ROC analysis of Arl8b levels derived from both AD patients and control individuals using GraphPad Prism software. The area under the curve (AUC) is 0.73 with a p-value of 0.0003
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