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. 2018 Jan 16;13(1):2.
doi: 10.1186/s13024-017-0234-4.

Molecular and Functional Signatures in a Novel Alzheimer's Disease Mouse Model Assessed by Quantitative Proteomics

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Free PMC article

Molecular and Functional Signatures in a Novel Alzheimer's Disease Mouse Model Assessed by Quantitative Proteomics

Dong Kyu Kim et al. Mol Neurodegener. .
Free PMC article

Abstract

Background: Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by the deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles. To understand the pathological mechanisms underlying AD, developing animal models that completely encompass the main features of AD pathologies is indispensable. Although mouse models that display pathological hallmarks of AD (amyloid plaques, neurofibrillary tangles, or both) have been developed and investigated, a systematic approach for understanding the molecular characteristics of AD mouse models is lacking.

Methods: To elucidate the mechanisms underlying the contribution of amyloid beta (Aβ) and tau in AD pathogenesis, we herein generated a novel animal model of AD, namely the AD-like pathology with amyloid and neurofibrillary tangles (ADLPAPT) mice. The ADLPAPT mice carry three human transgenes, including amyloid precursor protein, presenilin-1, and tau, with six mutations. To characterize the molecular and functional signatures of AD in ADLPAPT mice, we analyzed the hippocampal proteome and performed comparisons with individual-pathology transgenic mice (i.e., amyloid or neurofibrillary tangles) and wild-type mice using quantitative proteomics with 10-plex tandem mass tag.

Results: The ADLPAPT mice exhibited accelerated neurofibrillary tangle formation in addition to amyloid plaques, neuronal loss in the CA1 area, and memory deficit at an early age. In addition, our proteomic analysis identified nearly 10,000 protein groups, which enabled the identification of hundreds of differentially expressed proteins (DEPs) in ADLPAPT mice. Bioinformatics analysis of DEPs revealed that ADLPAPT mice experienced age-dependent active immune responses and synaptic dysfunctions.

Conclusions: Our study is the first to compare and describe the proteomic characteristics in amyloid and neurofibrillary tangle pathologies using isobaric label-based quantitative proteomics. Furthermore, we analyzed the hippocampal proteome of the newly developed ADLPAPT model mice to investigate how both Aβ and tau pathologies regulate the hippocampal proteome. Because the ADLPAPT mouse model recapitulates the main features of AD pathogenesis, the proteomic data derived from its hippocampus has significant utility as a novel resource for the research on the Aβ-tau axis and pathophysiological changes in vivo.

Keywords: 10-plex tandem mass tag; Alzheimer’s disease; Animal disease model; Aβ; Quantitative proteomics; Tau.

Conflict of interest statement

Ethics approval and consent to participate

Animals were treated and maintained as per the Helsinki Treaty, the Principles of Laboratory Animal Care (NIH publication No. 85–23, revised 1985), and the Animal Care and Use Guidelines of Seoul National University, Seoul, Korea. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Pathological characterization of a novel animal model of Alzheimer’s disease. a and c For detection of amyloid plaques, hippocampal regions of ADLP mice were immunostained with the biotin-4G8 antibody at 4-, 7-, and 10-month-old. The percentage of amyloid plaques area was not significantly different between ADLPAPP/PS1 and ADLPAPT mice (Student’s t-test, n = 3–4 per group). Scale bar represents 200 μm. b and d) The hippocampal CA1 layer of ADLP mice was stained with the AT8 antibody against phosphorylated tau (Ser202/Thr205). A significant increase in AT8 immunoreactivity was observed in ADLPAPT mice compared with age-matched ADLPTau mice (Student’s t-test, n = 3–4 per group). Scale bar represents 200 μm. e Sarkosyl-insoluble tau fractions from 7 and 10 months ADLP mice hippocampus were analyzed by western blot analysis using human tau specific antibody (Tau13). f Each distinct size of sarkosyl-insoluble tau was quantified in 10 months old ADLPTau and ADLPAPT mice (Chi-square test; n = 6 mice per genotypes). g The CA1 pyramidal neurons of ADLP mice were stained with anti-NeuN antibody to determine degrees of neuronal loss. Scale bars represents 100 μm or 50 μm (enlarged figures). h Quantification of the number of CA1 neurons in 7- and 10-month-old ADLP mouse model (one-way ANOVA in each age of ADLP mouse model). i ADLP model mice showed memory impairment compared with wild-type mice, which examined by the Y-maze test (one-way ANOVA in each age of ADLP mouse model, n = 9–11 per group). Results are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
Fig. 2
Fig. 2
Hippocampal proteome analysis of ADLP model mice. a Graphical illustration of the workflow used for our TMT-based proteomic analysis. The detailed sample assignments for TMT labeling are shown in Fig. S2A. b A total of 9814 protein groups were identified in our study; of them, 7022 protein groups were identified and quantified in all experimental sets. The “Not quantified” proteins were identified via search algorithm, but their reporter ions were not detected and were excluded from subsequent quantitative analysis. c A bar chart showing the number of proteins identified in the hippocampus of each ADLP model. d Dynamic range of protein abundance, spanning six orders of magnitude. Normalized reporter ion intensities of pooled samples in each experimental set were used. Proteins known as AD risk factors are annotated in the plot
Fig. 3
Fig. 3
Abundant and enriched proteins in the hippocampus of transgenic ADLP model mice. Scatter plot of relative protein expression levels in ADLP versus wild-type mice (y-axis). The x-axis represents the normalized protein abundance (ratio to pooled sample). Each graph was vertically placed along with ADLP model type and horizontally placed in order of age/disease progression. Significantly regulated proteins (Student’s T-test p-value <0.05 and fold-change cut-off: 1.25) are shown as orange circles. *APP = ADLPAPP/PS1, Tau = ADLPTau, APT = ADLPAPT mice
Fig. 4
Fig. 4
Protein quantitation overview and comparative pathway enrichment analysis of ADLP model mice. a Hierarchical clustering of differentially expressed proteins (DEPs) across different transgenic types and ages (ANOVA FDR < 0.05). Protein expression profiles were largely clustered into three patterns; The decreasing pattern (Cluster 2) and the increasing pattern (Cluster 3) of the mouse models with human APP (ADLP APP/PS1, ADLP APT) are prominent. The right panel shows the Z-normalized protein abundance according to the mouse samples as profile plots. b Canonical pathway enrichment for DEPs. The categories of nervous system signaling and immune response were overrepresented (~50%) and are highlighted. The significant pathways (Fisher’s exact test p-value <0.05) were deduced using Ingenuity Pathway Analysis (IPA) and their predictive activation/inhibition status is represented as the Z-score. *APP/PS1 = ADLPAPP/PS1, Tau = ADLPTau, APT = ADLPAPT mice
Fig. 5
Fig. 5
Protein clustering reveals major biological and molecular signatures of ADLPAPT mice. a Hierarchical clustering of differentially expressed proteins (DEPAPTs) between ADLPAPT and wild-type mice (732 proteins; ANOVA FDR < 0.05). The DEPsAPT were clustered into five types of expression pattern. The protein expression level is the normalized protein abundance represented after Z-normalization. Scatter plot of enriched Gene Ontology (GO) terms in protein cluster 2 (down-regulated proteins) and cluster 5 (up-regulated proteins) are shown in the right panel. The -log10 (P-value) is plotted against the fold-enrichment of each GO term. b Heatmaps showing the expression levels of the proteins involved in the GO terms, such as synapse proteins and cytoskeleton binding proteins, in cluster 2 (down-regulated proteins). c Heatmaps for the expression levels of co-regulated proteins corresponding to the GO terms, such as lysosome, endosome, leukocyte-mediated immunity, and phagocytosis, in cluster 5 (up-regulated proteins)
Fig. 6
Fig. 6
Prediction of molecular responses to AD pathology in ADLPAPT mice. a Hierarchical clustering of downstream biological functions (lower panel) and the upstream regulators (upper panel) assessed by IPA using 732 DEPsAPT (ANOVA FDR < 0.05). The predictive activation/inhibition status is shown as a Z-score. b Graphical representation of the protein expression levels of members of the leukocyte extravasation pathway and (c) the synaptic long-term potentiation pathway in 10-month-old ADLPAPT mice. Individual proteins’ fold-change values to the wild type were visualized in color (blue: down-regulation, red: up-regulation). Glu = Glutamate, PIP2 = Phosphatidylinositol 4,5-bisphosphate, DAG = Diacylglycerol, IP3 = Inositol 1,4,5-trisphosphate, ATP = Adenosine triphosphate, cAMP = Cyclic adenosine monophosphate
Fig. 7
Fig. 7
Exclusive DEPs in ADLPAPT mice and their related functional networks. a Proteins that were significantly altered in early or late ADLPAPT mice (Student’s t-test p-value <0.05 and fold-change >1.25) but not in the other models (fold change <1.25) were selected as exclusive DEPsAPT. b The biological functions derived from the GO analysis are shown along with their networking with associated proteins. c The knowledge database-derived protein network of App, Mapt, and exclusive DEPsAPT. The fold-change values of individual protein nodes were visualized in color circles (blue: down-regulation, red: up-regulation). The color of the inner circle is the fold-change of the 4-month ADLPAPT mouse, and the color of the outer circle represents that of the 10-month ADLPAPT mouse. d Three proteins that form bridge between App and Mapt (Hcls1, Ptprc and Abca1) were validated by western blot analysis. Representative western blot images are shown herein. The expression levels of the proteins are quantified compared with wild type mice (n = 3 per genotype for 4 and 10 months; Student’s t test). Results are expressed as the mean ± SEM. **P < 0.01; ***P < 0.001

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