Memantine Improves Cognitive Function and Alters Hippocampal and Cortical Proteome in Triple Transgenic Mouse Model of Alzheimer's Disease

Abstract

Memantine is a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist clinically approved for moderate-to-severe Alzheimer's disease (AD) to improve cognitive functions. There is no report about the proteomic alterations induced by memantine in AD mouse model yet. In this study, we investigated the protein profiles in the hippocampus and the cerebral cortex of AD-related transgenic mouse model (3×Tg-AD) treated with memantine. Mice (8-month) were treated with memantine (5 mg/kg/bid) for 4 months followed by behavioral and molecular evaluation. Using step-down passive avoidance (SDA) test, novel object recognition (NOR) test and Morris water maze (MWM) test, it was observed that memantine significantly improved learning and memory retention in 3xTg-AD mice. By using quantitative proteomic analysis, 3301 and 3140 proteins in the hippocampus and the cerebral cortex respectively were identified to be associated with AD abnormalities. In the hippocampus, memantine significantly altered the expression levels of 233 proteins, among which PCNT, ATAXIN2, TNIK, and NOL3 were up-regulated, and FLNA, MARK 2 and BRAF were down-regulated. In the cerebral cortex, memantine significantly altered the expression levels of 342 proteins, among which PCNT, PMPCB, CRK, and MBP were up-regulated, and DNM2, BRAF, TAGLN 2 and FRY1 were down-regulated. Further analysis with bioinformatics showed that memantine modulated biological pathways associated with cytoskeleton and ErbB signaling in the hippocampus, and modulated biological pathways associated with axon guidance, ribosome, cytoskeleton, calcium and MAPK signaling in the cerebral cortex. Our data indicate that memantine induces higher levels of proteomic alterations in the cerebral cortex than in the hippocampus, suggesting memantine affects various brain regions in different manners. Our study provides a novel view on the complexity of protein responses induced by memantine in the brain of AD.

Keywords: Alzheimer's disease (AD); MARK2; Memantine; Proteomic.

Fig. 1. Schematic diagram of experimental design. 3xTg-AD (AD) mice were treated with memantine (5 mg/kg, bid, ig) or equal volume of saline. The mice were sacrificed after behavioral assessment, and brain tissues were isolated for further proteomic analysis. Protein was pooled from six animals in each group (WT, AD or Memantine group) and was digested into peptides. The peptides were further labeled with 6-plex TMT and HPRP fractionation followed by LC-MS/MS analysis. Proteomics analysis was performed by using PEAKS 8.5 software, and bioinformatics analysis (Heat map, GO, STRING and Wiki path) was performed using DAVID 6.8 and Cytoscape software.

Fig. 2. Memantine ameliorated cognitive impairments of 3×Tg-AD (AD) mice. (A~B) Memantine significantly increased the step-down latency and reduced the number of errors made by AD mice in the step-down passive avoidance (SDA) test. (C) Memantine improved the novel object recognition abilities (measured as Discrimination Index, DI) in AD mice in the novel object recognition (NOR) test. Bar graphs show mean±SEM, n=13 each group, ^*p<0.05 or ^**p<0.01 vs. WT group, and ^#p<0.05 vs. AD group by two-tailed unpaired Student's t-test (ANOVA).

Fig. 3. Memantine ameliorated spatial learning and memory deficits in AD mice assessed by the Morris water maze (MWM) test. (A) The escape latency of mice in training session from day 1 to day 5. (B) The swimming trajectory of mice during the probe test. (C) The latency to cross platform location (the platform was removed) during probe test. (D) Number of platform site crossovers in target quadrant during probe test. Bar graphs show mean±SEM, n=13,^*p<0.05 or ^**p<0.01 vs. WT group, ^#p<0.05 vs. AD group by two-tailed unpaired Student's t-test (ANOVA).

Fig. 4. Heat map of altered proteins induced by memantine treatment in AD mice. (A) Hierarchical clustering of 233 changed proteins in the hippocampus between AD group and memantine group (criteria: ratio ≥1.2 represented up-regulation or ratio <0.83 represented down-regulation). (B) 40 proteins in the hippocampus related to cytoskeleton, angiogenesis, RNA processing and axon guidance were significantly altered between AD group and memantine group (ratio ≥1.5 or <0.67). (C) Hierarchical clustering of 342 changed proteins in the cerebral cortex between AD group and memantine group (criteria: ratio ≥1.2 or <0.83). (D) 50 proteins in the cerebral cortex related to cytoskeleton, mitochondrion function, oxidation reduction and DNA binding were significantly altered between AD group and memantine group (criteria: ratio ≥1.5 or <0.67). Different color stands for different level of protein expression, with red indicates increase and green indicates decrease when compared with WT group.

Fig. 5. Venn diagram of two repeated analysis of significantly changed protein in the hippocampus and the cerebral cortex. (A) Expressions of 24 proteins were significantly changed in both hippocampus and cerebral cortex after memantine treatment. (B) STING diagram showing the PPI network of the 24 proteins. The gene names of the altered proteins were shown. Only 8 proteins among the 24 altered proteins were observed to have interaction with each other.

Fig. 6. Bioinformatics analysis of differentially expressed proteins of the hippocampus and the cerebral cortex. 233 and 342 changed proteins in the hippocampus and the cerebral cortex, respectively, were analyzed by DAVID GO analysis and KEGG analysis. Proteins were functionally annotated in according to their biological process, cellular component and molecular function terms, and listed according to the −Log10 (p-value).

Fig. 7. Protein-protein interaction (PPI) analysis of significantly changed proteins in the hippocampus and the cerebral cortex by using STRING database and mapped by using Cytoscape 3.6.0. (A) PPI network of 233 differentially expressed proteins in the hippocampus. (B) PPI network of 342 differentially expressed proteins in the cerebral cortex. Circles indicate protein, gray lines indicate the interactions between two proteins, red nodes indicate up-regulated proteins, and blue nodes indicate down-regulated proteins.

Fig. 8. Visualization of proteins altered by memantine treatment in the hippocampus and the cerebral cortex that were associated with the ribosome pathway. All of the proteins identified by proteomics present in the hippocampus or the cerebral cortex were imported into Cytoscape software to map ribosome Wiki pathways based on published database. The red boxes indicate up-regulated protein, blue boxes indicate down-regulated protein and gray boxes indicate unidentified proteins.