SAMP8 mice have altered hippocampal gene expression in long term potentiation, phosphatidylinositol signaling, and endocytosis pathways

Abstract

The senescence-accelerated mouse (SAMP8) strain exhibits decreased learning and memory and increased amyloid beta (Aβ) peptide accumulation at 12 months. To detect differences in gene expression in SAMP8 mice, we used a control mouse that was a 50% cross between SAMP8 and CD-1 mice and which showed no memory deficits (50% SAMs). We then compared gene expression in the hippocampus of 4- and 12-month-old SAMP8 and control mice using Affymetrix gene arrays. At 12 months, but not at 4 months, pathway analysis revealed significant differences in the long term potentiation (6 genes), phosphatidylinositol signaling (6 genes), and endocytosis (10 genes) pathways. The changes in long term potentiation included mitogen-activated protein kinase (MAPK) signaling (N-ras, cAMP responsive element binding protein [CREB], protein phosphatase inhibitor 1) and Ca-dependent signaling (inositol triphosphate [ITP] receptors 1 and 2 and phospholipase C). Changes in phosphatidylinositol signaling genes suggested altered signaling through phosphatidylinositol-3-kinase, and Western blotting revealed phosphorylation changes in serine/threonine protein kinase AKT and 70S6K. Changes in the endocytosis pathway involved genes related to clathrin-mediated endocytosis (dynamin and clathrin). Endocytosis is required for receptor recycling, is involved in Aβ metabolism, and is regulated by phosphatidylinositol signaling. In summary, these studies demonstrate altered gene expression in 3 SAMP8 hippocampal pathways associated with memory formation and consolidation. These pathways might provide new therapeutic targets in addition to targeting Aβ metabolism itself.

Keywords: Clathrin-mediated endocytosis; Hippocampal gene expression; Long term potentiation; Memory loss; Phosphatidylinositol signaling; SAMP8 mouse.

Figure 1. Cognitive Deficits in SAMP8 vs. 50% SAMP8 Mice

T-maze acquisition (learning) and T-maze retention (memory) and object recognition were measured as previously described (see Farr et al., 2012 and Methods). The top row of graphs is from generations 3–4 and the bottom row is from generation 8 of the 50% SAMP8 mice. Bars are the mean ± SD of 8–10 animals from two separate experiments. Asterisks indicate significantly different from 4 months in post-hoc testing (P < 0.05). There was a significant strain x age effect in all measures.

Figure 2. Pathway Analysis of Age and Strain Differences

(A) Differential gene expression. There was differential expression of 194 genes in 4 month mice (87 up,107 down), 536 genes in 12 month mice (386 up,150 down), and 172 genes between 4 and 12 month old mice (81 up,91 down). (B) Pathway analysis of the genes in (A) using DAVID.

Figure 3. Relative Levels of Gene Expression in Signaling Pathways

From the array data, relative expression levels (Z-scores) were calculated for genes that showed significant changes in expression. Z-scores were calculated as previously described (Haren et al., 2011) and are a measure of relative gene expression. A positive Z-score indicates increased expression and a negative Z-score indicates decreased expression relative to the average gene expression in the hippocampus (zero line). Bars are the average of 4 independent biological replicates.

Figure 4. Phosphorylation of AKT and 70S6K in 12 Month Old Mice

Total protein and phosphorylated protein were measured using specific antibodies as previously described (Haren et al., 2011). Bars are the mean and standard error of 8 mice. Asterisk indicates that Control (50% SAMP8) mice are significantly different from SAMP8 mice (P < 0.05). Western blots of hippocampal samples from four individual animals are shown below each graph.

Figure 5. Relative Levels of Gene Expression in Clathrin-Mediated Endocytosis Pathway

From the array data, Z-scores (see Fig. 3) were calculated for specific categories of genes in the clathrin-mediated endocytosis pathway. Bars are the average of 4 independent biological replicates.

Figure 6. Inter-relationship of Hippocampal Signaling Pathways Altered in SAMP8 Mice

Observed changes in phosphatidylinositol signaling and related Ca signaling, along with the key changes observed in MAPK signaling, may affect initial long term potentiation and ultimately memory formation. Altered phosphatidylinositol signaling through PI3K and AKT/mTOR/70S6K may alter the protein synthesis and RNA processing necessary for memory consolidation. Finally, phosphatidylinositol signaling is a regulator of clathrin-related endocytosis, which is important in the receptor recycling necessary in memory formation and in APP processing itself. Dotted lines indicate proposed relationships.