Differentially Expressed Genes in the Brain of Aging Mice With Cognitive Alteration and Depression- and Anxiety-Like Behaviors

Abstract

Despite the great increase in human lifespan with improved medical care, the physiological and pathological changes such as memory and cognitive disorders and associated anxiety and depression are major concern with aging. Molecular mechanisms underlying these changes are little known. The present study examined the differentially expressed genes (DEGs) and the genes with differentially expressed isoforms in three brain regions, anterior cingulate cortex (ACC), amygdala and hippocampus, throughout the lifespan of mice. Compared to 2-month old mice, both 12- and 24-month old mice displayed memory and cognitive impairments in the Morris water maze, Y-maze, and novel object recognition tests and depression- and anxiety-like behaviors in the tail suspension, forced swimming, open field, and elevated plus maze tests. RNA sequencing analysis identified 634 and 1078 DEGs in ACC, 453 and 1015 DEGs in the amygdala and 884 and 1054 DEGs in hippocampus in the 12- and 24-month old mice, respectively. Similarly, many genes with differentially expressed isoforms were also identified in these three brain regions in the 12- and 24-month old mice. Further functional analysis revealed that many DEGs and the genes with differentially expressed isoforms in the ACC and amygdala were mapped to depression- and anxiety-related genes, respectively and that a lot of DEGs and the genes with differentially expressed isoforms in hippocampus were mapped to cognitive dysfunction-related genes from both 12- and 24-month old mice. All of these mapped DEGs and the genes with differentially expressed isoforms were closely related to neuroinflammation. Our findings indicate that these neuroinflammation-related DEGs and the genes with differentially expressed isoforms are likely new targets in the management of memory/cognitive impairment and emotional disorders during the aging.

Keywords: RNA sequencing; aging mice; anxiety; cognitive dysfunction; depression.

FIGURE 1

The cognitive dysfunction in aging mice. n = 8 for 2- or 12-month mice, n = 6 for 24-month mice. Data were shown as mean ± SEM. (A) The recognition indexes were significantly reduced in 12- and 24-month old mice as compared to 2-month old mice at 3 and 24 h after the training session in the novel object recognition test. ^∗∗P < 0.01 versus the corresponding 2-month old mice by two-way ANOVA with repeated measures followed by post hoc Tukey test. (B) The percentages of spontaneous alterations were significantly reduced in 12- and 24-month old mice as compared to 2-month old mice in the Y maze test. ^∗∗P < 0.01 versus the corresponding 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (C) The number of arm entries was significantly reduced in 24-month old mice, but not in 12-month old mice in the Y maze test. ^∗∗P < 0.01 versus the corresponding 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (D) The learning curve was examined for 5 days in the Morris water maze test. ^∗P < 0.05 versus 2-month old mice at the corresponding time point by two-way ANOVA with repeated measures followed by post hoc Tukey test. (E,F) Platform crossing times in the probe trial of the MWM test at 1 (E) and 24 h (F) after the training session. ^∗P < 0.01 versus 2-month old mice by two-tailed unpaired Student’s t-test. (G,H) The average escape latency to reach the previous platform location was examined at 1 (G) and 24 h (H) after the training session. ^∗∗P < 0.01 versus 2-month old mice by two-tailed unpaired Student’s t-test. (I,J) Total distance of movement was examined at 1 (I) and 24 h (J) after the training session. Two-tailed unpaired Student’s t-test.

FIGURE 2

Depression- and anxiety-like behavior in aging mice. n = 8 for 2- or 12-month mice, n = 6 for 24-month mice. Data were shown as mean ± SEM. (A,B) The immobility time was significantly increased in 12- and 24-month old mice in the forced swimming test (FST) (A) and tail suspension test (TST) (B) as compared to 2-month old mice. ^∗∗P < 0.01 versus 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (C) The total distance of 24-month-old mice in the open field test was less than that of 2-month-old mice. ^∗∗P < 0.01 versus 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (D–F) The percentage of distance in the central area (D), the time spent in the central area (E), and the entries in the central area (F) were significantly reduced in 12- and 24-month old mice in the open field test as compared to 2-month-old mice. ^∗∗P < 0.01 versus 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (G,H) The percentages of the time spent in open arms (G) and open arms entries (H) were both significantly reduced in 12- and 24-month old mice as compared to 2-month old mice in the elevated plus maze test. ^∗∗P < 0.01 versus 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (I) The total arm entries of 24-month old mice were less than those of 2-month old mice. ^∗∗P < 0.01 versus 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test.

FIGURE 3

Nociceptive response in different ages of mice. Paw withdrawal frequencies (PWF) in response to 0.07 g (A,B) and 0.4 g (C,D) von Frey filaments and paw withdrawal latency (PWL) on response to heat stimulation (E,F) on the left (A,C,E) and right (B,D,F) sides. n = 8 for 2- or 12-month mice, n = 6 for 24-month mice. Data were shown as mean ± SEM. One-way ANOVA with repeated measures followed by post hoc Tukey test.

FIGURE 4

Heatmaps of differentially expressed genes (DEGs) in the ACC (A), amygdala (B) and hippocampus (C) from 2-month old mice versus 12-month old mice (top) or 24-month old mice (bottom). Colors in the heatmaps indicate the Row Z-score among the different data sets. High expression is shown by the red color spectrum, and low expression is shown by the blue. N = 3 biological repeats (3 mice)/age.

FIGURE 5

Heatmaps of differentially expressed genes (DEGs) in the ACC (A), amygdala (B), and hippocampus (C) from both 12- and 24-month old mice versus 2-month old mice. Colors in the heatmaps indicate the Row Z-score among the different data sets. High expression is shown by the red color spectrum, and low expression is shown by the blue. N = 3 biological repeats (3 mice)/age.

FIGURE 6

Verification of the changes in expression of some differentially expressed genes using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. N = 3 biological repeats (3 mice)/age. Data were shown as mean ± SEM. (A,B) Expression of Igfbpl1 mRNA in the hippocampus (A) and Ostn mRNA in ACC (B) were significantly reduced in both 12- and 24-month old mice as compared to 2-month old mice. ^∗∗P < 0.01 versus the corresponding 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test. (C,D) Expression of Klk6 mRNA in amygdala (C) and Defb1 mRNA in ACC (D) were significantly increased in 12- and 24-month old mice as compared to 2-month old mice. ^*P < 0.05, ^∗∗P < 0.01 versus 2-month old mice by one-way ANOVA with repeated measures followed by post hoc Tukey test.

FIGURE 7

Functional enrichment analysis of the differentially expressed genes (DEGs) in aging mice. Analysis of the Gene Ontology database showed top 10 biological processes of the DEGs in the ACC (top), amygdala (middle), and hippocampus (bottom) from 12- and 24-month old mice according to the P-value of biological process. The DAVID database was used to do the GO enrichment analysis. Red and blue color bars represent 12- and 24-month old mice, respectively.

FIGURE 8

Establishment of a PPI network to analyze protein-protein interactions. Top 50 differentially expressed genes (DEGs) were selected according to the connection degree of genes in the ACC (A,B), amygdala (C,D), and hippocampus (E,F) of 12- (A,C,E) and 24- (B,D,F) month old mice. The size of the node is determined by the connection degree. The connection degree reflects the importance of the gene in the network as it represents the number of nodes connected. The red, blue, and purple circles represent the genes related to inflammation, apoptosis and both inflammation and apoptosis, respectively.