Long-Term Social Isolation-Induced Autophagy Inhibition and Cell Senescence Aggravate Cognitive Impairment in D(+)Galactose-Treated Male Mice

Abstract

Aging is associated with physiological and pathological changes and presents health complications, such as dementia. Isolation has also been associated with the experience of growing old. Both have been linked individually to the incidence of cognitive decline. In this present study, the effects of these two phenomena have been looked at in animal models where aging was induced with D(+)Galactose in mice who underwent long-term post-weaned social isolation (L-PWSI). Assessing cognitive function using Y-maze, Morris water maze (MWM), and passive avoidance tests (PATs) confirmed that cognition is impaired in either of the treatments but worsened when the D(+)Galactose mice were subjected to L-PWSI. Moreover, a synaptic protein, PSD95, and dendritic spines density were significantly reduced in the L-PWSI and D(+)Galactose-treated mice. Our previous study revealed that autophagy deficit is involved in cognitive impairment in the L-PWSI model. Here, we first report the inhibited cell cycle in L-PWSI, combined with the decreased autophagy, aggravates cognitive impairment in D(+)Galactose-treated mice. Beyond these, the autophagy and cell cycle mechanisms that link isolation and aging have been explored. The close association between isolation and aging in humans is very real and needs much research attention going forward for possible therapeutic interventions.

Keywords: D(+)Galactose; aging; autophagy; cell cycle; cognition; long-term post-weaned social isolation; memory.

FIGURE 1

Experimental timelines and open field test (OFT) results to reflex the exploratory activities and anxious behaviors. (A) Experimental timelines. The long-term post-weaned social isolation (L-PWSI) mice were individually housed from postnatal day 21 for 15 weeks, and the control mice were group housing. In the course of modeling the L-PWSI (8 weeks), mice were injected with D(+)Galactose till the end of 15 weeks of L-PWSI. After the modeling, the animals underwent weeks of behavioral testing (the L-PWSI mice were still isolated) i.e., Morris Water Maze, Y-maze, and Passive avoidance tests. The mice were allowed to rest every other day during the behavior testing. At the end of the experiment, animals were sacrificed and samples were collected for western-blot and Golgi. The results of the OFT show anxiety measures. (B) Means of distance moved, (C) time moved, (D) movement speed, and (E) the time spent in the center zone in the OFT. Data are presented as the mean ± SEM from nine mice in each group. * represents p < 0.05; N.S., represents no significance.

FIGURE 2

Spatial learning behavior is worsened in the long-term post-weaned social isolation (L-PWSI) and D(+)Galactose-treated mice in the Morris water maze (MWM). (A,B) The escape latencies and relative escape latencies were calculated for the various treatment and control groups of mice. The escape latencies of the four groups of mice on the first day were normalized to 1.0. The relative escape latencies on the subsequent days were calculated relative to those on the first day. (C) The total time spent in the targeted quadrant on the fifth day of the trial. (D) The cumulative distance (to the platform) during the training session (acquisition session). (E) The swimming speed of mice on the fifth day of the trial. (F) The average number of times that the four groups of mice swam across the target sites after retrieval of the platform (two trails). (G) The number of crossing the target sites in each trail after retrieval of the platform. Data are presented as the mean ± SEM. from 9 mice in each group. * represents p < 0.05; ** represents p < 0.01; N.S., represents no significance.

FIGURE 3

The results of the Y-maze-spontaneous alternation test and New arm test in the D(+)Galactose, long-term post-weaned social isolation (L-PWSI), L-PWSI + D(+) Galactose and control groups of mice. (A) Schematic representation of the Y-maze-spontaneous alternation test. Means of novel alternation (A1), total alternation (A2), and novel percentage (A3) in Spontaneous alternation test. (B) Schematic representation of the Y-maze new arm test in the consolidation memory test. Means of novel alternation (B1), total alternation (B2), and novel percentage (B3) in New arm test. Data are presented as the mean ± SEM. from 9 mice in each group. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; N.S., represents no significance.

FIGURE 4

Treatment with long-term post-weaned social isolation (L-PWSI) and D(+)Galactose affected escape learning behavior in PAT. The latency (A), frequency (B), and permanence time (C) to (in) the darkroom on the first day of PAT. The latency (D), frequency (E), and permanence time (F) to (in) the darkroom on the second day of PAT. Data are presented as the mean ± SEM. from 9 mice in each group. * represents p < 0.05; N.S., represents no significance.

FIGURE 5

Treatment with long-term post-weaned social isolation (L-PWSI) and D(+)Galactose decreased PSD-95 and dendritic spine density. Representative micrographs (A) of Western blot (protein expression) with densitometry analysis PSD-95 (B) in the hippocampus. The grouping of gels/blots is cropped from different parts of the same gel. (C) Representative micrographs of dendrites (D) and dendritic spines density in the hippocampus. Each data column represents the mean ± SEM obtained from 4 brain samples. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; N.S., represents no significance.

FIGURE 6

Treatment with long-term post-weaned social isolation (L-PWSI) and D(+)Galactose altered autophagy and cell-cycle molecules. Representative micrographs of Western blot (protein expression) with densitometry analysis of (A) phosphorylated mammalian target of rapamycin (p-mTOR), (B) mTOR, (C) p-ULK1, (D) ULK1, (E) p62, (F) Beclin 1, (G) p16, (H) cyclin D1, and (I) PCNA in the hippocampus. The grouping of gels/blots is cropped from different parts of the same gel. Each data column represents the mean ± SEM obtained from four brain samples. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; N.S., represents no significance.