Chronic social isolation-unpredictable stress induces early-onset cognitive deficits and exacerbates Aβ accumulation in the 5xFAD mouse model of Alzheimer's disease

Abstract

Aging and genetic predisposition are the primary risk factors for Alzheimer's disease (AD), while chronic stress represents a modifiable risk factor that can accelerate aging and drive AD progression. However, the complex interplay between aging, chronic stress and genetic underpinnings in AD pathogenesis remains poorly understood. Notably, cognitive phenotyping in AD mouse models has yielded inconsistent results. In this study, we characterized the age-dependent trajectory of phenotypes in 5xFAD mice on a congenic C57BL/6 J background. These mice harbor five familial AD (FAD)-related mutations in the amyloid precursor protein (APP) and presenilin 1 (PSEN1) genes. Aβ plaque deposition was detected in specific brain regions by 4 months of age, but cognitive performance remained intact at this stage. However, by 8-9 months, these mice developed impairments in spatial working memory, novel object recognition memory, and social recognition memory. By 11 months, they also showed metabolic alterations, including lower body weight, higher energy expenditure and increased locomotor activity. Furthermore, after 10 days of chronic social isolation-unpredictable stress, 4-month-old 5xFAD mice exhibited cognitive deficits, accompanied by increased Aβ accumulation in the medial prefrontal cortex, hippocampus, and entorhinal cortex. In contrast, age- and sex-matched wild-type littermate controls subjected to the same stress paradigm showed no significant cognitive changes. These observations suggest that Aβ deposition increases stress vulnerability in 5xFAD mice. We conclude that the phenotypic expression of AD-related gene mutations, including pathological changes and cognitive decline, progresses with age and can be induced by chronic psychological stress, underscoring the interactive effects of stress, aging, and genetic vulnerability on disease onset and severity.

Fig. 1. Age-dependent impairments in spatial working memory and object recognition memory in 5xFAD mice.

A Y-maze spontaneous alternation test. (A1) Schematic of the Y-maze test. (A2–A3) Spontaneous alternation rate (left) and total arm entries (right) of male 5xFAD mice and WT littermate control mice tested at 3–6-months old (A2) and 9–10-months old (A3). A2: WT, n = 19; 5xFAD, n = 26. A3: WT, n = 11; 5xFAD, n = 14. *P < 0.05, *P < 0.01 compared with WT mice. B Novel object recognition (NOR) test. (B1) Schematic of the experimental procedure of the NOR test. (B2) Trial 1 with a 2-h retention interval. Left, representative images of heat maps depicting the time spent by the mice in the test arena. Middle, exploration time between familiar (F) and novel object 1 (N1). Right, discrimination index. (B3) Trial 2 with a 24-h retention interval. Left, representative images of heat maps depicting the time spent by the mice in the test arena. Middle, exploration time between familiar (F) and novel object 2 (N2). Right, discrimination index. n = 7–11 male mice per group. For exploration time, **P < 0.01, paired t-test compared between familiar and novel object. For discrimination index, **P < 0.01 compared with WT mice.

Fig. 2. Age-dependent impairment of social recognition memory in 5xFAD mice.

A Schematic of the 3-chamber sociability and social recognition test. B Three-chamber social test on 4-month-old mice. n = 9 male mice per group. C Three-chamber social test on 8-month-old mice. n = 6–7 male mice per group. B, C Upper panel: Sociability test. Upper left, representative images of heat maps depicting the time spent in each chamber. Upper middle, time spent in chamber with novel object (NS) and stranger mouse 1 (S1). Upper right, social preference index. Lower panel: Social recognition test. Lower left, representative images of heat maps depicting the time spent in each chamber. Lower middle, time spent in chamber with stranger mouse 1 (S1) and stranger mouse 2 (S2). Lower right, social recognition index. For time in chamber, *P < 0.05, **P < 0.05, ***P < 0.001, paired t-test compared between NS and S1, or between S1 and S2. For social recognition index, *P < 0.05 compared with WT mice.

Fig. 3. Whole-brain analysis of Aβ plaque accumulation in 5xFAD mice.

A Staining of Aβ plaques on coronal brain sections at the indicated ages. The drawings of the sagittal planes were adapted from the Atlas of Paxinos and Franklin [91] with blue lines indicating the focal plane of coronal sections. PFC prefrontal cortex, CTX cortex, HIPP hippocampus, EC entorhinal cortex, Sub subiculum. B Schematic illustration of the iDISCO method for visualizing the whole-brain distribution of Aβ plaque accumulation. Illustrations were created with BioRender.com. C Each image stack was registered to the 3-D Allen Mouse Brain Atlas. D A single sagittal plane image showing the deposition of Aβ plaques in the brain at 4 and 8 months of age. E Quantification of Aβ plaques in eight major brain structures. The hippocampus here refers to the dentate gyrus and hippocampus proper (CA1-3), not including the subicular cortex. F Quantification of Aβ plaques for subregional comparisons between two age groups. Scale bar = 1000 μm. n = 3 male mice per group. *P < 0.05, **P < 0.01 at 8-month-old compared with 4-month-old. ACA anterior cingulate area, PL prelimbic cortex, IL infralimbic cortex, DG dentate gyrus, LEC lateral entorhinal cortex, MEC medial entorhinal cortex, BLA basolateral amygdalar nucleus, BMA basomedial amygdalar nucleus, CEA central amygdalar nucleus, BST bed nuclei of the stria terminalis, Xi xiphoid thalamic nucleus, MD mediodorsal nucleus of thalamus, PT parataenial nucleus, PVT paraventricular nucleus of the thalamus, SO supraoptic nucleus, ASO accessory supraoptic group, PVH paraventricular hypothalamic nucleus, ARH arcuate hypothalamic nucleus, PSTN parasubthalamic nucleus, PPN pedunculopontine nucleus, PB parabrachial nucleus, DMX dorsal motor nucleus of the vagus nerve, NTS nucleus of the solitary tract.

Fig. 4. Metabolic characterization of 4-month and 11-month-old 5xFAD mice.

A Left, body weight. Right, automated home-cage CLAMS monitoring system. B Accumulated food intake and total food intake during light and dark phase. C Accumulated water intake and total water intake during light and dark phase. D–H Real-time monitoring curve and the area under curve (AUC) quantification of D O₂ consumption, E CO₂ production, F respiratory exchange ratio (RER), G heat production, H horizontal activity, and I vertical activity in light and dark cycle. n = 5–8 male mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P = 0.07 compared with WT mice.

Fig. 5. Chronic social isolation-unpredictable stress induces the onset of cognition deficits in young 5xFAD mice.

A Timeline of experimental procedures. B Body weight changes before and after exposure to 10 days of social isolation and unpredictable stress (SIUS). C Spontaneous alternation rate (left) and total arm entries (right) in the Y-maze test. WT-Ctrl: male, n = 12, female, n = 9; WT-SIUS: male, n = 7, female, n = 10; 5xFAD-Ctrl: male, n = 7, female, n = 10; 5xFAD-SIUS: male, n = 7, female, n = 7. D Exploration time and discrimination index in the novel objection recognition test with a 2-h retention interval. E Exploration time and discrimination index in the novel objection recognition test with a 24-h retention interval. WT-Ctrl: male, n = 8, female, n = 7; WT-SIUS: male, n = 8, female, n = 9; 5xFAD-Ctrl: male, n = 7, female, n = 10; 5xFAD-SIUS: male, n = 7, female, n = 7. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P = 0.051. ns, not significant. Control, Ctrl; social isolation and unpredictable stress, SIUS.

Fig. 6. Chronic social isolation-unpredictable stress accelerates Aβ plaque deposition in 5xFAD mice.

Representative coronal brain sections showing staining of Aβ plaques and quantitative analysis of Aβ plaque load in the entorhinal cortex A, hippocampus B and mPFC C of 5xFAD mice under control (Ctrl) or chronic social isolation and unpredictable stress (SIUS) conditions. n = 8–10 sections from 4–5 mice per sex per group. Scale bar = 200 µm. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl mice.