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. 2023 Jul 11:32:100663.
doi: 10.1016/j.bbih.2023.100663. eCollection 2023 Oct.

Maternal separation differentially modulates early pathology by sex in 5xFAD Alzheimer's disease-transgenic mice

M G Garcia  1   2 , A Paulus  1   3 , S Vázquez-Reyes  1 , O Klementieva  3 , G K Gouras  2 , S Bachiller  1   4   5 , T Deierborg  1
Affiliations

Maternal separation differentially modulates early pathology by sex in 5xFAD Alzheimer's disease-transgenic mice

M G Garcia et al. Brain Behav Immun Health. .

Abstract

Alzheimer's disease (AD) is the most common neurodegenerative disease. Most cases of AD are considered idiopathic and likely due to a combination of genetic, environmental, and lifestyle-related risk factors. Despite occurring decades before the typical age of an AD diagnosis, early-life stress (ELS) has been suggested to have long-lasting effects that may contribute to AD risk and pathogenesis. Still, the mechanisms that underlie the role of ELS on AD risk remain largely unknown. Here, we used 5xFAD transgenic mice to study relatively short-term alterations related to ELS in an AD-like susceptible mouse model at 6 weeks of age. To model ELS, we separated pups from their dams for 3 h per day from postnatal day 2-14. Around 6 weeks of age, we found that maternally separated (MS) 5xFAD mice, particularly female mice, displayed increased amyloid-β-immunoreactivity in the anterior cingulate cortex (ACC) and basolateral amygdala (BLA). In anterior cingulate cortex, we also noted significantly increased intraneuronal amyloid-β-immunoreactivity associated with MS but only in female mice. Moreover, IBA1-positive DAPI density was significantly increased in relation to MS in ACC and BLA, and microglia in BLA of MS mice had significantly different morphology compared to microglia in non-MS 5xFAD mice. Cytokine analysis showed that male MS mice, specifically, had increased levels of neuroinflammatory markers CXCL1 and IL-10 in hippocampal extracts compared to non-MS counterparts. Additionally, hippocampal extracts from both male and female MS 5xFAD mice had decreased levels of synapse- and activity-related markers Bdnf, 5htr6, Cox2, and Syp in hippocampus. Lastly, we performed behavioral tests to evaluate anxiety- and depressive-like behavior and working memory but could not detect any significant differences between groups. Overall, we detected several sex-specific molecular and cellular alterations in 6-week-old adolescent 5xFAD mice associated with MS that may help explain the connection between ELS and AD risk.

Keywords: Alzheimer’s disease; Amyloid-β; Early-life stress; Maternal separation; Neuroinflammation.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Maternal separation exacerbates amyloid-β pathology in female 6-week-old 5xFAD mice. (A) Schematic with timeline pointing out maternal separation (MS) and endpoint behavioral testing and tissue collection. EPM = elevated plus maze. TST = tail suspension test. (B–D) Amyloid-β (Aβ)-immunoreactivity using anti-Aβ antibody MOAB2 was measured in anterior cingulate cortex (ACC, B), basolateral amygdala (BLA, C), and subiculum (D). Representative images (left) with inset images (upper right box, solid red line). Red dotted box indicates area blown up in the inset. Solid white arrowheads point to extra-NeuN Aβ-IR. Empty white arrowheads in inset boxes point to intraneuronal NeuN-positive Aβ. Very few/low intensity intraneuronal Aβ puncta were noted in ACC of males and non-MS females. Representative image scalebar = 40 μm. Inset scalebar = 10 μm. Aβ-IR was measured for the whole region (Aβ %area, left graph) and within a NeuN, region-specific mask (Aβ/NeuN %area, right graph). Data points represent an individual mouse. Unfilled black circles represent non-MS mice. Unfilled pink squares represent MS mice. n = 7–9 non-MS/sex/region, 3–4 MS/sex/region. P values from multiple comparisons testing are indicated at the tops of graphs. Error bars indicate mean ± SD
Fig. 2
Fig. 2
Maternal separation alters IBA1 morphology in 6-week-old 5xFAD mice. (AC) IBA1-immunoreactive microglia were measured in anterior cingulate cortex (ACC, A), basolateral amygdala (BLA, B), and subiculum (C). Representative images are maximum intensity projections. Red square indicates a blow up of a representative IBA1- IR microglial cell. Lower magnification image scale bar = 100 μm. Blow up image scalebar = 20 μm. Data points represent an individual mouse. Unfilled black circles represent non-MS mice. Unfilled pink squares represent MS mice. n = 4–9 nonMS/sex/region, 3–5 MS/sex/region. P values from multiple comparisons testing are indicated at the tops of graphs. Error bars indicate mean ± SD
Fig. 3
Fig. 3
Maternal separation alters cytokine levels in 6-week-old male 5xFAD mice. Multiplex immunoassay results using total protein extracted from hippocampal tissue. Data points represent an individual mouse. Unfilled black circles represent non-MS mice. Unfilled pink squares represent MS mice. n = 4–6 non-MS/sex, 3–5 MS/sex. P values from multiple comparisons testing are indicated at the tops of graphs. Error bars indicate mean ± SD
Fig. 4
Fig. 4
Maternal separation reduces synapse and activity-related gene expression in hippocampus of 6-week-old 5xFAD mice. Gene expression levels of 5ht6r (A), Bdnf (B), Cox2 (C), Gfap (D), and Syp (E) were measured from total mRNA extracted from hippocampal tissue from 6-week-old 5xFAD mice by RT-qPCR. Data points represent an individual mouse. Unfilled black circles represent non-MS mice. Unfilled pink squares represent MS mice. n = 4–7 non-MS/sex, 3 MS/sex. P values from multiple comparisons testing are indicated at the tops of graphs. Error bars indicate mean ± SD
Fig. 5
Fig. 5
Maternal separation does not affect behavioral outcomes in 6-week-old 5xFAD mice. (A) Elevated plus maze was used to assess anxiety-like behavior via anxiety index. (B) Tail suspension test was used to evaluate depressive-like behavior by measuring percent of time spent immobile. (C) Spatial working memory was assessed using Y-maze and analyzed by spontaneous alterations. Data points represent an individual mouse. Unfilled black circles represent non-MS mice. Unfilled pink squares represent MS mice. n = 6–8 non-MS/sex, 7–10 MS/sex. P values from multiple comparisons testing are indicated at the tops of graphs. Error bars indicate mean ± SD
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