Sex-Specific Effects of Anxiety on Cognition and Activity-Dependent Neural Networks: Insights from (Female) Mice and (Wo)Men

Abstract

Introduction: Neuropsychiatric symptoms (NPS), such as depression and anxiety, are observed in 90% of Alzheimer's disease (AD) patients, two-thirds of whom are women. NPS usually manifest long before AD onset creating a therapeutic opportunity. Here, we examined the impact of anxiety on AD progression and the underlying brain-wide neuronal mechanisms.

Methods: To gain mechanistic insight into how anxiety impacts AD progression, we performed a cross-sectional analysis on mood, cognition, and neural activity utilizing the ArcCreER^T2 x enhanced yellow fluorescent protein (eYFP) x APP/PS1 (AD) mice. The ADNI dataset was used to determine the impact of anxiety on AD progression in human subjects.

Results: Female AD mice exhibited anxiety-like behavior and cognitive decline at an earlier age than control (Ctrl) mice and male mice. Brain-wide analysis of c-Fos⁺ revealed changes in regional correlations and overall network connectivity in AD mice. Sex-specific memory trace changes were observed; female AD mice exhibited impaired memory traces in dorsal CA3 (dCA3), while male AD mice exhibited impaired memory traces in the dorsal dentate gyrus (dDG). In the ADNI dataset, anxiety predicted transition to dementia. Female subjects positive for anxiety and amyloid transitioned more quickly to dementia than male subjects.

Conclusions: While future studies are needed to understand whether anxiety is a predictor, a neuropsychiatric biomarker, or a comorbid symptom that occurs during disease onset, these results suggest that AD network dysfunction is sexually dimorphic, and that personalized medicine may benefit male and female AD patients rather than a one size fits all approach.

Keywords: Arc; engrams; hippocampus; immediate early gene; memory; sex differences.

Fig. 1.. Female AD mice exhibit earlier anxiety-like behavior and cognitive decline.

(A) Experimental timeline. (B) Ctrl and AD female mice exhibit a similar percent distance in the center of the OF. (C) Female Ctrl and AD mice spend less time in the open arms of the EPM with age. (D) Female AD mice bury a larger percentage of marbles at 4 months of age compared to Ctrl mice. (E) Female AD mice spend less activity on the light side of the box at 2 months of age compared to Ctrl mice. Female Ctrl and AD mice display less activity on the light side with age. (F-G) In the NSF, latency to the pellet is similar between female Ctrl and AD mice. (H) Female AD mice exhibit cognitive deficits at 2 months of age compared to Ctrl mice in the CFC test. (I) Freezing (%) is correlated with increased marbles buried in 6-month-old female AD mice. (J) Male Ctrl and AD mice exhibit similar percent distance in the center of the OF. (K) Male AD mice spend less time in the open arms of the EPM with age. (L) Male Ctrl and AD male mice bury more marbles with age. (M) Age significantly impacts time spent on the light side in the LDT. (N-O) Latency time to the food pellet is similar in all groups when graphed in a survival plot; however; Age influence latency to eat in the RMANOVA. (P) Male AD mice are impaired in CFC freezing (%) at 6 months of age. (Q) Freezing (%) is not correlated with increased marbles buried in 6-month-old male AD mice. (n=4–18 mice per group). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001. OF, open field; EPM, elevated plus maze; MB, marble burying; LDT, light dark test; NSF, novelty suppressed feeding; CFC, contextual fear conditioning; RE, re-exposure; C, center; P, periphery; dist, distance; sec, seconds; Ctrl, control; AD, Alzheimer’s disease.

Fig. 2. AD alters the correlated activity between brain regions during fear memory retrieval.

(A) Experimental timeline. (B-C) Representative c-Fos⁺ images in Ctrl and AD mice, with Methoxy-X04 staining amyloid plaques. (D-E) Brain-wide heat map correlations include the hippocampus, amygdala, thalamus, and hypothalamus. In female mice, pink indicates strong positive correlations while grey indicates strong negative correlations. (F-G) Brain-wide heat map correlations include the hippocampus, amygdala, thalamus, and hypothalamus. In male mice, blue indicates strong positive correlations while grey indicates strong negative correlations. (H) Volcano plot highlighting a subset of regional correlations with a correlation difference higher than 1 between Ctrl and AD mice. (I) Parallel plots confirm the pattern observed in the heat, female AD mice exhibit a greater number of negative correlations compared to positive correlations whereas female Ctrl mice show more positive correlations. (J-K) Volcano plot highlighting a subset of regional correlations with a correlation difference higher than 1 between Ctrl and AD mice. Male AD mice display increased positive correlations while male Ctrl mice exhibit increased negative correlations throughout the brain. (L-M) Cluster map analysis revealed sparse connectivity in female AD mice, while female Ctrl mice exhibit an integrated network of regions. (N-O) The cluster map is highly interconnected in male Ctrl mice, while the cluster map is less cohesive in male AD mice. (P) The regions that most contribute to freezing behavior include the RSP, ECT, MEA, vDG, and LP. (n=4–7 mice per group). Error bars represent ± SEM. *p<.05, **p<.01, ***p<.001. CFC, contextual fear conditioning; RE, re-exposure; iDISCO, immunolabeling-enabled imaging of solvent-cleared orgrans; Ctrl, control; AD, Alzheimer’s disease; all other brain regions are defined in supplemental tables.

Fig 3.. Sex-specific changes in brain-wide functional connectivity during memory retrieval in AD mice.

(A-B) The fear retrieval network in female AD mice is sparser when compared to female Ctrl mice (grey = negative correlations; pink=positive correlations). Thicker lines indicate stronger correlations and larger circles represent an increased degree of nodes. (C-D) In male mice, an opposite pattern is observed. Male AD mice networks are more positively correlated and connected compared to male Ctrl mice. (E-F) Male Ctrl and AD mice, and female AD mice exhibit a decrease in mean degree and global efficiency compared to female Ctrl mice. (G) Male Ctrl mice have a decreased mean clustering coefficient when compared to female Ctrl mice. (H) Mean betweenness centrality is similar among the groups. (I) Frequency for each level of degree among the groups. (J-M) Betweenness scores for each region reveal the importance of the vDG in females Ctrl and male Ctrl mice. (N) Male mice have increased activation in the subiculum whereas female mice show increased c-Fos⁺ activation in the entorhinal cortex. (O) In the dHPC, Female Ctrl and AD mice, and male AD mice exhibit decreased c-Fos⁺ activation in the dDG compared to females Ctrl mice. Similar levels are observed in dCA3 and dCA1 between the groups. (P) In the vHPC, c-Fos⁺ levels are similar in the vDG but are elevated in female AD mice in vCA3. There is a significant effect of Sex in vCA1. (Q) c-Fos⁺ activation is similar throughout the amygdalar regions except in the COA where female mice exhibit increased levels compared to male mice. (n=4–7 mice per group). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001. Ctrl, control; AD, Alzheimer’s disease; mm, millimeters; Sub, subiculum; Ent, entorhinal cortex; dDG, dorsal dentate gyrus; dCA3, dorsal cornu ammonis 3; dCA1, dorsal cornu ammonis 1; vDG, ventral dentate gyrus; vCA3, ventral cornu ammonis 3; vCA1, ventral cornu ammonis 1; COA, cortical amygdala area; BMA, basomedial amygdala; PAA, piriform-amygdala area; BLA, basolateral amygdala; LA, lateral amygdala; MEA, medial amygdala; IA, intercalated amygdala; CEA, central amygdala; all other brain regions are defined in supplemental tables.

Fig 4.. Female AD mice exhibit a decrease in memory traces in the dorsal CA3.

(A) Genetic design. (B) Experimental timeline. (C-D) Female and male AD mice exhibit decreased freezing behavior at 6 months of age compared to Ctrl mice. (E-F) Representative images confirming plaque pathology in AD mice using Methoxy-X04. (G-I) Representative images of indelible memory tagging in the dHPC. (J-L) Female Ctrl and AD mice exhibit comparable levels of eYFP⁺-tagged cells in the dDG, dCA3, and dCA1. (M) In the dDG, c-Fos⁺ activation is decreased in female AD mice, and Ctrl and AD male mice. (N-O) c-Fos⁺ activation is similar among the groups in dCA3 and dCA1. (P) In the dDG, female mice exhibit a greater co-labeled / eYFP (%) when compared to male AD mice. (Q) In dCA3, female AD, and Ctrl and AD male mice a decreased co-labeled / eYFP (%) when compared to Ctrl female mice. (R) In dCA1, female mice exhibit a greater co-labeled / eYFP (%) when compared to male mice. (S) in dDG, there are no differences in female groups for co-labeled / c-Fos⁺ (%). Male AD mice exhibit a decreased co-labeled / c-Fos⁺ (%) when compared to male Ctrl mice. (T-U) Memory trace activation is similar among the groups in dCA3 and dCA1. (n=4–7 mice per group). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001. pA, polyadenylation; ChR2, channelrhodopsin-2; eYFP, enhance yellow fluorescent protein; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element ; 4-OHT, 4-hydroxytamoxifen; d, days; h, hours; sac, sacrifice; Ctrl, control; AD, Alzheimer’s disease; mm, millimeter; dDG, dorsal dentate gyrus; dCA3, dorsal cornu ammonis; dCA1, dorsal cornu ammonis; CA1.

Fig 5.. Encoding and retrieval cell activation are increased in the ventral hippocampus of AD female mice.

(A-F) Representative images of eYFP⁺, c-Fos⁺, and memory traces in the vHPC. (G-H) eYFP expression was comparable in the groups in the vDG and vCA3. (I) In vCA1, female AD mice exhibit increased eYFP⁺ activation compared to male Ctrl and AD mice. (J-K) c-Fos⁺ expression was comparable in the groups in the vDG and vCA3. (L) In vCA1, female AD mice exhibited increased c-Fos⁺ expression compared to female Ctrl mice. Male Ctrl mice exhibited increased c-Fos⁺ activation when compared to female Ctrl mice. (M-O) In vDG, vCA3, and vCA1, the co-labeled / eYFP (%) was comparable between all groups. (P) Male mice exhibited a greater co-labeled / c-Fos⁺ (%) when compared to female mice. (Q) In vCA3, the co-labeled / c-Fos⁺ (%) was comparable between all groups. (R) In vCA1, male mice exhibited a greater co-labeled / c-Fos⁺ (%) than female mice. (n=4–7 mice per group). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001. eYFP, enhanced yellow fluorescent protein; mm, microns; mm, millimeter; vDG, ventral dentate gyrus; vCA3, ventral cornu ammonis 3; vCA1, ventral cornu ammonis 1; Ctrl, control; AD, Alzheimer’s disease.

Fig. 6. Female patients with anxiety and amyloid transition more quickly to dementia.

(A) ADNI data collection pipeline. (B-C) The percentage of subjects with anxiety is increased in those with MCI and AD in both females and males. (D) The odds ratio indicates that female AD subjects are eight times more likely to have anxiety compared to CN subjects, and male AD subjects are 24 times more likely to have anxiety compared to CN subjects. (E-F) Female subjects with amyloid and anxiety transition faster to dementia than males. (G) Bar graph representation of the survival plots in E-F indicates that subjects with amyloid positivity and anxiety transition to dementia at a faster rate. (n=1661). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001. CN, cognitively normal; MCI, mild cognitive impairment; AD, Alzheimer’s disease; OR, odds ratio of anxiety; Aβ, amyloid beta.

Fig. 7. Sex-specific effect of anxiety on brain volume in ADNI participants.

(A) fMRI representative images of female-male, female, and male brain volumes using a t-value scale (red= increased volume, blue=decreased volume). (B) Anxiety is the greatest predictor of dementia transition in male and female subjects compared to brain atrophy and age. (C-D) Female subjects with anxiety have decreased inferior temporal gray volume and amygdala volume compared to female subjects without anxiety. Male subjects with anxiety have increased inferior temporal gray and amygdala volume compared to male subjects without anxiety. (E) Male subjects with anxiety have increased ventral diencephalon volume in the left hemisphere compared to male subjects without anxiety. There is no significant difference in the female groups. (F) Male subjects with anxiety have increased ventral diencephalon volume in the right hemisphere compared to male subjects without anxiety. There is no significant difference in the female groups, although it is trending. (G) In the right hemisphere, male subjects with anxiety have increased insula gray volume. There is no significant difference in the female groups, although it is trending. (H) Female subjects with anxiety have decreased hippocampal volume. There is no difference between the groups for male subjects in the hippocampus. (n=1650). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001.