FKBP51 in glutamatergic forebrain neurons promotes early life stress inoculation in female mice

Abstract

Early life stress (ELS) can increase vulnerability to psychiatric disorders, but also trigger resilience. FKBP51 has been associated with an increased risk for developing psychiatric disorders, specifically in interaction with ELS exposure. Here, the contribution of FKBP51 in glutamatergic forebrain neurons to the long-term consequences of ELS was investigated in both sexes. In female wild-type Fkbp5^lox/lox mice, ELS exposure led to an anxiolytic phenotype and improved memory performance in a stressful context, however this ELS effect was absent in Fkbp5^Nex mice. These interactive FKBP51 x ELS effects in female mice were also reflected in reduced brain region volumes, and on structural and electrophysiological properties of CA1 pyramidal neurons of the dorsal hippocampus. In contrast, the behavioral, structural and functional effects in male ELS mice were less pronounced and independent of FKBP51. RNA sequencing of the hippocampus revealed the transcription factor 4 (TCF4) as a potential regulator of the female interactive effects. Cre-dependent viral overexpression of TCF4 in female Nex-Cre mice led to similar beneficial effects on behavior as the ELS exposure. This study demonstrates a sex-specific role for FKBP51 in mediating the adaptive effects of ELS on emotional regulation, cognition, and neuronal function, implicating TCF4 as a downstream effector.

Fig. 1. FKBP51 in glutamatergic forebrain neurons and early life stress exposure have an interactive effect on behaviour in female mice.

A Offspring of Fkbp5^lox/lox WT mice and mice lacking FKBP51 in glutamatergic forebrain neurons (Fkbp5^Nex) were exposed to limited bedding and nesting (LBN) early life stress (ELS) paradigm from postnatal day 2 (P2) to P9. At 6 months of age, mice were exposed to a behavioural protocol, including tests assessing anxiety-like behaviour and (spatial) memory performance in a neutral or stressful context. Panel (B–F) shows data of female mice only, data on male mice can be found in Supplementary Fig. 2. Genotype affected locomotor behaviour of female mice independent of ELS exposure (B). Data from the open field (OF) test (B) and elevated plus maze (EPM) test (C) revealed a strong beneficial effect of ELS exposure on anxiety-like behaviour. The fold change of the ELS effect (vs. the control condition) on EPM parameters was significantly larger in WT mice than in KO mice (D). Further, memory performance in a neutral context (E) revealed a deteriorating effect of ELS, in interaction with FKBP5 genotype. Interestingly, for assessing memory function in a stressful context (F) with the Morris water maze (MWM), ELS had a beneficial effect on spatial memory performance in Fkbp5^lox/lox mice only. ELS: n = 10 Fkbp5^Nex and n = 11 Fkbp5^lox/lox; control: n = 10 Fkbp5^Nex and n = 9 Fkbp5^lox/lox. Error bars represent mean + S.E.M. Panels B,C,E,F: 2-way ANOVA. Panel (D): 2-sided t test. Panel (F): repeated measures ANOVA. *effect of ELS p < 0.05; **effect of ELS p < 0,01; *** effect of ELS p < 0.001; ^# effect of genotype p < 0.05; ^$ interaction effect ELS x genotype p < 0.05. Images of MWM, mouse brain and MRT scanner created in BioRender. Schmidt, M. (2025) https://BioRender.com/p00d573.

Fig. 2. ELS and Fkbp5 genotype lead to separate and interactive changes in brain volume in female mice.

A Deformation-based morphometry analyses of the female brains revealed main effects of genotype (Gen, T-contrast WT > KO) in grey matter (GM) volumes of the cortex (right somatosensory cortex and bilateral visual cortex) and in the ventral subiculum. Overall white matter (WM) deformations were found for genotype in the bilateral dorsal hippocampal commissure. In addition, interactive effects of Fkbp5 genotype and early life stress (ELS) exposure (GxE, T-contrast, pronounced negative effect of stress in KO mice compared to WT), were found in various cortical regions (right somatosensory cortex, bilateral visual cortex and bilateral retrosplenial cortex). B Post-hoc t tests revealed reductions in brain volume following ELS, in Fkbp5^Nex mice (KO) only, within bilateral cortical areas and increased volumes in the third ventricle. C Furthermore, within mice that underwent ELS, Fkbp5^Nex mice had smaller GM volumes in cortical areas and reduced volumes in WM structures of the dorsal hippocampal commissure. Scales represent Z-scores. WT = wild-type, KO = Fkbp5^Nex, C = control, ELS = early life stress, Gen = genotype, GxE = gene by environment interaction. ELS: Fkbp5^lox/lox n = 7, Fkbp5^Nex n = 10;control: Fkbp5^lox/lox n = 11 and Fkbp5^Nex n = 8.

Fig. 3. FKBP51 in glutamatergic forebrain neurons and early life stress exposure have an interactive effect on neuronal structure and function in female mice.

A second cohort (A) of female Fkbp5^Nex and Fkbp5^lox/lox offspring was exposed to limited bedding and nesting (LBN) early life stress (ELS) paradigm, and a Golgi Cox staining was performed at the hippocampus of the 8-month old female mice. A very similar interaction effect, as was observed for behaviour, was also found for apical spine densities in pyramidal neurons of the CA1 region (ELS: Fkbp5^Nex n = 15 neurons vs. Fkbp5^lox/lox n = 12 neurons; Control: Fkbp5^Nex n = 15 neurons vs. Fkbp5^lox/lox n = 15 neurons). Moreover, an interaction effect and main effect of ELS was found for dendritic complexity (B), where dendritic complexity of CA1 pyramidal neurons was increased following ELS exposure, particularly in Fkbp5^lox/lox mice (ELS: Fkbp5^Nex 30 neurons vs. Fkbp5^lox/lox 40 neurons; Control: Fkbp5^Nex 21 neurons vs. Fkbp5^lox/lox 33 neurons). In addition, data from electrophysiological LTP measurements, again demonstrated interactive effects of the Fkbp5 genotype with an ex vivo stress exposure, in the form of a corticosterone application (C; CORT: Fkbp5^Nex: n = 4 mice, n = 11 brain slices; Fkbp5^lox/lox: n = 5 mice, n = 11 brain slices, vehicle: Fkbp5^Nex: n = 5 mice, n = 9 brain slices; Fkbp5^lox/lox: n = 6 mice, n = 9 brain slices), and with ELS exposure (D; ELS: Fkbp5^Nex: n = 4 mice, n = 11 brain slices; Fkbp5^lox/lox: n = 5 mice, n = 11 brain slices; control: Fkbp5^Nex: n = 5 mice, n = 9 brain slices; Fkbp5^lox/lox: n = 6 mice, n = 9 brain slices). Error bars represent mean + S.E.M. Panel (A): 2-way ANOVA. Panel (B, C): repeated measures ANOVA. **effect of ELS p < 0,01; *** effect of ELS or CORT p < 0.001; ^$ interaction effect ELS x genotype p < 0.05; ^$$ interaction effect ELS x genotype p < 0.01. Image of neuron created in BioRender. Schmidt, M. (2025) https://BioRender.com/o64z476.

Fig. 4. RNA bulk sequencing reveals transcription factor 4 as a potential regulator of early life stress-induced effects on the brain that interact with FKBP51 in glutamatergic forebrain neurons.

Bulk mRNA sequencing was performed on the hippocampus of female mice of the first cohort. A A clear differential expression profile was found for the effects of genotype. Furthermore, a weighted gene co-expression analysis (WGCNA) revealed 18 co-expressed gene modules that were associated with effects of genotype, ELS exposure or their interaction (B). One of these modules was associated not only with ELS exposure but was also associated with the interaction of ELS and Fkbp5 genotype. C Subsequently, a transcription factor enrichment analysis was performed for the dark orange module, which resulted in 10 enriched transcription factors. Using the software Knowing01, all genes that are regulated by the enriched transcription factors were overlaid with datasets from human psychiatric GWAS studies and the hub genes of the dark orange module (D). The right panel (D) shows which genes are regulated by specific enriched transcription factors and their resulting (indirect) overlap with the datasets. This revealed that the transcription factor 4 (TCF4) regulates the largest number of genes that had an overlap with any of the datasets. Moreover, it is the only enriched transcription factor that regulates a gene that was associated with early life adversity in females (Slc17a6; yellow dot).

Fig. 5. Tcf4 overexpression leads to similar beneficial effects on behaviour as ELS exposure in Fkbp5^lox/lox mice.

To investigate whether TCF4 in glutamatergic neurons of the hippocampus is indeed the underlying molecular target regulating the beneficial effects of ELS exposure on behaviour, we overexpressed Tcf4 in these neurons by injecting an AVV Cre-dependent Tcf4 overexpression (OE) virus in Nex-Cre female mice of 6 months of age and compared them to female mice that were injected with an AVV GFP control virus (A). Interestingly, TCF4 OE indeed leads to (B) an anxiolytic phenotype on the elevated plus maze (EPM) test, like was observed upon ELS exposure in Fkbp5^lox/lox mice. Furthermore, data from the MWM showed that TCF4 OE mice had an improved memory of the platform location on the 3rd training day, which was a less strong but similar effect to as was observed for ELS-exposed female Fkbp5^lox/lox mice. n = 15 per group. Error bars represent mean + S.E.M. Panel B: Wilcoxon signed-rank test. Panel (C): repeated measures ANOVA. *p < 0.05; **p < 0,01; ***p < 0.001. Images of mouse head and MWM created in BioRender. Schmidt, M. (2025) https://BioRender.com/o64z476.