FKBP51-Hsp90 Interaction-Deficient Mice Exhibit Altered Endocrine Stress Response and Sex Differences Under High-Fat Diet

Abstract

FK506-binding protein 51 kDa (FKBP51), encoded by Fkbp5 gene, gained considerable attention as an important regulator of several aspects of human biology including stress response, metabolic dysfunction, inflammation, and age-dependent neurodegeneration. Its catalytic peptidyl-prolyl isomerase (PPIase) activity is mediated by the N-terminal FK506-binding (FK1) domain, whereas the C-terminal tetratricopeptide motif (TPR) domain is responsible for FKBP51 interaction with molecular chaperone heat shock protein 90 (Hsp90). To understand FKBP51-related biology, several mouse models have been created. These include Fkbp5 complete and conditional knockouts, overexpression, and humanized models. To dissect the role of FKBP51-Hsp90 interaction in FKBP51 biology, we have created an interaction-deficient mouse (Fkbp5^TPRmut) by introducing two-point mutations in the TPR domain of FKBP51. FKBP51-Hsp90 interaction-deficient mice are viable, fertile and show Mendelian inheritance. Intracellular association of FKBP51 with Hsp90 is significantly reduced in homozygous mutants compared to wild-type animals. No behavioral differences between genotypes were seen at 2 months of age, however, sex-dependent differences were detected in Y-maze and fear conditioning tests at the age of 12 months. Moreover, we have found a significant reduction in plasma levels of corticosterone and adrenocorticotropic hormone in Fkbp5^TPRmut mice after acute stress. In contrast to Fkbp5 knockout mice, females of Fkbp5^TPRmut showed increased body weight gain under high-fat diet treatment. Our data confirm the importance of FKBP51-Hsp90 interactions for stress-related endocrine signaling. Also, Fkbp5^TPRmut mice can serve as a useful in vivo tool to discriminate between Hsp90-dependent and independent functions of FKBP51.

Keywords: FKBP51-Hsp90 interaction; Metabolism; Mouse knock-in model; Stress.

Fig. 1

A Schematic view of CRISPR/Cas9-mediated gene editing. Mutations (*-*) carried in the targeting oligo were introduced into the wild-type allele of FKBP5 exon 10 via homology-directed repair using gRNAs and CRISPR/Cas9. Forward (F1) and reverse (R1) sequencing sites are also indicated. B DNA sequences flanking mutation sites (marked in shadow). Bgl I recognition site of the mutant allele is underlined. C Agarose gel electrophoresis image of the PCR products from wild-type (WT), Fkbp5^TPRmut heterozygous (Het), and Fkbp5^TPRmut homozygous (Hom) mice cleaved with Bgl I restriction enzyme. WT mice displayed one band of intact PCR product at approx. 500 bp, homozygous KO mice displayed two bands at approx. 200 and 300 bp resulting from cleavage products, while heterozygotes displayed all three bands

Fig. 2

Lack of interactions between Hsp90 and FKBP51 in Fkbp5^TPRmut mice. A Images of primary mouse embryonic fibroblasts (MEFs) from wild-type (WT) and Fkbp5^TPRmut homozygous (Hom) embryos, taken by Zeiss Cell observer inverted microscope (40×). Scale bars, 50 μm. B Quantification of the PLA signal/Nuclei in WT and Fkbp5^TPRmut groups. Values represent mean ± SEM. ^**p < 0.01, Mann-Whitney U test (two-tailed). C Coomassie-stained SDS-PAGE showing isolated GST (lane 1) and GST-Hsp90 (lane 2). D Western blot with anti-FKBP51 antibodies. Arrow indicates position of FKBP51

Fig. 3

No behavioral impairments in Fkbp5^TPRmut mice at 2 months of age. A battery of behavioral tests was conducted. Distance moved (A) and the number of rearings (B) in the open field test. C Time spent in the light compartment in light-dark test. D Time spent in the open arms in elevated plus maze test. E Percentage of spontaneous alternations in the Y-maze test. F Discrimination index in the novel object recognition test with 30-min or 24-h interval. Percentage of freezing time in the context (G) and cue (H) tests in fear conditioning. I immobility time in forced swimming test. J Latency to fall in rotarod test. Values represent mean ± SEM (n = 11–14). There are no statistically significant differences between genotypes. A, B, G, H and J, two-way ANOVA followed by Sidak’s multiple comparison test, ^****p < 0.0001. C, D, E, F and I, unpaired Student’s t test (two-tailed)

Fig. 4

Behavioral assessment of Fkbp5^TPRmut mice at 12 months of age. Distance moved (A) and the number of rearings (B) in the open field test. C Time spent in the light compartment in light-dark test. D Time spent in the open arms in elevated plus maze test. E Percentage of spontaneous alternations in the Y-maze test. F Discrimination index in the novel object recognition test with 30-min or 24-h interval. Percentage of freezing time in the context (G) and cue (H) tests in fear conditioning. I immobility time in forced swimming test. J Latency to fall in rotarod test. Values represent mean ± SEM (n = 11–14). A, B, G, H and J, two-way ANOVA followed by Sidak’s multiple comparison test, ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001. C, D, E, F and I, unpaired Student’s t test (two-tailed), ^*p < 0.05, ^**p < 0.01

Fig. 5

Body weight assessment of WT and Fkbp5^TPRmut mice at various ages. A 2 months old, B 5,5 months old, C 12 months old. Values represent mean ± SEM (n = 9–14). Statistical significance between WT and Fkbp5^TPRmut was determined by using Unpaired Student’s t test (two-tailed). p < 0.05 was considered statistically significant. ^*, ^** and ^*** represent p < 0.05, p < 0.01 and p < 0.001, respectively

Fig. 6

Reduced levels of corticosterone (CORT) and adrenocorticotropic hormone (ACTH) in Fkbp5^TPRmut mice after acute stress. After 15-min restraint stress followed by 30-min recovery, blood was collected by heparin tubes and ELISA was performed to detect plasma CORT levels (A) and ACTH levels (B) in male and female mice from WT and Fkbp5^TPRmut groups. Values represent mean ± SEM (n = 9–10). A ^****p < 0.0001, Mann-Whitney U test (two-tailed). B ^*p < 0.05, Unpaired Student’s t test (two-tailed)

Fig. 7

qPCR gene expression measurement in primary mouse embryonic fibroblasts (MEFs) isolated from WT and Fkbp5^TPRmut mice 4 h after dexamethasone (Dexa) treatment. Values represent mean ± SEM (n = 3). Statistical significance between groups was tested by two-way ANOVA followed by Sidak’s multiple comparison test. p < 0.05 was considered statistically significant. ^*p < 0.05 and ^****p < 0.0001

Fig. 8

Body weight gain and glucose tolerance in WT and Fkbp5^TPRmut mice under HFD. A Body weight gain in WT and Fkbp5^TPRmut mice treated with CD or HFD for 5 weeks. Data are represented as mean ± SEM (n = 11–12). The whole-body fat mass (B) and lean mass (C) measured by EchoMRI-100™ after 5-week treatments. Data are represented as mean ± SEM (n = 11–12). Fasting glucose levels (D), glucose AUC (E) and the curve of glucose tolerance test (F) were detected after 5-week treatments. Values represent mean ± SEM (n = 5–6). (A)-(F), Two-way ANOVA followed by Sidak’s multiple comparison test. A ⁺p < 0.05, ⁺⁺p < 0.01, Fkbp5^TPRmut + HFD vs. WT + HFD; ^***p < 0.001, ^****p < 0.0001, Fkbp5^TPRmut + HFD vs. Fkbp5^TPRmut + CD; ^##p < 0.01, ^###p < 0.001, WT + HFD vs. WT + CD. B-F, ns, not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001

Fig. 9

Genotype-dependent differential composition of Hsp90-TPR proteins heterocomplexes associated with glucocorticoid (GR) and androgen (AR) receptors. Whole brains from wild-type (WT) or Fkbp51^TPRmut homozygote mice (Hom) were lysed and incubated with empty agarose beads or with agarose beads coupled to GR and AR antibodies. Protein levels of GR, AR, and associated Hsp90 and TPR co-chaperones were determined by Western blot with respective antibodies