Analysis of the cerebellar molecular stress response led to first evidence of a role for FKBP51 in brain FKBP52 expression in mice and humans

Abstract

As the cerebellar molecular stress response is understudied, we assessed protein expression levels of hypothalamic-pituitary-adrenal (HPA) axis regulators and neurostructural markers in the cerebellum of a male PTSD mouse model and of unstressed vs. stressed male FK506 binding protein 51 (Fkbp5) knockout (KO) vs. wildtype mice. We explored the translatability of our findings in the Fkbp5 KO model to the situation in humans by correlating mRNA levels of candidates with those of FKBP5 in two whole transcriptome datasets of post-mortem human cerebellum and in blood of unstressed and stressed humans. Fkbp5 deletion rescued the stress-induced loss in hippocampal, prefrontal cortical, and, possibly, also cerebellar FKBP52 expression and modulated post-stress cerebellar expression levels of the glucocorticoid receptor (GR) and possibly (trend) also of glial fibrillary acidic protein (GFAP). Accordingly, expression levels of genes encoding for these three genes correlated with those of FKBP5 in human post-mortem cerebellum, while other neurostructural markers were not related to Fkbp5 either in mouse or human cerebellum. Also, gene expression levels of the two immunophilins correlated inversely in the blood of unstressed and stressed humans. We found transient changes in FKBP52 and persistent changes in GR and GFAP in the cerebellum of PTSD-like mice. Altogether, upon elucidating the cerebellar stress response we found first evidence for a novel facet of HPA axis regulation, i.e., the ability of FKBP51 to modulate the expression of its antagonist FKBP52 in the mouse and, speculatively, also in the human brain and blood and, moreover, detected long-term single stress-induced changes in expression of cerebellar HPA axis regulators and neurostructural markers of which some might contribute to the role of the cerebellum in fear extinction.

Keywords: Cerebellum; FKBP5 knockout; FKBP52; HPA axis; PTSD mouse model.

Fig. 1

Expression of neurostructural marker proteins in the cerebellum of the PTSD mouse model (A) Mouse model of posttraumatic stress disorder (PTSD): brains were dissected two, 28 or 60 days (d2, d28, d60) after mock – or foot-shock treatment (n = 14 per group except from synaptophysin n = 7 per group). We previously described foot-shock exposure to robustly induce a PTSD-like syndrome. Graphs show results of two-tailed unpaired t-tests of relative protein expression levels of (B) neurofilament H (NF–H), (C) synaptophysin, (D) HOMER1 b/c, (E) microtubule-associated protein 2 (MAP-2) and (F) glial fibrillary acidic protein (GFAP) after normalization to glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) in lysates of total cerebellum in foot-shocked (shock (s)) vs. mock-treated (no shock (ns)) mice including unadjusted and Benjamini-Hochberg (FDR)-adjusted significant p-values. Plotted immunoblot data represent means of two technical replicates ± standard error of the mean (SEM). Representative immunoblots showing expression levels of candidate proteins (B–F) and GAPDH are presented in (G). Significances are indicated with t, p ≤ 0.01; *, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001. Statistical details of significant results are reported in the results section.

Fig. 2

Expression of neurostructural marker proteins in the cerebellum of the Fkbp5 KO mouse model (A) Fkbp5 KO-WT mouse batch: FK 506 binding protein (Fkbp5) knockout (KO) and wildtype (WT) mice have been either subjected to different combination stressors (RSI and RSII) or remained unstressed (no stress); n = 6 per group. For details, see methods chapter. We previously reported Fkbp5 KO mice of the same Fkbp5 KO-WT mouse batch to exhibit a diminished endocrine and behavioral stress response (Touma et al., 2011). Graphs show results of the two-way ANOVA (followed by Bonferroni-adjusted post-hoc tests) of relative protein expression levels of (B) neurofilament H (NF–H), (C) synaptophysin, (D) HOMER1 b/c, (E) microtubule-associated protein 2 (MAP-2) and (F) glial fibrillary acidic protein (GFAP) after normalization to glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) in lysates of total cerebellum. Plotted immunoblot data represent means of three technical replicates ± standard error of the mean (SEM). Representative immunoblots showing expression levels of candidate proteins (B–F) and GAPDH are presented in (G). Note that the sequence of experimental groups in blot inserts differs from that shown in the corresponding graphs since blots were not cut apart. Main effects (G, Genotype; S, Stressor) and their interaction (G x S) were FDR-corrected for multiple testing and their unadjusted and FDR-adjusted p-values are depicted below the graphs (s, significant; ns, not significant; t, trend). Significances of post-hoc tests are indicated with *, p ≤ 0.05 (between stress group significance), #; p ≤ 0.05 (between-genotype significance). Statistical details of significant results are reported in the results section.

Fig. 3

Expression of HPA axis regulating proteins in the PTSD and the Fkbp5 KO mouse models plus hippocampal and prefrontal cortical FKBP52 in the Fkbp5 KO mouse model. Mouse models are described in detail in the methods section and graphically outlined in Fig. 1A and 2A. PTSD mouse model: n = 14 per group except from FKBP51 d28 and d60 (n = 6) and HSP90 d60 (n = 12); FK 506 binding protein (Fkbp5) KO mouse model: n = 6 per group. (A–D): PTSD mouse model: Graphs show results of two-tailed unpaired t-tests of relative protein expression levels of (A) glucocorticoid receptor (GR), (B) FKBP 51, (C) FKBP52 and (D) heat shock protein 90 (HSP90) after normalization to glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) in lysates of total cerebellum including unadjusted and Benjamini-Hochberg False Discovery Rate (FDR)-adjusted significant p-values. (E–J): Fkbp5 KO mouse model: Graphs show results of the two-way ANOVA (followed by Bonferroni-adjusted post-hoc tests) of relative protein expression levels of (E) mineralocorticoid receptor (MR), (F) GR, (G) FKBP51 (not FDR-corrected) and (H) FKBP52 as well as of (I) hippocampal and (J) prefrontal cortical FKBP52 after normalization to GAPDH in lysates of (E–H) total cerebellum, (I) bilaterally pooled hippocampus and (J) total prefrontal cortex. Main effects (G, Genotype; S, Stressor) and their interaction (G x S) were corrected for multiple testing employing FDR and their unadjusted and FDR-adjusted p-values are depicted below the graphs (s, significant; ns, not significant; t, trend). Plotted immunoblot data represent means of three (Fkbp5 KO model) or two (PTSD mouse model) technical replicates ± standard error of the mean (SEM). Inserts depict corresponding representative immunoblots showing expression levels of analyzed proteins. Note that the sequences of experimental groups in blot inserts belonging to H-J differ from those shown in the corresponding graphs since blots were not cut apart. Significances of t-tests and post-hoc tests are indicated with t, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001 for between-stress group significances and #, p ≤ 0.05; ##, p ≤ 0.01 for between-genotype significances. Outliers: (D) HSP90 d28: 1 no shock; HSP90 d60: 1 no shock and 1 shock. Statistical details of significant results are reported in the results section.

Fig. 4

Glucocorticoid-responsive elements (GREs) in the FKBP4 promoters of humans and mice In silico search revealed a potential GRE in both the human and the mouse FKBP4 gene promoter. A, the program LASAGNA (Lee and Huang, 2013) was used to identify predicted GREs in the promoter of the FKBP4 gene. Depicted are the GREs with the highest score identified by this program, along with the previously identified CAAT boxes (Scammell et al., 2003) and transcription start sites. B, comparison of the identified GREs with the consensus sequence for NR3C1 binding sites (human, rat, mouse, JASPAR id MAO113.1, (http://jaspar.genereg.net/matrix/MAO113.1/). For further details, see methods chapter and Suppl.Fig. 1.