Journal: Molecular Psychiatry

Article Title: The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

doi: 10.1038/s41380-021-01044-x

Figure Lengend Snippet: A Cre-LoxP based generation of the Fkbp5 PVN−/− mouse line. B Validation of Fkbp5 mRNA expression in the PVN via in situ hybridization (ISH) and RNAscope (for mRNA quantification see Supplementary Fig. ). C Fkbp5 PVN-/- mice ( n = 16) presented reduced body weight, lowered adrenal weights, and increased thymus weights under non-stressed conditions compared to their WT littermates ( n = 15). D Corticosterone levels were significantly reduced following a 15 min restraint stress until at least 60 min after stress onset. E A combined Dex/CRH test showed a significantly pronounced response to a low dose of dexamethasone as well as a dampened response to CRH injection. Data are received from mice between 16 and 20 weeks of age and are presented as mean ± SEM. All data were analyzed with a student’s t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following primary antibodies were used: Actin (1:5000, Santa Cruz Biotechnologies, sc-1616), GR (1:1000, Cell Signaling Technology, #3660), p-GR Ser211 (1:500, Sigma, SAB4503820), p-GR Ser226 (1:1000, Sigma, SAB4503874), p-GR 203 (1:500, Sigma, SAB4504585), FKBP51 (1:1000, Bethyl, A301-430A).

Techniques: Biomarker Discovery, Expressing, In Situ Hybridization, RNAscope, Injection

Journal: Molecular Psychiatry

Article Title: The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

doi: 10.1038/s41380-021-01044-x

Figure Lengend Snippet: A Overexpression of Fkbp5 in the PVN was achieved by bilateral viral injections. B Validation of Fkbp5 mRNA overexpression in the PVN by ISH (see Supplementary Fig. for mRNA quantification). C Fkbp5 PVN OE mice ( n = 20) showed significantly increased adrenal weights and a reduced thymus weight under non-stressed conditions compared to the controls ( n = 20). D Fkbp5 overexpression resulted in heightened corticosterone levels during the day. E Fifteen and 30 min after stress onset, Fkbp5 PVN OE mice displayed significantly higher corticosterone levels. F Fkbp5 PVN OE mice showed significantly elevated corticosterone 6 h after dexamethasone treatment. The following CRH injection further significantly increased the corticosterone release compared to controls. Data are presented as mean ± SEM. All data were received from mice between 14 and 20 weeks of age and analyzed a with student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant.

Article Snippet: The following primary antibodies were used: Actin (1:5000, Santa Cruz Biotechnologies, sc-1616), GR (1:1000, Cell Signaling Technology, #3660), p-GR Ser211 (1:500, Sigma, SAB4503820), p-GR Ser226 (1:1000, Sigma, SAB4503874), p-GR 203 (1:500, Sigma, SAB4504585), FKBP51 (1:1000, Bethyl, A301-430A).

Techniques: Over Expression, Biomarker Discovery, Injection

Journal: Molecular Psychiatry

Article Title: The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

doi: 10.1038/s41380-021-01044-x

Figure Lengend Snippet: A Experimental procedure. B Validation of successful Fkbp5 rescue by ISH (see Supplementary Fig. for mRNA quantification) and comparable WT Fkbp5 expression. C The reinstatement of Fkbp5 in the PVN resulted in increased adrenal weights and elevated morning corticosterone levels under basal conditions D . Furthermore, Fkbp5 re-instated animals displayed a significantly higher corticosterone response after restraint stress E . For comparison of Fkbp5 Frt/Frt and WT see control experiments in Supplementary Fig. . Data are presented as mean ± SEM. All data were received from mice between 16 and 20 weeks of age and analyzed a with student’s t -test. * p < 0.05.

Article Snippet: The following primary antibodies were used: Actin (1:5000, Santa Cruz Biotechnologies, sc-1616), GR (1:1000, Cell Signaling Technology, #3660), p-GR Ser211 (1:500, Sigma, SAB4503820), p-GR Ser226 (1:1000, Sigma, SAB4503874), p-GR 203 (1:500, Sigma, SAB4504585), FKBP51 (1:1000, Bethyl, A301-430A).

Techniques: Biomarker Discovery, Expressing, Comparison, Control

Journal: Molecular Psychiatry

Article Title: The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

doi: 10.1038/s41380-021-01044-x

Figure Lengend Snippet: A Animals lacking Fkbp5 in the PVN showed significantly lower phosphorylation at pGR Ser203 and higher levels of ( B ) pGR Ser211 and ( C ) pGR Ser234 compared to the control animals. D Fkbp5 PVN−/− mice showed no differences in total GR. E Fkbp5 PVN OE animals showed the opposite effect on GR phosphorylation with higher phosphorylation on pGr Ser203 . F In addition, we observed significantly lower phosphorylation at the GR sites Ser 211 and ( G ) Ser 234 . H Fkbp5 overexpression had no effect on total GR protein level. Representative blots are shown in ( D ) and ( H ). Group size for ( A )–( H ): 6 vs. 6. Data were received from animals between 16 and 20 weeks of age and are presented as relative fold change to control condition, mean ± SEM, and were analyzed with a two-way ANOVA. *significant genotype effect, (* p < 0.05, ** p < 0.01, *** p < 0.001). +significant genotype × stress interaction ( + p < 0.05), # significant stress effect ( # p < 0.05, ## p < 0.01).

Article Snippet: The following primary antibodies were used: Actin (1:5000, Santa Cruz Biotechnologies, sc-1616), GR (1:1000, Cell Signaling Technology, #3660), p-GR Ser211 (1:500, Sigma, SAB4503820), p-GR Ser226 (1:1000, Sigma, SAB4503874), p-GR 203 (1:500, Sigma, SAB4504585), FKBP51 (1:1000, Bethyl, A301-430A).

Techniques: Phospho-proteomics, Control, Over Expression

Journal: Molecular Psychiatry

Article Title: The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

doi: 10.1038/s41380-021-01044-x

Figure Lengend Snippet: A Single-cell sequencing depicted several different cell types. With the majority being neurons (38%), ependymal cells (25%), and astrocytes (14%). Fkbp5 + cells are highlighted. B Diversity of Fkbp5 + cell population. C Neurons could be divided mostly into GABAergic (66%) and glutamatergic (Glut, 11%) cells. Furthermore, the well-known stress markers corticotropin-releasing hormone ( Crh , 6%), somatostatin ( Sst , 5%), oxytocin ( Oxt , 2%), and vasopressin ( Avp , 1%) could be detected under basal conditions. D Fkbp5 + cells of selected neuronal subpopulations under basal conditions. E RNAscope validation of Fkbp5 mRNA expression in Crh + neurons. F Quantity of Fkbp5 + cells, as well as Fkbp5 mRNA levels, are significantly increased after stress. Data are were received from animals between 8 and 12 weeks of age and are presented as mean ± SEM. For ( E ): data were analyzed with student’s t -test. *** p < 0.001.

Article Snippet: The following primary antibodies were used: Actin (1:5000, Santa Cruz Biotechnologies, sc-1616), GR (1:1000, Cell Signaling Technology, #3660), p-GR Ser211 (1:500, Sigma, SAB4503820), p-GR Ser226 (1:1000, Sigma, SAB4503874), p-GR 203 (1:500, Sigma, SAB4504585), FKBP51 (1:1000, Bethyl, A301-430A).

Techniques: Sequencing, RNAscope, Biomarker Discovery, Expressing

Journal: Molecular Psychiatry

Article Title: The co-chaperone Fkbp5 shapes the acute stress response in the paraventricular nucleus of the hypothalamus of male mice

doi: 10.1038/s41380-021-01044-x

Figure Lengend Snippet: A Crh- specific overexpression of Fkbp5 in the PVN was achieved by bilateral injections of a Cre-dependent Fkbp5 overexpression virus in the PVN of CRH-ires-CRE/Ai9 mice. B Validation of Crh -specific Fkbp5 mRNA overexpression in the PVN by ISH and RNAscope. Arrowheads pointing at viral Fkbp5 expressing Crh + neurons (For mRNA quantification see Supplementary Fig. ). C Fkbp5 CRH OE mice showed significantly increased adrenal weights ( n = 10) and reduced thymus weights ( n = 9) under non-stressed conditions compared to the controls ( n = 10). D Corticosterone levels were mildly, but not significantly increased in Fkbp5 CRH OE at 15, 30, and 60 min post-stress ( Fkbp5 CRH OE n = 10; Control n = 9). E Fkbp5 CRH OE mice displayed a decrease in CRH neuronal mEPSC frequency accompanied by an increased amplitude ( Fkbp5 CRH OE n mouse = 4; n neuron = 29; Control n mouse = 4; n neuron = 25), which is reflected in two representative recording traces. Data are presented as mean ± SEM. All data were received from animals between 30 and 34 weeks of age and analyzed with the student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, T = 0.05 < p < 0.1, n.s . not significant.

Article Snippet: The following primary antibodies were used: Actin (1:5000, Santa Cruz Biotechnologies, sc-1616), GR (1:1000, Cell Signaling Technology, #3660), p-GR Ser211 (1:500, Sigma, SAB4503820), p-GR Ser226 (1:1000, Sigma, SAB4503874), p-GR 203 (1:500, Sigma, SAB4504585), FKBP51 (1:1000, Bethyl, A301-430A).

Techniques: Over Expression, Virus, Biomarker Discovery, RNAscope, Expressing, Control