Journal: bioRxiv

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

doi: 10.1101/824664

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. (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 minutes restraint stress until at least 60 minutes 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 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: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: Fkbp5 deletion in the PVN has no effect basal CORT levels ( Fkbp5 PVN−/− n = 16; Fkbp5 lox/lox n =15) (A). ACTH levels under basal ( Fkbp5 PVN−/− n = 16; Fkbp5 lox/lox n =15) (B) and 30 minutes after stress onset were unaltered ( Fkbp5 PVN−/− n = 16; Fkbp5 lox/lox n =15) (C) . (D) mRNA changes of stress responsive genes within the PVN ( Fkbp5 PVN−/− n = 16; Fkbp5 lox/lox n =15). (E) Fkbp5 protein levels were not detectable under basal conditions. All data are presented as mean ± SEM and were analyzed with a student’s t-test. n.d. = not detectable; n.s. = not significant; T = 0.05 < p < 0.1.

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:

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: (A) Animals with an age of 10 weeks had no alterations in body physiology. (B) Morning and evening corticosterone levels were unchanged. (C) Fkbp5 PVN−/− had significant lower corticosterone levels 15 minutes after stress onset. (Group size for A-C: Fkbp5 PVN−/− n = 9; Fkbp5 lox/lox n =9). All data are presented as mean ± SEM and were analyzed with a student’s t-test. n.s. = not significant; T = 0.05 < p < 0.1; * = 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:

Journal: bioRxiv

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

doi: 10.1101/824664

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. (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) 15 and 30 minutes 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 analyzed 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: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: (A-B) ACTH levels were significantly higher in Fkbp5 PVN OE mice under basal (n = 20 vs. 20) and unchanged 30 minutes after stress (n = 12 vs. 12= onset compared to their controls (C) . We did not detect any differences in corticosterone levels 90 minutes after stress onset (n = 20 vs. 20). (D) Fkbp5 overexpression resulted in significant increase in mRNA levels of Fkbp5 , N1c3 and Crh . Avp levels stayed unchanged (n = 20 vs. 20). (E) Viral overexpression resulted in a 4-fold Fkbp5 protein upregulation in the PVN (n = 20 vs. 20). All data are presented as mean ± SEM and were analyzed with a student’s t-test (A-D) or with a two way ANOVA (E). n.s. = not significant; T = 0.05 < p < 0.1. * = p < 0.05, *** = 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: Over Expression

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: (A) Experimental procedure. (B) Validation of successful Fkbp5 rescue by ISH. (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) . Data are presented as mean ± SEM. All data were analyzed 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

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: Fkbp5 reinstatement had no effect on thymus weights (A) . Basal ACTH and evening corticosterone levels were unaltered (B & C) . (D) Rescue of endogenous Fkbp5 in global knock-out animals had no significant effect on the Dex/CRH test and on stress responsive mRNA levels in the brain. (E) mRNA levels in Fkbp5 Rescue animals compared to global knock-out littermates. Group sizes for A-E: Fkbp5 Rescue n = 10; Fkbp5 Frt/Frt n =9) All data are presented as mean ± SEM and were analyzed with a student’s t-test. 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: Knock-Out

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: (A) Animals lacking Fkbp5 in the PVN showed significantly lower phosphorylation at pGR Ser203 and higher levels of pGR Ser211 (B) and pGR Ser234 (C) compared to the control animals. Fkbp5 PVN OE animals showed the opposite effect on GR phosphorylation with higher phosphorylation on pGr Ser203 (E) . Additionally, we observed a significantly lower phosphorylation at the GR sites Ser 211 (F) and Ser 234 (G) . Representative blots are shown in (D) and (H) . Group size for A-H: 6 vs. 6. Data are presented as mean ± SEM and were analyzed with a two-way ANOVA. * = significant genotype effect, (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). + = significant genotype x 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

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: (A) GR protein levels were unchanged in Fkbp5 PVN−/− animals (n = 6) and (B) in Fkbp5 PVN OE animals (n = 6) under basal and stressed conditions. All data were analyzed with a two-way ANOVA.

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:

Journal: bioRxiv

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

doi: 10.1101/824664

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) 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. (C) Diversity of Fkbp5 + cell population.

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

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: In comparison to single-cell sequencing, RNAscope revealed a significant higher co-localization ratio of Fkbp5 in oxytocin ( Oxt ), corticotropin-releasing hormone ( Crh ), vasopressin ( Avp ), somatostatin ( Sst ) and thyronine releasing hormone ( Trh ) neurons. Overnight food deprivation increased Fkbp5 mRNA in all cell populations.

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: Comparison, Sequencing, RNAscope

Journal: bioRxiv

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

doi: 10.1101/824664

Figure Lengend Snippet: Under basal conditions Fkbp5 mRNA signal was detected in 83% of Oxt + neurons, 65% Of Crh + neurons, 68% in Avp + and 84% of Sst + neurons. We monitored an increased in Fkbp5 mRNA expression in all neuronal populations. Interestingly, only Crh + neurons showed a significant increase of Fkbp5 mRNA following stress. All data are presented as mean ± SEM and were analyzed with a student’s t-test. ** = 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: Expressing