FKBP5 Exacerbates Impairments in Cerebral Ischemic Stroke by Inducing Autophagy via the AKT/FOXO3 Pathway

Abstract

Cerebral ischemic stroke is regarded as one of the most serious diseases in the human central nervous system. The secondary ischemia and reperfusion (I/R) injury increased the difficulty of treatment. Moreover, the latent molecular regulating mechanism in I/R injury is still unclear. Based on our previous clinical study, we discovered that FK506 binding protein 5 (FKBP5) is significantly upregulated in patients, who suffered acute ischemic stroke (AIS), with high diagnostic value. Levels of FKBP5 were positively correlated with patients' neurological impairments. Furthermore, a transient middle cerebral artery occlusion (tMCAO) model of mice was used to confirm that FKBP5 expression in plasma could reflect its relative level in brain tissue. Thus, we hypothesized that FKBP5 participated in the regulation of cerebral I/R injury. In order to explore the possible roles FKBP5 acted, the oxygen and glucose deprivation and reoxygenation (OGD/R) model was established to mimic I/R injury in vitro. FKBP5 expressing levels were changed by plasmid stable transfection. The altered expression of FKBP5 influenced cell viability and autophagy after OGD/R injury notably. Besides, AKT/FOXO3 cascade was involved in the FKBP5-regulating process. In the present study, FKBP5 was verified upregulated in cerebral I/R injury, related to the severity of ischemia and reperfusion injury. Additionally, our analyses revealed that FKBP5 regulates autophagy induced by OGD/R via the downstream AKT/FOXO3 signaling pathway. Our findings provide a novel biomarker for the early diagnosis of ischemic stroke and a potential strategy for treatment.

Keywords: FKBP5; FOXO3; autophagy; cerebral ischemic stroke; ischemia and reperfusion injury.

Figure 1

FK506 binding protein 5 (FKBP5) was upregulated notably in acute ischemic stroke (AIS) patients. (A) Expression of FKBP5 in plasma of AIS patients (n = 50) and healthy controls (n = 50) examined by real-time qPCR. (B) Expression of FKBP5 in plasma of AIS patients (n = 50) and healthy controls (n = 50) tested by ELISA. Data are presented as the mean ± SD. *P < 0.05 vs. health control group. (C) Linear regression analysis was applied in each individual between the FKBP5 expression and National Institute of Health Stroke Scale (NIHSS) score, **P < 0.01. (D) Receiver-operator characteristic (ROC) curve of FKBP5 in the diagnosis of AIS (n = 50). (E) Linear regression analysis of the relationship between FKBP5 expression and diffusion-weighted imaging (DWI) volume of lesion, **P < 0.01.

Figure 2

FKBP5 was significantly upregulated in transient middle cerebral artery occlusion (tMCAO) mice. (A,B) Infarct volume in the brain tissues of tMCAO and sham mice (n = 6 each group). (C,D) Expression of FKBP5 in plasma and brain tissue of tMCAO and sham mice detected by real-time qPCR (n = 10 each group). (E,F) Expression of FKBP5 in the brain tissue of tMCAO and sham mice detected by Western blot assay (n = 10 each group). IDV is short for integrated density values. Data are presented as the mean ± SD. *P < 0.05 vs. sham group. (G) Linear regression analysis was performed in each tMCAO mouse between the FKBP5 expression in brain tissue and plasma (n = 10 each group), **P < 0.01.

Figure 3

FKBP5 was upregulated in oxygen and glucose deprivation and reoxygenation (OGD/R) cells and establishments of stable transfected cell lines. (A) Expression of FKBP5 in OGD/R and control group quantified by real-time qPCR. (B,C) Expression of FKBP5 in OGD/R and control group determined by Western blot assay. *P < 0.05 vs. control group. (D,E) Protein levels of FKBP5 after stably transfecting plasmids with FKBP5(+), FKBP5(−), and their relative noncoding (NC) sequences. Data are presented as the mean ± SD. *P < 0.05 vs. FKBP5(+)-NC group. ^#P < 0.05 vs. FKBP5(−)-NC group. For (A–E), n = 3 in each group.

Figure 4

FKBP5 aggravated cell damage via autophagy. (A) CCK-8 assay was performed to assess the influences of FKBP5 and autophagy on cell viability. **P < 0.01 vs. control group. ^#P < 0.05 vs. OGD/R + FKBP5(+)-NC group. ^▴P < 0.05 vs. OGD/R + FKBP5(−)-NC group. ^&P < 0.05 vs. OGD/R group. ^▾P < 0.05 vs. OGD/R + 3-MA+FKBP5(+)-NC group. (B–D) Western blot analysis of SQSTM1 and LC3B II expression in FKBP5 stably transfected cell lines. GAPDH was regarded as an endogenous control. Data are presented as the mean ± SD. Statistical analysis was conducted with nonparametric Mann–Whitney test. **P < 0.01 vs. control group. ^#P < 0.05 vs. OGD/R group. (E) TEM was used to observe the autophagosomes in OGD/R cells with altered FKBP5 expression. Arrows point to autophagic vacuoles (AVs) with double membranes. Scale bars represent 1 μm. For (A–E), n = 3 in each group.

Figure 5

FKBP5-mediated autophagy induced by OGD/R. (A) The mRFP-GFP-LC3B adenovirus was infectious for autophagy flux to be monitored in OGD/R cells with FKBP5 expression alteration. Scale bars represent 10 μm. (B) During each experiment, more than 100 cells were counted. Data are presented as the mean ± SD. (C) Immunofluorescence staining assay with red dots for LC3B expression in the OGD/R group changing with FKBP5 expression. Scale bars represent 40 μm. (D) More than 100 cells were taken into account. Experiments were repeated three times. *P < 0.05 vs. control group. ^#P < 0.05 vs. OGD/R group. For (A–D), n = 3 in each group.

Figure 6

FKBP5-regulated AKT/FOXO3 signaling pathway. (A–C) Protein levels of p-FOXO3/FOXO3 and p-AKT/AKT in the OGD/R group with altered FKBP5 expression. *P < 0.05 vs. control group. ^#P < 0.05 vs. OGD/R + FKBP5(+)-NC group. ^▴P < 0.05 vs. OGD/R + FKBP5(−)-NC group. (D–F) Western blot analysis of p-FOXO3, FOXO3, p-AKT, and AKT expression in OGD/R cells with the treatment of FKBP5(−) and LY294002. GAPDH was regarded as an endogenous control. *P < 0.05 vs. control group. ^#P < 0.05 vs. OGD/R + FKBP5(−)-NC group. ^▴P < 0.05 vs. OGD/R + FKBP5(−) group. For (A–F), n = 3 in each group.