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Knockout of programmed cell death 5 (PDCD5) gene attenuates neuron injury after middle cerebral artery occlusion in mice
Brain Research 1650 (2016) 152–161
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Research report
Knockout of programmed cell death 5 (PDCD5) gene attenuates neuron injury after middle cerebral artery occlusion in mice Jianfei Lu a, Zhao Jiang a, Yingyu Chen b, Changman Zhou a, Chunhua Chen a,n a b
Department of Anatomy and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China Peking University Center for Human Disease Genomics, Beijing 100191, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 May 2016 Received in revised form 30 August 2016 Accepted 4 September 2016 Available online 5 September 2016
Loss of von Hippel-Lindau tumor suppressor protein (VHL) or hypoxia results in nuclear relocalization of PDCD5 and subsequent mouse double minute 2 homolog (Mdm2) degradation. Thus, VHL may involved in the PDCD5 mediated apoptosis and autophagy after MCAO. In the present study, using PDCD5 knockout (PDCD5-/-) mice, we aimed to demonstrate that knockout of PDCD5 gene could protect the brain from ischemic injury by inhibiting the PDCD5-VHL pathway. 24 h post MCAO surgery, PDCD5 gene knockout mice presented obvious improved brain blood flow, improved neurological behavior and decreased cerebral infarction compared with wild type mice. The levels of apoptotic and autophagic proteins were increased both in wild type and PDCD5 knockout mice, whereas they were more pronounced in the wild type mice. We observed decrease in the expression of VHL in wild type mice after MCAO. Reduced expression of VHL may result in increased expression of hypoxia-inducible factor 1α(HIF-1α) and (BCL2/ adenovirus E1B 19 kDa protein-interacting protein 3) BNIP3. However, mice lacking PDCD5 had no changes in the expression of VHL and have slighter increases in the expression of HIF-1α and BNIP3, suggesting that PDCD5 may regulate apoptosis and autophagy through VHL-HIF-1α-BNIP3 pathway. & 2016 Elsevier B.V. All rights reserved.
Keywords: Apoptosis Autophagy Knockout mice Middle cerebral artery occlusion (MCAO) Programmed cell death 5 (PDCD5)
1. Introduction Stroke is a worldwide burden with high occurrence of morbidity, mortality and disability. Among all types of stroke, 87% are ischemic stroke (Feigin et al., 2015). Ischemic brain injury results from a complex sequence of pathophysiological events that evolve over time and space. In the core area, cells are killed rapidly because of bioenergetic failure and breakdown of ion homeostasis. The penumbra, which lies between damaged core and normal brain, is an area of constrained blood flow with partially preserved energy metabolism (Dirnagl et al., 1999). Many researchers focus on this area for the salvage of a reversible brain damage. Programmed cell death 5 (PDCD5), formerly designated as TFAR19 (TF-1-cell apoptosis-related gene 19), is an apoptosis accelerating gene but it could not induce apoptosis solely (Liu et al., 1999). Under apoptosis, PDCD5 protein is significantly increased and translocates rapidly from the cytoplasm to the nucleus of cells Abbreviations: PDCD5, programmed cell death 5; RhPDCD5, recombinant human programmed cell death 5; MCAO, middle cerebral artery occlusion; VHL, von Hippel-Lindau tumor suppressor protein; Mdm2, mouse double minute 2 homolog; HIF-1α, hypoxia-inducible factor 1 alpha; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; WT, wild type; KO, knockout n Corresponding author. E-mail address: [email protected] (C. Chen). http://dx.doi.org/10.1016/j.brainres.2016.09.005 0006-8993/& 2016 Elsevier B.V. All rights reserved.
(Chen et al., 2001). PDCD5 is expressed in a wide variety of tissues both in human and mouse (Liu et al., 1999), yet a decreased expression has been detected in various human tumors, such as lung cancer (Spinola et al., 2006), gastric cancer (Yang et al., 2006), acute and chronic myelogenous leukemia (Ruan et al., 2006), prostate cancer (Du et al., 2009), epithelial ovarian carcinomas (Zhang et al., 2011), astrocytic gliomas (Li et al., 2008) and chondrosarcoma (Chen et al., 2010). Functional studies found that PDCD5 could bind to Tip60, enhance its stability and function as a Tip60 coactivator to promote apoptosis after DNA damage (Xu et al., 2009). PDCD5 also could enhance the stability of p53 by dissociating the interaction between p53 and Mdm2 and interact with Mdm2 to promote its degradation (Xu et al., 2012). Our previous study indicated that the expression of PDCD5 increased in ischemic neurons after MCAO in vivo (Chen et al., 2013). Meanwhile, reduction of expression of PDCD5 has a neuronal protective role after stroke and the effect is related to the inhibition of apoptosis and autophagy (Jiang et al., 2014). PDCD5siRNA could reduce the expression of some apoptosis and autophagy related protein such as p53, bax, caspase-3, LC3 and Beclin-1. But the direct relationship between PDCD5 and these proteins are still unknown. Recent studies mentioned that PDCD5 could directly bind to von Hippel–Lindau (VHL) which is a tumor suppressor gene and its mutation is related to some cancers (Essers et al., 2015). VHL could
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regulate the ubiquitination of HIF-1α and subsequent proteasomal degradation (Jaakkola et al., 2001; Park et al., 2014). Former study demonstrated that HIF-1α is involved in the regulation of apoptosis followed by MCAO (Chen et al., 2009). Other vitro studies found HIF-1α regulate autophagy in hypoxia condition (Zhang et al., 2008; Wu et al., 2015). Thus, PDCD5 might regulate programmed cell death through VHL and HIF-1α pathway. In this study, we used PDCD5 knockout and wild type mice to conduct the middle cerebral artery occlusion and reperfusion (MCAO) model. Then, brain infarction, blood–brain barrier (BBB) disruption, and neurological deficits were observed. We used western blot and immunohistochemistry techniques to evaluate the expression of endogenous PDCD5, VHL, HIF-1α, BNIP3 and tried to explore the mechanism of PDCD5 mediated apoptosis and autophagy.
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knockout mice (Fig. 1b). The expression of PDCD5 was detected by Immunofluorescence and Western blot analysis at 24 h after MCAO. We found that PDCD5 significantly increased in the penumbra area 24 h after MCAO compared with Sham group (Fig. 1c&d). 2.2. Knock out of PDCD5 gene significantly improved brain blood flow after MCAO During the ischemic period, the blood flow of the core area of MCA was blocked. After reperfusion, the blood flow of the area would recover temporarily. When ischemia occurred, the blood flow among MCAO, PDCD5 KO and KOþRhPDCD5 groups has no significant difference. But 24 h after stroke, the blood flow of all the three groups improved in various degrees. But PDCD5 KO group has a more significant improvement compared with other groups (Fig. 2a&b).
2. Results
2.3. Knockout of PDCD5 gene reduced cerebral infarction
2.1. PDCD5 protein does not express in the neurons of PDCD5 knockout mouse
The cerebral infarction (white colored area) at 24 h after stroke was shown in Fig. 3a. The ratio of the lesion in four different groups was shown in Fig. 3b. Compared with the Sham group, all the mice after MCAO have severe infarction. But the lesion area of the PDCD5 KO group was reduced about 15.1% compared to the wild type mice. The infarction ratio of the PDCD5 KO mice that were injected with rhPDCD5 protein has a significant increase.
Double Immunofluorescence labeling indicated that in the hippocampus and cortex area of wild type mice, PDCD5 (green) colocalized with the neuronal marker MAP-2 (red, microtubule-associated protein 2) (Fig. 1a). But it rarely expressed in neurons of PDCD5
Fig. 1. Expression and localization of PDCD5 after MCAO. (a) Double immunofluorescence staining showed the colocalization of PDCD5 (green) with MAP2 (red) in hippocampus and cortex. (b) Immunofluorescene staining using FITC-conjugated PDCD5 antibody was detected in the penumbra area. The expression of PDCD5 in MCAO group is stronger than sham mice and no positive cell was observed in PDCD5 knockout mice. (c, d) Western blot showed that the expression of PDCD5 increased at 24 h and the PDCD5 knockout mice rarely expressed PDCD5. *p o 0.05 versus Sham WT. Bar ¼100 mm. Values are the mean 7 SEM.
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Fig. 2. Effects of Knock out PDCD5 gene on cerebral blood flow after MCAO. (a) The representative images of cerebral blood flow (CBF) of the whole brain in different groups. The magnitude of CBF is represented by different colors, with blue to red denoting low to high. (b) Quantitative analysis of CBF in different groups. 24 h after reperfusion, The CBF of the mice suffering stroke improved. *p o 0.05. PDCD5 KO mice had a more pronounced improvement compared to other two surgery groups. #p o 0.05 versus PDCD5 KO perfusion 24 h. Values are the mean 7 SEM.
2.4. Knockout of PDCD5 gene improved neurological score The neurological score was 15.83371.169 in Sham group, 6.00071.414 in MCAO group, 8.66771.366 in PDCD5 KO group and 5.33371.211 in PDCD5 KOþRhPDCD5 group. Knock out of PDCD5 gene could improve the neurological behavior outcome (Fig. 3c). 2.5. Knockout of PDCD5 gene decreased apoptosis and autophagy levels after MCAO Previous studies have confirmed that PDCD5 was involved in the apoptosis and autophagy processes. Lower expression of PDCD5 could attenuate apoptosis and autophagy levels (Jiang et al., 2014; Chen et al., 2013). We detected autophagy related protein such as Beclin 1, LC3, LC3-ll, P62 and apoptosis related protein Bax in the three groups. As showed in Fig. 4a, knock out of PDCD5 gene could attenuate the expression of Beclin 1, LC3, LC3-ll, P62 and Bax compared with MCAO group. The results of TUNEL staining verified this outcome. Seldom positive cells were observed in Sham group. In MCAO and PDCD5 KO groups, positive cells could be observed in the penumbra area. The axons and dendrites disappeared and nuclei were disrupted in MCAO group, but rarely in PDCD5 KO mice (Fig. 8). As double immunofluorescence labeling showed, all these proteins (Red) colocalized with PDCD5 (Green). These results indicated that PDCD5 may have effects on apoptosis and autophagy (Fig. 4b).
2.6. Knockout of PDCD5 gene attenuated the decreased expression of VHL and the increased expression of HIF-1α after MCAO To gain insight into the relationship between PDCD5 and VHL, we examined the expression of VHL and HIF-1α in various groups. Western blot (Figs. 5 and 6) and immunohistochemistry staining (Fig. 8) indicated that the expression of VHL decreased in the penumbra area of wild type mice after MCAO. But in the similar area of PDCD5 KO mice, there was no significant difference compared to Sham group. HIF-1α had an increased expression both in wild type mice and PDCD5 KO mice after MCAO. But the enhancement was slighter in KO mice. We speculated that PDCD5 may regulate the expression of HIF-1α via VHL and then promote apoptosis and autophagy.
2.7. PDCD5 regulated apoptosis and autophagy processes via BNIP3 Previous research confirmed that HIF-1α could regulate apoptosis and autophagy through BNIP3. Our results revealed that the expression of BNIP3 increased in MCAO group and PDCD5 KO group. But there was a significant reduction in PDCD5 KO mice compared to wild type mice (Figs. 7 and 8) which indicated that PDCD5 may influence the expression of BNIP3 via VHL and HIF-1α, and then regulate apoptosis and autophagy.
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Fig. 3. Effects of Knock out PDCD5 gene on infarction size and neurological function after MCAO. (a) Representative images of brain slices stained by TTC in different groups at 24 h after reperfusion. Normal brain tissue is stained red, while the infarction remains unstained. (b) Quantitative analysis of infarct volume in different groups. *po 0.05 versus Sham. #po 0.05 versus PDCD5 KO. (c) Neurological scores of mice in different groups evaluated at 24 h after reperfusion. *p o 0.05 versus Sham. #p o0.05 versus PDCD5 KO. Values were expressed as mean 7SEM.
3. Discussion In the present study, we demonstrated that the PDCD5 knockout mice are resistant to ischemia and reperfusion injury compared to wild type mice. And this resistance was compromised after the injection of rhPDCD5. PDCD5 is a well-established protein associated with apoptosis. Chen et al. (2001) found that PDCD5 alone could not induce apoptosis, but over expression of PDCD5 could enhance apoptosis independent of types of cells and apoptosis inductions. Cell suicide is the predominant mechanism that follows MCAO, particularly within the ischemic penumbra. Hence, it’s a potential therapeutic application to block or inhibit this procedure (Dirnagl et al., 1999). For that reason, we applied PDCD5siRNA to reduce the expression of PDCD5. Results showed that i.c. v injection of PDCD5siRNA could reduce the infarction volume, improve neurological behavior and maintain the integrity of BBB after MCAO (Chen et al., 2013). In this study, we found that knockout of PDCD5 gene has a neuroprotective effect after MCAO consistently. In addition to infarction volume and behavior changes, we also found that inhibition of PDCD5 could reduce the expression of
apoptotic-related proteins such as p53, Bax and caspase-3 (Chen et al., 2013). And recent study found that PDCD5 was involved in autophagy as well. Intracerebroventricularly injection of PDCD5siRNA could reduce the expression of Beclin 1 and LC3, revealing that PDCD5 mediated the autophagic cell death via Beclin 1 and LC3 following MCAO injury (Jiang et al., 2014). We observed that knockout of PDCD5 gene could decrease ischemia induced apoptosis and autophagy concomitantly, and affect the related genes especially Bax, LC3-ll and P62. These insights are important to further understand the role of PDCD5 in ischemia and reperfusion injury. PDCD5 could directly bind to pVHL and knockout of VHL could induce the nuclear translocation and subsequent degradation of MDM2 to maintain the stability of P53 (Essers et al., 2015). VHL is a tumor suppressor gene and is involved in the ubiquitination of HIF1α and subsequent proteasomal degradation via the VHL ubiquitination complex (Jaakkola et al., 2001; Park et al., 2014). In normoxia, HIF-1α is degraded through the VHL ubiquitination pathway. HIF-1α functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, HIF-1α activates the
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Fig. 4. Effects of knock out PDCD5 gene on autophagy and apoptosis levels after MCAO. (a) Autophagy and apoptosis related protein Beclin 1, LC3, LC3-ll, P62 and Bax were detected in the penumbra area. Almost no Beclin 1, LC3, LC3-ll and Bax were observed in Sham group. Slight P62 positive cells were observed in Sham mice. Massive immunoreactivity of these proteins was localized in the penumbra area. However, the extent of these proteins lessened in PDCD5 KO mice. (b) Double immunofluorescence labeling showed Beclin 1, LC3, LC3-ll, P62 and Bax (Red) were all colocalized with PDCD5 (Green). Bar ¼100 mm.
transcription of many apoptosis and autophagic genes including BNIP3 (Sowter et al., 2001). BNIP3 is a pro-apoptotic mitochondrial protein and Bcl-2 family member (Chen et al., 1997). BNIP3 associates with anti-apoptotic family members Bcl-2, Bcl-xL, and the adenovirus homolog E1B-19 kDa. Different from other Bcl-2 family members, the TM domain of BNIP3 instead of the BH3 domain is critical for heterodimerization with Bcl-2, Bcl-XL and pro-apoptotic activity (Ray et al., 2000). In addition to apoptosis, BNIP3 has been implicated in autophagy. In hypoxic conditions, BNIP3 can induce mitochondrial autophagy (mitophagy) by displacing Beclin 1 from Bcl-XL and Bcl-2 (Azad et al., 2008; Bellot et al., 2009). BNIP3 can also promote mitophagy by mediating the translocation of the E3 ubiquitin ligase Parkin to the mitochondria or by directly interacting with LC3 on the autophagosome (Lee et al., 2011; Hanna et al., 2012; Shi et al., 2014). Based on the above, we provided evidence that showed the expression of VHL was significantly reduced after MCAO in wild type mice. But knockout of PDCD5 gene could weaken this effect. Robust interaction between pVHL and PDCD5 was observed via tandem-affinity purification (Essers et al., 2015). It seems that VHL was degraded or inhibited through PDCD5 after MCAO. We further demonstrated that knockout of PDCD5 gene resulted in significant inhibition of HIF-1α and BNIP3. That may related to VHL regulated ubiquitination and subsequent proteasomal degradation. These results suggesting VHL, HIF-1α and BNIP3 were associated with PDCD5 in the regulation of apoptosis and autophagy. Chen et al. (2001) found PDCD5 translocates to the nucleus in cells undergoing apoptosis. But the occurrence time of translocation varied depends on the cell types and apoptosis inductions. Former research found this phenomenon in rats 24 h after MCAO. But in
this study, we didn’t find the translocation in mice at 24 h after MCAO. The translocation of PDCD5 occurred at 48 h after MCAO or later (data not shown). That means PDCD5 probably participate apoptosis and autophagy in two phases, the cytoplasm and the nuclear. The difference might come from different species which needed further exploration. Our data demonstrated that there has a crosstalk between apoptosis and autophagy to co-mediate the cell death. Apoptosis is regarded as suicide in the penumbra, but the specific role of autophagy in brain ischemia and reperfusion injury is still controversial. The common view is that moderate autophagy has the protective role but severe autophagy may destroy the cell. Studies investigating cellular death following neonatal hypoxia–ischemia have shown that autophagy has neuroprotective role. In our results, knockout of PDCD5 gene could reduce the autophagy level after MCAO and have a protective role which indicated that PDCD5 mediated apoptosis and autophagy might have a synergy effect. Our further study will focus on the direct interaction between PDCD5 and VHL-HIF-1α-BNIP3 pathway. PDCD5 can also dissociate p53 and Mdm2 and maintain the basal level of p53. Upon DNA damage, PDCD5 can mediate p21 transactivation and G1 phase arrest and allow cells to initiate productive DNA repair processes (Xu et al., 2012). PDCD5 together with Tip60 can also promote p53K120 acetylation and transactivation of Bax expression in order to initiate an apoptotic response (Xu et al., 2009). We have verified P53 involved in PDCD5 mediated apoptosis and autophagy after MCAO (Chen et al., 2013). The interaction between P53 and PDCD5 in ischemia and reperfusion injury is another direction of our future research.
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Fig. 5. Western blot and immunofluorescence labeling for the detection of VHL. (a) Western blot indicated the expression of VHL decreased in MCAO group. No significant difference was found between Sham group and PDCD5 KO group. (b) Quantitative analysis of VHL in different groups. *p o 0.05 versus Sham. Values are the mean7 SEM. (c) Double immunofluorescence labeling showed VHL (red) localized in cytoplasm and was colocalized with PDCD5 (green). Bar¼ 100 mm.
Our results indicated that knockout of PDCD5 gene had a direct neuronal protective effect that reduced programmed cell death of neurons under the hypoxia condition and increased the survival rate of neurons on penumbra area. In addition, we can test the effects of knockout PDCD5 on white matters and vascular endothelial cells in the further study. In conclusion, PDCD5 participated in the process of both apoptosis and autophagy after MCAO. Knock out of the PDCD5 gene could attenuate neuron injury and the role was related to VHL-HIF-1α-BNIP3 pathway. Thus, inhibiting or silencing PDCD5 might be a new strategy applied to the therapy of ischemia and reperfusion injury.
surgery, animals were anesthetized using compound anesthetic agent (3 ml/kg) with a mixture of 5% chloral hydrate, 1% pentobarbital sodium and 2% magnesium sulfate. Under operating microscope, the right common carotid, internal carotid and external carotid arteries were surgically exposed. Then the external carotid artery was isolated and ligated. A 6-0 nylon suture with silicon rubber coated (coating length 3 mm, diameter 0.21 mm) was inserted into the internal carotid artery through the external carotid artery stump and gently advanced to occlude the MCA. After 1 h of MCAO, the nylon suture was carefully removed and the blood flow reperfused. The body (rectal) temperature was carefully maintained at 37.0 °C by a heating pad until the animal completely recovered from the anesthesia.
4. Experimental procedures
4.2. In vivo rhPDCD5 protein transfer
4.1. Animals and treatments
The stereotaxic coordinates were 0.4 mm posterior, 1.0 mm lateral to the bregma and 2.5 mm ventral to the surface of the skull. RhPDCD5 protein (70 μg/kg; Beijing Biosea Biotechnology Co., China) was injected intracerebroventricularly using a Hamilton microsyringe under guidance of stereotaxy instrument within 30 min after reperfusion.
All experimental protocol in this study was evaluated and approved by the Animal and Ethics Review Committee at Peking University Health Science Center in Beijing, China. Mice were purchased from the Center of Experimental Animals of Peking University Health Science Center and were kept under standard conditions with free access to food and water. Thirty six C57BL/6N male mice and twenty four C57BL/6N PDCD5 knockout mice weighing 20–24 g were randomly assigned to four groups: Sham surgery (N ¼ 18), MCAO (N ¼18), PDCD5 knockoutþ MCAO (KO) (N ¼ 18) and PDCD5 knockoutþMCAO þ RhPDCD5 (KO þRhPDCD5) (N ¼ 6). MCAO model was induced by Longa et al. (1989) with some added modifications (Longa et al., 1989; Chen et al., 2013). During
4.3. Infarct volume measurement 2,3,7-Triphenyltetrazolium chloride (TTC) staining for infarction volume measurement was conducted as described previously (Chen et al., 2009). Brains were sliced into 2-mm-thick coronal sections in an adult mouse brain matrix (Kent Scientific Corporation). The slices were stained in 1% 2,3,5-triphenyltetrazolium chloride solution (TTC; Sigma-Aldrich, St Louis, MO, USA) for
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Fig. 6. Western blot and immunofluorescence labeling for the detection of HIF-1α. (a) Western blot indicated the expression of HIF-1α increased both in MCAO and PDCD5 KO groups. Slighter expression of HIF-1α was found in PDCD5 KO group. (b) Quantitative analysis of HIF-1α in different groups. *p o0.05 versus Sham. #p o 0.05 versus MCAO ipsilateral hemisphere. Values are the mean 7 SEM. (c) Double immunofluorescence labeling showed HIF-1α (red) was colocalized with PDCD5 (green) in Sham group. HIF-1α mainly localized in the cytoplasm. After MCAO, it showed nuclear translocation in the infarction regions. Bar ¼100 mm.
20 min at 37 °C. The infarction tissue was stained white and the healthy part was pink. The infarcted areas of each section were measured by Image J. The infarction rate was measured with: infarcted area of the ipsilateral hemisphere/whole brain. 4.4. Neurological deficits The neurological scores were performed in a blinded fashion at 24 h after MCAO based on the scoring system reported by Garcia et al. (1995). The minimum neurological score was 3 and the maximum was 18. A lower score represented a more severe injury. 4.5. Cerebral blood flow measurement Cerebral blood flow (CBF) was measured using Laser Doppler perfusion image system (PeriScan PIM3 System; PERIMED, Stockholm, Sweden) (N ¼6) as previously described (Huang et al., 2012). We made an incision through the scalp and exposed the skull. The cerebral cortex was positioned in parallel to the computer-controlled optical scanner at a distance of 18.5 cm. The cerebral blood flow was measured at 1 h after ischemia and 24 h after reperfusion. A color-coded image to denote specific relative perfusion levels was displayed on a video monitor and the ischemia core area in all images was evaluated with the software LDPIwin 3.1 (PeriScan PIM3 System; PERIMED, Stockholm, Sweden). 4.6. Western blot Western blot analysis was performed as described previously (Chen et al., 2009). Tissues from ischemic penumbra area of ipsilateral hemisphere were homogenized. Bicinchoninic acid (BCA; Applygen Technologies) was used to detect the protein concentration
of each sample (Ashwal et al., 1998). Protein samples (50 μg) were loaded onto polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane (Hybond-C, Amersham Biosciences, USA). The nitrocellulose membranes were then blocked by incubation with the primary antibodies overnight at 4 °C. The primary antibodies were applied as follows: rabbit anti-VHL, rabbit anti-HIF-1α, rabbit anti-BNIP3 (Cell Signaling, Beverly, Massachusetts, USA). A monoclonal antibody against β-actin (Cell Signaling, Beverly, Massachusetts, USA) was used as a control group for protein gel loading. Immunoblots were processed with corresponding secondary antibodies (Applygen Technologies) for 1 h at room temperature. Immunoblots were probed and the bands were visualized by enhanced chemiluminescence detection reagents (Applygen Technologies). The optical density was detected by Bio-Rad image analysis. 4.7. Immunohistochemistry, double fluorescence labeling and TUNEL staining For the histological analysis, animals (N¼6) were anesthetized at 24 h after operation. After perfusion with 40 ml 0.01 M PBS and 40 ml of 4% paraformaldehyde in 0.1 M PB (pH¼7.4), brains were removed and postfixed with formalin, and cryoprotected in 30% sucrose in 0.1 M PB for over 24 h at 4 °C. Coronal brain sections (10 mm) were sliced using a cryostat (Leica CM3050 S). Immunohistochemistry was performed as described previously (Chen et al., 2009). After triple wash with PBS and blocking with 3% normal goat serum at room temperature for 1 h, the slices were incubated with primary antibodies diluted in PBS overnight at 4 °C. The mouse anti-PDCD5 monoclonal antibody and FITC-labeled anti-PDCD5 antibody have been described previously (Chen et al., 2001). Other primary antibodies were applied: rabbit anti-bax, mouse anti-MAP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-LC3,
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Fig. 7. Western blot and immunofluorescence labeling for the detection of BNIP3. (a) Western blot indicated the expression of BNIP3 increased both in MCAO and PDCD5 KO groups. Slighter expression of BNIP3 was found in PDCD5 KO group. (b) Quantitative analysis of BNIP3 in different groups. *p o0.05 versus Sham. #p o0.05 versus MCAO ipsilateral hemisphere. Values are the mean 7 SEM. (c) Double immunofluorescence labeling showed BNIP3 (red) was colocalized with PDCD5 (green). Bar ¼100 mm.
Fig. 8. Immunoreactivity staining and TUNEL assay. Results showed the expression of VHL reduced in MCAO and PDCD5 KO groups, whereas it was more pronounced in MCAO group. The expression of HIF-1α and BNIP3 were increased both in MCAO and PDCD5 KO groups. However, they had slighter increase in PDCD5 KO mice. Bar ¼ 100 mm. Rarely detectable TUNEL-positive cells were found in Sham mice. In MCAO and PDCD5 KO groups, positive cells could be observed in penumbra area. The axons and dendrites disappeared and nucleus disrupted in MCAO group, but rarely in PDCD5 KO mice. Bar ¼ 100 mm. The arrows showed the positive cells.
rabbit anti-Beclin1 (MBL, Woburn, MA, USA), rabbit anti-LC3-ll, rabbit anti-P62 (abgent, San Diego, CA, USA) and then treated with PV kit. Peroxidase activity was revealed by dipping the sections in a mixture
containing 3-diaminobenzidine (DAB) and H2O2 at room temperature. The brain sections were dehydrated and cover-slipped, and photographed under an OLYMPUS BX51 microscope.
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The methods for double fluorescence labeling have been described previously (Zhou et al., 2005). Brain coronal sections were incubated with primary antibodies overnight at 4 °C. The brain sections were then incubated with goat anti-rabbit IgG-tetramethylrhodamine isothiocyanate (TRITC, Red) (Jackson Immuno Research Inc., Pennsylvania, USA). Mounting medium with DAPI (blue) (Jackson Immuno Research Inc., Pennsylvania, USA) was applied to stain all the nuclei. The images were photographed under a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany). The fragmentation of nuclear DNA in cells has been identified extensively with The In Situ Cell Death Detection Kit, POD (TUNEL) (Sigma-Aldrich, St Louis, MO, USA). TUNEL-positive cells were observed under an OLYMPUS BX51 microscope. 4.8. Statistical analysis All data were expressed as mean 7SEM. Statistical significance was verified with one-way ANOVA followed by the Tukey test for multiple comparisons. The clinical behavior scores were compared with Kruskal-Wallis one-way ANOVA followed by multiple comparison procedures by Dunn’s method. A probability value of p o0.05 was considered statistically significant.
Conflict of interest The authors declare no actual or potential conflict of interest.
Author contributions JL, YC, CZ and CC conceived and designed the experiments, YC and CZ contributed reagents and materials, JL and ZJ performed the experiments, JL and CC analyzed the data, JL and CC wrote the paper.
Acknowledgements This work was supported by NIH grants 81471186. We thank Prof. Yingyu Chen for advice and providing PDCD5 antibody. All authors have disclosed that they do not have any potential conflicts of interest.
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Research report
Knockout of programmed cell death 5 (PDCD5) gene attenuates neuron injury after middle cerebral artery occlusion in mice Jianfei Lu a, Zhao Jiang a, Yingyu Chen b, Changman Zhou a, Chunhua Chen a,n a b
Department of Anatomy and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China Peking University Center for Human Disease Genomics, Beijing 100191, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 May 2016 Received in revised form 30 August 2016 Accepted 4 September 2016 Available online 5 September 2016
Loss of von Hippel-Lindau tumor suppressor protein (VHL) or hypoxia results in nuclear relocalization of PDCD5 and subsequent mouse double minute 2 homolog (Mdm2) degradation. Thus, VHL may involved in the PDCD5 mediated apoptosis and autophagy after MCAO. In the present study, using PDCD5 knockout (PDCD5-/-) mice, we aimed to demonstrate that knockout of PDCD5 gene could protect the brain from ischemic injury by inhibiting the PDCD5-VHL pathway. 24 h post MCAO surgery, PDCD5 gene knockout mice presented obvious improved brain blood flow, improved neurological behavior and decreased cerebral infarction compared with wild type mice. The levels of apoptotic and autophagic proteins were increased both in wild type and PDCD5 knockout mice, whereas they were more pronounced in the wild type mice. We observed decrease in the expression of VHL in wild type mice after MCAO. Reduced expression of VHL may result in increased expression of hypoxia-inducible factor 1α(HIF-1α) and (BCL2/ adenovirus E1B 19 kDa protein-interacting protein 3) BNIP3. However, mice lacking PDCD5 had no changes in the expression of VHL and have slighter increases in the expression of HIF-1α and BNIP3, suggesting that PDCD5 may regulate apoptosis and autophagy through VHL-HIF-1α-BNIP3 pathway. & 2016 Elsevier B.V. All rights reserved.
Keywords: Apoptosis Autophagy Knockout mice Middle cerebral artery occlusion (MCAO) Programmed cell death 5 (PDCD5)
1. Introduction Stroke is a worldwide burden with high occurrence of morbidity, mortality and disability. Among all types of stroke, 87% are ischemic stroke (Feigin et al., 2015). Ischemic brain injury results from a complex sequence of pathophysiological events that evolve over time and space. In the core area, cells are killed rapidly because of bioenergetic failure and breakdown of ion homeostasis. The penumbra, which lies between damaged core and normal brain, is an area of constrained blood flow with partially preserved energy metabolism (Dirnagl et al., 1999). Many researchers focus on this area for the salvage of a reversible brain damage. Programmed cell death 5 (PDCD5), formerly designated as TFAR19 (TF-1-cell apoptosis-related gene 19), is an apoptosis accelerating gene but it could not induce apoptosis solely (Liu et al., 1999). Under apoptosis, PDCD5 protein is significantly increased and translocates rapidly from the cytoplasm to the nucleus of cells Abbreviations: PDCD5, programmed cell death 5; RhPDCD5, recombinant human programmed cell death 5; MCAO, middle cerebral artery occlusion; VHL, von Hippel-Lindau tumor suppressor protein; Mdm2, mouse double minute 2 homolog; HIF-1α, hypoxia-inducible factor 1 alpha; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; WT, wild type; KO, knockout n Corresponding author. E-mail address: [email protected] (C. Chen). http://dx.doi.org/10.1016/j.brainres.2016.09.005 0006-8993/& 2016 Elsevier B.V. All rights reserved.
(Chen et al., 2001). PDCD5 is expressed in a wide variety of tissues both in human and mouse (Liu et al., 1999), yet a decreased expression has been detected in various human tumors, such as lung cancer (Spinola et al., 2006), gastric cancer (Yang et al., 2006), acute and chronic myelogenous leukemia (Ruan et al., 2006), prostate cancer (Du et al., 2009), epithelial ovarian carcinomas (Zhang et al., 2011), astrocytic gliomas (Li et al., 2008) and chondrosarcoma (Chen et al., 2010). Functional studies found that PDCD5 could bind to Tip60, enhance its stability and function as a Tip60 coactivator to promote apoptosis after DNA damage (Xu et al., 2009). PDCD5 also could enhance the stability of p53 by dissociating the interaction between p53 and Mdm2 and interact with Mdm2 to promote its degradation (Xu et al., 2012). Our previous study indicated that the expression of PDCD5 increased in ischemic neurons after MCAO in vivo (Chen et al., 2013). Meanwhile, reduction of expression of PDCD5 has a neuronal protective role after stroke and the effect is related to the inhibition of apoptosis and autophagy (Jiang et al., 2014). PDCD5siRNA could reduce the expression of some apoptosis and autophagy related protein such as p53, bax, caspase-3, LC3 and Beclin-1. But the direct relationship between PDCD5 and these proteins are still unknown. Recent studies mentioned that PDCD5 could directly bind to von Hippel–Lindau (VHL) which is a tumor suppressor gene and its mutation is related to some cancers (Essers et al., 2015). VHL could
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regulate the ubiquitination of HIF-1α and subsequent proteasomal degradation (Jaakkola et al., 2001; Park et al., 2014). Former study demonstrated that HIF-1α is involved in the regulation of apoptosis followed by MCAO (Chen et al., 2009). Other vitro studies found HIF-1α regulate autophagy in hypoxia condition (Zhang et al., 2008; Wu et al., 2015). Thus, PDCD5 might regulate programmed cell death through VHL and HIF-1α pathway. In this study, we used PDCD5 knockout and wild type mice to conduct the middle cerebral artery occlusion and reperfusion (MCAO) model. Then, brain infarction, blood–brain barrier (BBB) disruption, and neurological deficits were observed. We used western blot and immunohistochemistry techniques to evaluate the expression of endogenous PDCD5, VHL, HIF-1α, BNIP3 and tried to explore the mechanism of PDCD5 mediated apoptosis and autophagy.
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knockout mice (Fig. 1b). The expression of PDCD5 was detected by Immunofluorescence and Western blot analysis at 24 h after MCAO. We found that PDCD5 significantly increased in the penumbra area 24 h after MCAO compared with Sham group (Fig. 1c&d). 2.2. Knock out of PDCD5 gene significantly improved brain blood flow after MCAO During the ischemic period, the blood flow of the core area of MCA was blocked. After reperfusion, the blood flow of the area would recover temporarily. When ischemia occurred, the blood flow among MCAO, PDCD5 KO and KOþRhPDCD5 groups has no significant difference. But 24 h after stroke, the blood flow of all the three groups improved in various degrees. But PDCD5 KO group has a more significant improvement compared with other groups (Fig. 2a&b).
2. Results
2.3. Knockout of PDCD5 gene reduced cerebral infarction
2.1. PDCD5 protein does not express in the neurons of PDCD5 knockout mouse
The cerebral infarction (white colored area) at 24 h after stroke was shown in Fig. 3a. The ratio of the lesion in four different groups was shown in Fig. 3b. Compared with the Sham group, all the mice after MCAO have severe infarction. But the lesion area of the PDCD5 KO group was reduced about 15.1% compared to the wild type mice. The infarction ratio of the PDCD5 KO mice that were injected with rhPDCD5 protein has a significant increase.
Double Immunofluorescence labeling indicated that in the hippocampus and cortex area of wild type mice, PDCD5 (green) colocalized with the neuronal marker MAP-2 (red, microtubule-associated protein 2) (Fig. 1a). But it rarely expressed in neurons of PDCD5
Fig. 1. Expression and localization of PDCD5 after MCAO. (a) Double immunofluorescence staining showed the colocalization of PDCD5 (green) with MAP2 (red) in hippocampus and cortex. (b) Immunofluorescene staining using FITC-conjugated PDCD5 antibody was detected in the penumbra area. The expression of PDCD5 in MCAO group is stronger than sham mice and no positive cell was observed in PDCD5 knockout mice. (c, d) Western blot showed that the expression of PDCD5 increased at 24 h and the PDCD5 knockout mice rarely expressed PDCD5. *p o 0.05 versus Sham WT. Bar ¼100 mm. Values are the mean 7 SEM.
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Fig. 2. Effects of Knock out PDCD5 gene on cerebral blood flow after MCAO. (a) The representative images of cerebral blood flow (CBF) of the whole brain in different groups. The magnitude of CBF is represented by different colors, with blue to red denoting low to high. (b) Quantitative analysis of CBF in different groups. 24 h after reperfusion, The CBF of the mice suffering stroke improved. *p o 0.05. PDCD5 KO mice had a more pronounced improvement compared to other two surgery groups. #p o 0.05 versus PDCD5 KO perfusion 24 h. Values are the mean 7 SEM.
2.4. Knockout of PDCD5 gene improved neurological score The neurological score was 15.83371.169 in Sham group, 6.00071.414 in MCAO group, 8.66771.366 in PDCD5 KO group and 5.33371.211 in PDCD5 KOþRhPDCD5 group. Knock out of PDCD5 gene could improve the neurological behavior outcome (Fig. 3c). 2.5. Knockout of PDCD5 gene decreased apoptosis and autophagy levels after MCAO Previous studies have confirmed that PDCD5 was involved in the apoptosis and autophagy processes. Lower expression of PDCD5 could attenuate apoptosis and autophagy levels (Jiang et al., 2014; Chen et al., 2013). We detected autophagy related protein such as Beclin 1, LC3, LC3-ll, P62 and apoptosis related protein Bax in the three groups. As showed in Fig. 4a, knock out of PDCD5 gene could attenuate the expression of Beclin 1, LC3, LC3-ll, P62 and Bax compared with MCAO group. The results of TUNEL staining verified this outcome. Seldom positive cells were observed in Sham group. In MCAO and PDCD5 KO groups, positive cells could be observed in the penumbra area. The axons and dendrites disappeared and nuclei were disrupted in MCAO group, but rarely in PDCD5 KO mice (Fig. 8). As double immunofluorescence labeling showed, all these proteins (Red) colocalized with PDCD5 (Green). These results indicated that PDCD5 may have effects on apoptosis and autophagy (Fig. 4b).
2.6. Knockout of PDCD5 gene attenuated the decreased expression of VHL and the increased expression of HIF-1α after MCAO To gain insight into the relationship between PDCD5 and VHL, we examined the expression of VHL and HIF-1α in various groups. Western blot (Figs. 5 and 6) and immunohistochemistry staining (Fig. 8) indicated that the expression of VHL decreased in the penumbra area of wild type mice after MCAO. But in the similar area of PDCD5 KO mice, there was no significant difference compared to Sham group. HIF-1α had an increased expression both in wild type mice and PDCD5 KO mice after MCAO. But the enhancement was slighter in KO mice. We speculated that PDCD5 may regulate the expression of HIF-1α via VHL and then promote apoptosis and autophagy.
2.7. PDCD5 regulated apoptosis and autophagy processes via BNIP3 Previous research confirmed that HIF-1α could regulate apoptosis and autophagy through BNIP3. Our results revealed that the expression of BNIP3 increased in MCAO group and PDCD5 KO group. But there was a significant reduction in PDCD5 KO mice compared to wild type mice (Figs. 7 and 8) which indicated that PDCD5 may influence the expression of BNIP3 via VHL and HIF-1α, and then regulate apoptosis and autophagy.
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Fig. 3. Effects of Knock out PDCD5 gene on infarction size and neurological function after MCAO. (a) Representative images of brain slices stained by TTC in different groups at 24 h after reperfusion. Normal brain tissue is stained red, while the infarction remains unstained. (b) Quantitative analysis of infarct volume in different groups. *po 0.05 versus Sham. #po 0.05 versus PDCD5 KO. (c) Neurological scores of mice in different groups evaluated at 24 h after reperfusion. *p o 0.05 versus Sham. #p o0.05 versus PDCD5 KO. Values were expressed as mean 7SEM.
3. Discussion In the present study, we demonstrated that the PDCD5 knockout mice are resistant to ischemia and reperfusion injury compared to wild type mice. And this resistance was compromised after the injection of rhPDCD5. PDCD5 is a well-established protein associated with apoptosis. Chen et al. (2001) found that PDCD5 alone could not induce apoptosis, but over expression of PDCD5 could enhance apoptosis independent of types of cells and apoptosis inductions. Cell suicide is the predominant mechanism that follows MCAO, particularly within the ischemic penumbra. Hence, it’s a potential therapeutic application to block or inhibit this procedure (Dirnagl et al., 1999). For that reason, we applied PDCD5siRNA to reduce the expression of PDCD5. Results showed that i.c. v injection of PDCD5siRNA could reduce the infarction volume, improve neurological behavior and maintain the integrity of BBB after MCAO (Chen et al., 2013). In this study, we found that knockout of PDCD5 gene has a neuroprotective effect after MCAO consistently. In addition to infarction volume and behavior changes, we also found that inhibition of PDCD5 could reduce the expression of
apoptotic-related proteins such as p53, Bax and caspase-3 (Chen et al., 2013). And recent study found that PDCD5 was involved in autophagy as well. Intracerebroventricularly injection of PDCD5siRNA could reduce the expression of Beclin 1 and LC3, revealing that PDCD5 mediated the autophagic cell death via Beclin 1 and LC3 following MCAO injury (Jiang et al., 2014). We observed that knockout of PDCD5 gene could decrease ischemia induced apoptosis and autophagy concomitantly, and affect the related genes especially Bax, LC3-ll and P62. These insights are important to further understand the role of PDCD5 in ischemia and reperfusion injury. PDCD5 could directly bind to pVHL and knockout of VHL could induce the nuclear translocation and subsequent degradation of MDM2 to maintain the stability of P53 (Essers et al., 2015). VHL is a tumor suppressor gene and is involved in the ubiquitination of HIF1α and subsequent proteasomal degradation via the VHL ubiquitination complex (Jaakkola et al., 2001; Park et al., 2014). In normoxia, HIF-1α is degraded through the VHL ubiquitination pathway. HIF-1α functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, HIF-1α activates the
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Fig. 4. Effects of knock out PDCD5 gene on autophagy and apoptosis levels after MCAO. (a) Autophagy and apoptosis related protein Beclin 1, LC3, LC3-ll, P62 and Bax were detected in the penumbra area. Almost no Beclin 1, LC3, LC3-ll and Bax were observed in Sham group. Slight P62 positive cells were observed in Sham mice. Massive immunoreactivity of these proteins was localized in the penumbra area. However, the extent of these proteins lessened in PDCD5 KO mice. (b) Double immunofluorescence labeling showed Beclin 1, LC3, LC3-ll, P62 and Bax (Red) were all colocalized with PDCD5 (Green). Bar ¼100 mm.
transcription of many apoptosis and autophagic genes including BNIP3 (Sowter et al., 2001). BNIP3 is a pro-apoptotic mitochondrial protein and Bcl-2 family member (Chen et al., 1997). BNIP3 associates with anti-apoptotic family members Bcl-2, Bcl-xL, and the adenovirus homolog E1B-19 kDa. Different from other Bcl-2 family members, the TM domain of BNIP3 instead of the BH3 domain is critical for heterodimerization with Bcl-2, Bcl-XL and pro-apoptotic activity (Ray et al., 2000). In addition to apoptosis, BNIP3 has been implicated in autophagy. In hypoxic conditions, BNIP3 can induce mitochondrial autophagy (mitophagy) by displacing Beclin 1 from Bcl-XL and Bcl-2 (Azad et al., 2008; Bellot et al., 2009). BNIP3 can also promote mitophagy by mediating the translocation of the E3 ubiquitin ligase Parkin to the mitochondria or by directly interacting with LC3 on the autophagosome (Lee et al., 2011; Hanna et al., 2012; Shi et al., 2014). Based on the above, we provided evidence that showed the expression of VHL was significantly reduced after MCAO in wild type mice. But knockout of PDCD5 gene could weaken this effect. Robust interaction between pVHL and PDCD5 was observed via tandem-affinity purification (Essers et al., 2015). It seems that VHL was degraded or inhibited through PDCD5 after MCAO. We further demonstrated that knockout of PDCD5 gene resulted in significant inhibition of HIF-1α and BNIP3. That may related to VHL regulated ubiquitination and subsequent proteasomal degradation. These results suggesting VHL, HIF-1α and BNIP3 were associated with PDCD5 in the regulation of apoptosis and autophagy. Chen et al. (2001) found PDCD5 translocates to the nucleus in cells undergoing apoptosis. But the occurrence time of translocation varied depends on the cell types and apoptosis inductions. Former research found this phenomenon in rats 24 h after MCAO. But in
this study, we didn’t find the translocation in mice at 24 h after MCAO. The translocation of PDCD5 occurred at 48 h after MCAO or later (data not shown). That means PDCD5 probably participate apoptosis and autophagy in two phases, the cytoplasm and the nuclear. The difference might come from different species which needed further exploration. Our data demonstrated that there has a crosstalk between apoptosis and autophagy to co-mediate the cell death. Apoptosis is regarded as suicide in the penumbra, but the specific role of autophagy in brain ischemia and reperfusion injury is still controversial. The common view is that moderate autophagy has the protective role but severe autophagy may destroy the cell. Studies investigating cellular death following neonatal hypoxia–ischemia have shown that autophagy has neuroprotective role. In our results, knockout of PDCD5 gene could reduce the autophagy level after MCAO and have a protective role which indicated that PDCD5 mediated apoptosis and autophagy might have a synergy effect. Our further study will focus on the direct interaction between PDCD5 and VHL-HIF-1α-BNIP3 pathway. PDCD5 can also dissociate p53 and Mdm2 and maintain the basal level of p53. Upon DNA damage, PDCD5 can mediate p21 transactivation and G1 phase arrest and allow cells to initiate productive DNA repair processes (Xu et al., 2012). PDCD5 together with Tip60 can also promote p53K120 acetylation and transactivation of Bax expression in order to initiate an apoptotic response (Xu et al., 2009). We have verified P53 involved in PDCD5 mediated apoptosis and autophagy after MCAO (Chen et al., 2013). The interaction between P53 and PDCD5 in ischemia and reperfusion injury is another direction of our future research.
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Fig. 5. Western blot and immunofluorescence labeling for the detection of VHL. (a) Western blot indicated the expression of VHL decreased in MCAO group. No significant difference was found between Sham group and PDCD5 KO group. (b) Quantitative analysis of VHL in different groups. *p o 0.05 versus Sham. Values are the mean7 SEM. (c) Double immunofluorescence labeling showed VHL (red) localized in cytoplasm and was colocalized with PDCD5 (green). Bar¼ 100 mm.
Our results indicated that knockout of PDCD5 gene had a direct neuronal protective effect that reduced programmed cell death of neurons under the hypoxia condition and increased the survival rate of neurons on penumbra area. In addition, we can test the effects of knockout PDCD5 on white matters and vascular endothelial cells in the further study. In conclusion, PDCD5 participated in the process of both apoptosis and autophagy after MCAO. Knock out of the PDCD5 gene could attenuate neuron injury and the role was related to VHL-HIF-1α-BNIP3 pathway. Thus, inhibiting or silencing PDCD5 might be a new strategy applied to the therapy of ischemia and reperfusion injury.
surgery, animals were anesthetized using compound anesthetic agent (3 ml/kg) with a mixture of 5% chloral hydrate, 1% pentobarbital sodium and 2% magnesium sulfate. Under operating microscope, the right common carotid, internal carotid and external carotid arteries were surgically exposed. Then the external carotid artery was isolated and ligated. A 6-0 nylon suture with silicon rubber coated (coating length 3 mm, diameter 0.21 mm) was inserted into the internal carotid artery through the external carotid artery stump and gently advanced to occlude the MCA. After 1 h of MCAO, the nylon suture was carefully removed and the blood flow reperfused. The body (rectal) temperature was carefully maintained at 37.0 °C by a heating pad until the animal completely recovered from the anesthesia.
4. Experimental procedures
4.2. In vivo rhPDCD5 protein transfer
4.1. Animals and treatments
The stereotaxic coordinates were 0.4 mm posterior, 1.0 mm lateral to the bregma and 2.5 mm ventral to the surface of the skull. RhPDCD5 protein (70 μg/kg; Beijing Biosea Biotechnology Co., China) was injected intracerebroventricularly using a Hamilton microsyringe under guidance of stereotaxy instrument within 30 min after reperfusion.
All experimental protocol in this study was evaluated and approved by the Animal and Ethics Review Committee at Peking University Health Science Center in Beijing, China. Mice were purchased from the Center of Experimental Animals of Peking University Health Science Center and were kept under standard conditions with free access to food and water. Thirty six C57BL/6N male mice and twenty four C57BL/6N PDCD5 knockout mice weighing 20–24 g were randomly assigned to four groups: Sham surgery (N ¼ 18), MCAO (N ¼18), PDCD5 knockoutþ MCAO (KO) (N ¼ 18) and PDCD5 knockoutþMCAO þ RhPDCD5 (KO þRhPDCD5) (N ¼ 6). MCAO model was induced by Longa et al. (1989) with some added modifications (Longa et al., 1989; Chen et al., 2013). During
4.3. Infarct volume measurement 2,3,7-Triphenyltetrazolium chloride (TTC) staining for infarction volume measurement was conducted as described previously (Chen et al., 2009). Brains were sliced into 2-mm-thick coronal sections in an adult mouse brain matrix (Kent Scientific Corporation). The slices were stained in 1% 2,3,5-triphenyltetrazolium chloride solution (TTC; Sigma-Aldrich, St Louis, MO, USA) for
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Fig. 6. Western blot and immunofluorescence labeling for the detection of HIF-1α. (a) Western blot indicated the expression of HIF-1α increased both in MCAO and PDCD5 KO groups. Slighter expression of HIF-1α was found in PDCD5 KO group. (b) Quantitative analysis of HIF-1α in different groups. *p o0.05 versus Sham. #p o 0.05 versus MCAO ipsilateral hemisphere. Values are the mean 7 SEM. (c) Double immunofluorescence labeling showed HIF-1α (red) was colocalized with PDCD5 (green) in Sham group. HIF-1α mainly localized in the cytoplasm. After MCAO, it showed nuclear translocation in the infarction regions. Bar ¼100 mm.
20 min at 37 °C. The infarction tissue was stained white and the healthy part was pink. The infarcted areas of each section were measured by Image J. The infarction rate was measured with: infarcted area of the ipsilateral hemisphere/whole brain. 4.4. Neurological deficits The neurological scores were performed in a blinded fashion at 24 h after MCAO based on the scoring system reported by Garcia et al. (1995). The minimum neurological score was 3 and the maximum was 18. A lower score represented a more severe injury. 4.5. Cerebral blood flow measurement Cerebral blood flow (CBF) was measured using Laser Doppler perfusion image system (PeriScan PIM3 System; PERIMED, Stockholm, Sweden) (N ¼6) as previously described (Huang et al., 2012). We made an incision through the scalp and exposed the skull. The cerebral cortex was positioned in parallel to the computer-controlled optical scanner at a distance of 18.5 cm. The cerebral blood flow was measured at 1 h after ischemia and 24 h after reperfusion. A color-coded image to denote specific relative perfusion levels was displayed on a video monitor and the ischemia core area in all images was evaluated with the software LDPIwin 3.1 (PeriScan PIM3 System; PERIMED, Stockholm, Sweden). 4.6. Western blot Western blot analysis was performed as described previously (Chen et al., 2009). Tissues from ischemic penumbra area of ipsilateral hemisphere were homogenized. Bicinchoninic acid (BCA; Applygen Technologies) was used to detect the protein concentration
of each sample (Ashwal et al., 1998). Protein samples (50 μg) were loaded onto polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane (Hybond-C, Amersham Biosciences, USA). The nitrocellulose membranes were then blocked by incubation with the primary antibodies overnight at 4 °C. The primary antibodies were applied as follows: rabbit anti-VHL, rabbit anti-HIF-1α, rabbit anti-BNIP3 (Cell Signaling, Beverly, Massachusetts, USA). A monoclonal antibody against β-actin (Cell Signaling, Beverly, Massachusetts, USA) was used as a control group for protein gel loading. Immunoblots were processed with corresponding secondary antibodies (Applygen Technologies) for 1 h at room temperature. Immunoblots were probed and the bands were visualized by enhanced chemiluminescence detection reagents (Applygen Technologies). The optical density was detected by Bio-Rad image analysis. 4.7. Immunohistochemistry, double fluorescence labeling and TUNEL staining For the histological analysis, animals (N¼6) were anesthetized at 24 h after operation. After perfusion with 40 ml 0.01 M PBS and 40 ml of 4% paraformaldehyde in 0.1 M PB (pH¼7.4), brains were removed and postfixed with formalin, and cryoprotected in 30% sucrose in 0.1 M PB for over 24 h at 4 °C. Coronal brain sections (10 mm) were sliced using a cryostat (Leica CM3050 S). Immunohistochemistry was performed as described previously (Chen et al., 2009). After triple wash with PBS and blocking with 3% normal goat serum at room temperature for 1 h, the slices were incubated with primary antibodies diluted in PBS overnight at 4 °C. The mouse anti-PDCD5 monoclonal antibody and FITC-labeled anti-PDCD5 antibody have been described previously (Chen et al., 2001). Other primary antibodies were applied: rabbit anti-bax, mouse anti-MAP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-LC3,
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Fig. 7. Western blot and immunofluorescence labeling for the detection of BNIP3. (a) Western blot indicated the expression of BNIP3 increased both in MCAO and PDCD5 KO groups. Slighter expression of BNIP3 was found in PDCD5 KO group. (b) Quantitative analysis of BNIP3 in different groups. *p o0.05 versus Sham. #p o0.05 versus MCAO ipsilateral hemisphere. Values are the mean 7 SEM. (c) Double immunofluorescence labeling showed BNIP3 (red) was colocalized with PDCD5 (green). Bar ¼100 mm.
Fig. 8. Immunoreactivity staining and TUNEL assay. Results showed the expression of VHL reduced in MCAO and PDCD5 KO groups, whereas it was more pronounced in MCAO group. The expression of HIF-1α and BNIP3 were increased both in MCAO and PDCD5 KO groups. However, they had slighter increase in PDCD5 KO mice. Bar ¼ 100 mm. Rarely detectable TUNEL-positive cells were found in Sham mice. In MCAO and PDCD5 KO groups, positive cells could be observed in penumbra area. The axons and dendrites disappeared and nucleus disrupted in MCAO group, but rarely in PDCD5 KO mice. Bar ¼ 100 mm. The arrows showed the positive cells.
rabbit anti-Beclin1 (MBL, Woburn, MA, USA), rabbit anti-LC3-ll, rabbit anti-P62 (abgent, San Diego, CA, USA) and then treated with PV kit. Peroxidase activity was revealed by dipping the sections in a mixture
containing 3-diaminobenzidine (DAB) and H2O2 at room temperature. The brain sections were dehydrated and cover-slipped, and photographed under an OLYMPUS BX51 microscope.
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The methods for double fluorescence labeling have been described previously (Zhou et al., 2005). Brain coronal sections were incubated with primary antibodies overnight at 4 °C. The brain sections were then incubated with goat anti-rabbit IgG-tetramethylrhodamine isothiocyanate (TRITC, Red) (Jackson Immuno Research Inc., Pennsylvania, USA). Mounting medium with DAPI (blue) (Jackson Immuno Research Inc., Pennsylvania, USA) was applied to stain all the nuclei. The images were photographed under a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany). The fragmentation of nuclear DNA in cells has been identified extensively with The In Situ Cell Death Detection Kit, POD (TUNEL) (Sigma-Aldrich, St Louis, MO, USA). TUNEL-positive cells were observed under an OLYMPUS BX51 microscope. 4.8. Statistical analysis All data were expressed as mean 7SEM. Statistical significance was verified with one-way ANOVA followed by the Tukey test for multiple comparisons. The clinical behavior scores were compared with Kruskal-Wallis one-way ANOVA followed by multiple comparison procedures by Dunn’s method. A probability value of p o0.05 was considered statistically significant.
Conflict of interest The authors declare no actual or potential conflict of interest.
Author contributions JL, YC, CZ and CC conceived and designed the experiments, YC and CZ contributed reagents and materials, JL and ZJ performed the experiments, JL and CC analyzed the data, JL and CC wrote the paper.
Acknowledgements This work was supported by NIH grants 81471186. We thank Prof. Yingyu Chen for advice and providing PDCD5 antibody. All authors have disclosed that they do not have any potential conflicts of interest.
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