- Email: [email protected]
DAXX signaling pathway in rats
Neuroscience Letters 651 (2017) 207–215
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Necrostatin-1 protects hippocampal neurons against ischemia/reperfusion injury via the RIP3/DAXX signaling pathway in rats Rongli Yang a,1 , Kun Hu a,1 , Jieyun Chen a , Shiguang Zhu b , Lei Li a , Hailong Lu a , Pingjing Li a , Ruiguo Dong b,∗ a b
Department of Geriatrics, Affiliated Hospital of Xuzhou Medical University, 99 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China Department of Neurology, Affiliated Hospital of Xuzhou Medical University, 99 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China
h i g h l i g h t s • • • •
Nec-1 attenuates cerebral I/R induced cognitive deficits. Nec-1 preserves CA1 hippocampal neuronal survival under cerebral I/R condition. Nec-1 inhibits RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction. Nec-1 blocks DAXX translocation to cytoplasm, which results in the inactiviation of DAXX.
a r t i c l e
i n f o
Article history: Received 10 February 2017 Received in revised form 16 April 2017 Accepted 8 May 2017 Available online 10 May 2017 Keywords: Global cerebral ischemia/reperfusion Receptor-interacting protein 3 The death-associated protein Necrostatin-1
a b s t r a c t Global cerebral ischemia/reperfusion (I/R) induces selective neuronal injury in CA1 region of hippocampus, leading to severe impairment in behavior, learning and memory functions. However, the molecular mechanism underlying the processes was not elucidated clearly. RIP3 is a key molecular switch connecting apoptosis, necrosis and necroptosis. DAXX, as a novel substrate of RIP3, plays a vital role in ischemia-induced neuronal death. The aim of this study is to investigate the role and mechanism of RIP3/DAXX signaling pathway on neurons in CA1 region of the rat hippocampus after cerebral I/R. Global cerebral ischemia was induced by the method of four-vessel occlusion. RIP1 specific inhibitor Necrostatin1 was administered by intracerebroventricular injection 1 h before ischemia. Open-field, closed-field, and Morris water maze tests were performed respectively to examine the anxiety and cognitive behavior in each group. Hematoxylin and eosinstaining was used to examine the survival of hippocampal CA1 pyramidal neurons. Western blot or immunoprecipitation were carried to detect protein expression, phosphorylation, and interaction. We found that pre-treatment with Nec-1 protected locomotive ability, relieved anxiety behavior, and improved cognitive ability in the rats subjected to cerebral I/R. In addition Moreover, Nec-1 decreased significantly the dead rate of neurons in hippocampal CA1 region after cerebral I/R through suppressing RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction, and then blocked DAXX translocation from nucleaus to cytoplasm, which resulted in the inactiviation of DAXX. We concluded that pre-treatment with Nec-1 can protect neurons in the hippocampal CA1 region against ischemic damage through the RIP3-DAXX signaling pathway. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: I/R, ischemia/reperfusion; RIP3, receptor-interacting protein 3; DAXX, death-associated protein; Nec-1, necrostatin-1; HE, hematoxylin and eosin; PML-NBs, promyelocytic leukemia protein-nuclear bodies; FADD, fas-associated with death domain protein; DMSO, dimethylsulfoxide; RHIM, C-terminal RIP homotypic interaction motif; ANOVA, analysis of variance. ∗ Corresponding author at: Department of Neurology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, PR China. E-mail address: [email protected] (R. Dong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2017.05.016 0304-3940/© 2017 Elsevier B.V. All rights reserved.
Ischemic stroke, is the most common type of stroke and remains the leading cause of death and permanent disability in adults worldwide [1]. The mechanisms underlying cerebral ischemic injury are complicated. This condition often drives neuronal damage in the ischemic tissue [2]. The hippocampus is the cerebral region most vulnerable to ischemia [3]. Damages to this area result in deficits in spatial learning and memory, thereby seriously impacting the quality of life for the patients. There is currently no
208
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
effective approach for prevention and treatment of this memory and cognitive impairment [4]. Ischemia induces hippocampal neurons damage via excitotoxicity of N-methyl-d-aspartate (NMDA), Ca2+ overload, free radical generation, and so on [5,6]. Although current clinical treatment for ischemic stroke is to restore the blood flow to the ischemic area through thrombolytic therapy, the reperfusion injury and the narrow time window and a certain amount of risk for thrombolysis restricts its application for the majority of stroke patients [7]. Receptor-interacting protein 3 (RIP3) is a member of RIP Ser/Thr kinase family, which functions as an essential sensor and regulates cell survival, apoptosis and necrosis to pathogen infection or inflammation [8,9]. RIP3 binds to RIP1 through its C-terminal RIP homotypic interaction motif (RHIM) domain [10]. The kinase activity of RIP3 is required for the caspase-independent cell death [11]. To date, however, the mechanism underlying the RIP3-mediated neuronal death in hippocampal CA1 during global cerebral I/R remains unclear. Death associated protein (DAXX), a kind of nucleoprotein, is a component of promyelocytic leukemia protein-nuclear bodies (PML-NBs), playing a direct role in the transcriptional regulation [12]. Previous study validated that DAXX is a novel substrate of RIP3 [13], and is translocated from the neuronal cytoplasm into the nucleus under stress, which promotes neuronal death [14]. In the present study, we explored whether DAXX could be mediated by RIP3 after cerebral ischemia and its possible influence in this process. Necrostatin-1 (Nec-1), a small molecule inhibitor of RIP1 kinase, can inhibit RIP1-RIP3 interaction and decrease the oxidative injury and programmed necrosis in neonatal hypoxia–ischemia (HI) [15]. We investigated whether Nec-1 can protect rat hippocampal neurons against cerebral I/R injury via the RIP3/DAXX signaling pathway. 2. Experimental procedures 2.1. Materials Antibodies, including rabbit anti-RIP1 (1:200, sc-7881), rabbit anti-p-Ser (1:200, sc-81514) and mouse anti-Actin (1:1000, sc-47778), were purchased from Santa Cruz Biotechnology. Rabbit anti-RIP3 (1:500, ab58828) and Rabbit anti-Histone H3 (1:1000, ab8898) were purchased from Abcam. Rabbit anti-DAXX (1:1000, NB100-56136SS) antibody was obtained from NOVUS Biotechnology. Necrostatin-1 (N9037) was obtained from Sigma Biotechnology. All other chemicals were purchased from Sigma unless indicated otherwise. 2.2. Drug administration Nec-1 was dissolved in 1% DMSO with 0.1 M phosphate buffer solution (PBS). Animals were given Nec-1 (1 ug) or vehicle (0.1 M PBS + 1% DMSO) by means of unilateral intracerebroventricular infusion (anteroposterior, 0.8 mm; lateral, 1.5 mm; depth, 3.5 mm) 1 h before ischemia in a total volume of 5 l at 0.5 l/min. The needle was maintained in place for an additional 5 min before withdrawal to prevent fluid reflux. 2.3. Groups and animal model of ischemia Adult male Sprague-Dawley (SD) rats weighing 250–300 g were used (Shanghai Experimental Animal Center, Chinese Academy of Science). All rats were randomly divided into four groups: sham group, I/R group, DMSO group, Nec-1 group (13 rats in each group). The experimental procedures were approved by local legislation for ethics of experiments on animals. Transient cerebral
ischemia was induced by four-vessel occlusion, as described previously [16]. Briefly, under anesthesia with sodium pentobarbital (30 mg/kg, intraperitoneal), vertebral arteries were electrocauterized, and common carotid arteries were exposed. On the following day, anesthesia was induced with 4% isoflurane and both carotid arteries were occluded with aneurysm clamps to induce cerebral ischemia. After 15 min of occlusion, the aneurysm clamps were removed for reperfusion. Rats that lost their lighting reflex were selected for the experiments. Rectal temperature was maintained at about 37 ◦ C throughout the procedure. Rats with seizures were discarded. Rats were moved to an incubator to keep the proper temperature until they were fully awake. Sham controls were performed using the same surgical procedures, except that the carotid arteries were not occluded. 2.4. Morris water maze testing Evaluation of memory and learning capacity was started on the 5th day after reperfusion following 15 min of ischemia. Morris water maze testing of spatial learning capacity was performed as described previously [17]. The Morris water maze consisted of a large circular black pool (210 cm diameter, 50 cm height, filled to a depth of 30 cm with water at 22 ± 2 ◦ C), which was placed in a darkened room, illuminated by dim light. Within the pool, a submerged platform (black, round, 8 cm diameter, 1 cm below surface) was hidden in a fixed location, 55 cm from the edge of the pool. The rat could climb on the plat-form to escape from the necessity of swimming. The rats were given 3 swimming trials per day on 4 consecutive days with a different starting position in each trial. The rat was given a maximum of 120 s to find the hidden platform and was allowed to stay on it for 20 s. Rats that failed to locate the platform were put onto it by the experimenter for 20 s. After 4 days of training, the platform was removed from the pool and the animal was released from the quadrant opposite where the platform had been located. The length of the trial was 120 s, after which, the rat was taken out of the pool. The proportion of time and swim distance the rat spent searching for the platform in the training quadrant where the previous location of the platform was, were recorded and used as a measure of memory. Morris maze performance was analyzed for latency to find the platform using the ANY-maze video tracking system (Stoelting, Wood Dale, IL, USA) with a CCD camera. 2.5. Open-Field test and closed-Field test Five days after intracerebroventricular injection of Nec-1 or DMSO, the locomotive activity in all the rats were evaluated using an open-field test or a closed-field test. Each rat was placed in the center of an open-field apparatus (W50 × D50 × H30 cm) or a closed-field apparatus (W50 × D50 × H30 cm) and acclimated for 3 min. Afterward, the free-moving behavior was monitored for 5 min and analyzed using the ANY-maze Video Tracking System (Stoelting, Wood Dale, IL, USA) with a CCD camera. The total distance traveled and the crossing lines were analyzed. 2.6. Sample preparation Rats were decapitated immediately 6 h after reperfusion, and then the hippocampal CA1 region was isolated and quickly frozen in liquid nitrogen. Tissues were homogenized in an ice-cold homogenization buffer (50 mM MOPS (pH 7.4), 100 mM KCl, 320 mM sucrose, 50 mM NaF, 0.5 mM MgCl2 , 0.2 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 1 mM Na3 VO4 , 20 mM sodium pyrophosphate, 20 mM -phosphoglycerol, 1 mM p-nitrophenyl phosphate, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 5 g/ml of leupeptin, aprotinin, and pepstatin A), and then centrifuged at 12,000 × g for 15 min at 4 ◦ C. Supernatants were collected and
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
209
protein concentration was determined by the method of Lowry. Samples were stored at −80 ◦ C for future use [18]. 2.7. Immunoprecipitation and immunoblotting Tissue homogenates (400 g of protein) were diluted to the same concentration with RIPA lysis and extraction buffer. Samples were pre-cleared by incubation with protein G beads (20 l; Amersham Biosciences) for 2 h, followed by incubation with 1–2 g of primary antibodies overnight at 4C. Protein G beads were added to the tube for further incubation for 2 h. Samples were then centrifuged at 10,000g for 1 min at 4 ◦ C and the pellets were washed three times with immunoprecipitation buffer. For immunoblotting, bound proteins were eluted by boiling at 100 ◦ C for 10 min in SDS-PAGE loading buffer and then isolated by centrifugation. The supernatants were separated on SDS-PAGE gels and then electrotransferred to a PVDF membrane. After being blocked, membranes were incubated with primary antibodies overnight at 4 ◦ C and incubated with horse-radish peroxidase-conjugated secondary antibodies for 1 h at room temperature, followed by development with ECL Western blotting detection reagents.
Fig. 1. The escape latency of rats in the different groups in the Morris water maze test during 4 consecutive training days. Data are presented as the mean ± SD (n = 8). * P < 0.05 versus respective sham group; # P < 0.05 versus respective I/R group.
Table 1 The escape latency of rats in the different groups in the Morris water maze test during 4 consecutive training days. Latency (sec)
2.8. HE-staining The rats subjected to 5 days of reperfusion were perfusionfixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffin-embedded brain sections (5 m) were prepared and stained with hematoxylin and eosin. Histological evaluations were performed with HE-staining for assessment of neuronal damage in the hippocampus. An initial dissector frame was positioned randomly in hippocampal sectors and cells in every 10th section throughout the entire hippocampus. The number of surviving hippocampal CA1 pyramidal cells per 1 mm of length was counted as the neuronal density. Five independent rats were used in each group and both sides of hippocampi were counted. In brief, cell counts were performed at × 400 magnification with the use of an Olympus BH-2 microscope connected to a Sony charge-coupled device video camera, a motorize stage system, and commercial stereology software. 2.9. Statistical analysis Five animals were randomly selected as samples in all groups for western blotting, immunoprecipitation, and histology assays. Image J (Version 1.30 v) analysis software was used for semiquantitative analysis of the bands. Values were expressed as the means ± SD. Data were analyzed using one-way or two-way ANOVA followed by Tukey’s test, or by t-test. P values of <0.05 were considered significant.
Sham I/R DMSO Nec-1
Day 1
Day 2
Day 3
Day 4
89.25 ± 6.48 107.25 ± 4.53* 108.62 ± 5.24* 92.63 ± 4.24#
68.6 ± 7.07 80.88 ± 6.94* 80.25 ± 5.65* 63.83 ± 4.89#
39.75 ± 6.04 56.25 ± 8.29* 54.38 ± 6.19* 40.88 ± 5.25#
25.75 ± 4.95 37.75 ± 6.54* 36.5 ± 5.10* 31.37 ± 4.53#
Values are mean ± SD (n = 8). * P < 0.05 versus respective sham group. # P < 0.05 versus respective I/R group.
significantly increased the swimming distance and the number of crossings compared with I/R group (P < 0.05, Fig. 2). 3.2. Nec-1 improved locomotor impairment and anxiety-like behavior induced by cerebral I/R The results from open- and closed-field tests showed that the total distance traveled by the rats in the sham group in an open field was significantly longer than that in I/R group or DMSO group (P < 0.05; Fig. 3), and was shorter in I/R group or DMSO group compared with Nec-1 group (P < 0.05; Fig. 3). These changes were consistent with the total distance traveled in the closed field, in which the total distance traveled by the Nec-1 group was longer than that in I/R group or DMSO group with regard to crossing the lines in the closed field. In the closed-field test, the rats in I/R group showed a shorter travelling distance and fewer crossings (P < 0.05). Treatment with Nec-1 increased the travelling distance and number of crossings compared with I/R group (P < 0.05).
3. Results 3.1. Nec-1 improved memory deficit and cognitive impairment induced by cerebral I/R In Morris water maze, during the training period, the escape latency in each group gradually decreased (Fig. 1 and Table 1). The latencies in I/R or DMSO group were significantly higher than that in sham groups, but decreased significantly in the Nec-1 group compared with I/R group or DMSO group (P < 0.05; Fig. 1 and Table 1). Spatial memory was measured on the 5th day. During this test, the platform was removed, the swimming distance and the number of crossings in I/R group or DMSO group were greatly decreased compared with sham group (P < 0.05, Fig. 2). Pre-treatment with Nec-1
3.3. Nec-1 protected hippocampal CA1 neurons against global cerebral I/R injury In HE-staining slides, normal pyramidal cells in the sham group (Fig. 4A and B) showed round and pale stained nuclei, whereas dead cells in the I/R group and DMSO group (Fig. 4C and F) displayed pyknotic nuclei. In Nec-1 group (Fig. 4G and H), the neuronal degeneration significantly decreased compared with I/R group. In the four groups, the surviving pyramidal cells counted within a 1-mm length of the CA1 region were 207.67 ± 18.06 in sham group, 31.50 ± 2.88 in I/R group, 31.67 ± 5.54 in DMSO group, 121.67 ± 6.74 in Nec-1 group, corresponding to Fig. 4B, D, F, and H, respectively. Taken together, the results indicated that treat-
210
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Fig. 2. Nec-1 improved memory deficit and cognitive impairment induced by cerebral I/R. (A) The track maps of rats in the different groups on Day 5 without the platform. (B) Total swimming distance traveled in 120 s for each group. (C) Number of crossings in 120 s for each group. Data are presented as the mean ± SD (n = 8). * P < 0.05 versus respective sham group; # P < 0.05 versus respective I/R group.
ment with Nec-1 had a neuroprotective effect against cerebral I/R-induced neuronal cell death in vivo.
3.4. Nec-1 inhibited the interaction of RIP3 with RIP1, reducing RIP3 expression and phosphorylation To explore the role of Nec-1 on RIP3-medicated pathway, interaction of RIP3 with RIP1, expression of RIP1 and RIP3, phosphorylation of RIP3 were measured by immunoblotting and Immunoprecipitation. As shown in Fig. 5, interaction of RIP3 with RIP1, expression of RIP1 and RIP3, phosphorylation of RIP3 in I/R group were increased significantly when compared with that of sham group (P < 0.05). Nec-1 blocked significantly RIP1-RIP3 interaction, RIP1 and RIP3 expression, RIP3 phosphorylation compared with that of I/R group (P < 0.05).
Besides, DAXX subdivision in the cells was measured by immunoblotting (Fig. 6B). As illustrated in Fig. 6B, in I/R group, nuclear DAXX protein level was increased obviously, while cytoplasmic DAXX protein level was decreased distinctly, which indicated that cerebral ischemia reperfusion could induce DAXX translocation to cytoplasm from nucleus. Nec-1 reversed significantly DAXX translocation; this was corresponding to decreased DAXX protein level in the nucleus and its increased protein level in the cytoplasm, which suggested that Nec-1 could block DAXX translocation from nucleaus to cytoplasm during cerebral ischemia stress. In addition, phosphorylation of DAXX in cytoplasm was measured by immunoprecipitation. As shown in Fig. 6C, DAXX phosphorylation in I/R group were increased significantly compared with that of sham group (P < 0.05), and Nec-1 reduced DAXX phosphorylation compared with that of I/R group (P < 0.05). 4. Discussion
3.5. Nec-1 inhibited RIP3-DAXX interaction and translocation of DAXX from nucleus to cytoplasm, resulting in inactivation of DAXX In current study, we used immunoprecipitation to detect interaction of RIP3 with DAXX in each group. As shown in Fig. 6A, interaction of RIP3 with DAXX in I/R group were increased significantly compared with that of sham group (P < 0.05). Nec-1 could block RIP3-DAXX interaction compared with that of I/R group (P < 0.05).
Hippocampus is vulnerable to global cerebral I/R injury. The pattern of behavior, learning, and memory impairments attribute to hippocampal damage [3,19]. Previous studies indicated that most extensive neuronal damage in the CA1 layer of the hippocampus was caused by cerebral ischemia, further resulting in impairment of cognitive ability and other neurological deficits [20]. Our findings suggested that Nec-1 enhanced the survival rate of hippocampal neurons and reduced the impairment of behavior, learning, and
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
211
Fig. 3. Nec-1 improved locomotor impairment and anxiety-like behavior induced by cerebral I/R. (A) The track maps of different groups. (B) Total distance traveled during 5 min. (C) Crossing lines during 5 min. (B, C) Data are presented as the mean ± SD (n = 8). * P < 0.05 versus respective sham group; # P < 0.05 versus respective I/R group.
memory induced by cerebral ischemia, which involved the RIP3DAXX signaling pathway. The family members of RIP are critical for cell necrosis and apoptosis, in which RIP1 and RIP3 are involved in the signaling pathways for cell death induced by FasL and TNF␣. RIP3 binds to RIP1 through its C-terminal RIP homotypic interaction motif (RHIM) domain in response to TNF␣ [10]. TNF␣ stimulation leads to the formation of TNF receptor 1 (TNFR1) signaling complex I, and complex II containing RIP1, RIP3, FADD (Fas-associated with death domain protein) and caspase-8, of which the activation initiates ischemic neuron death [21]. Although the other complex II components such as FADD and caspase-8 contribute to the size of complex II, the amyloid complex of RIP1 and RIP3 are the core of complex II [22]. Both RIP1 and RIP3 are cleaved by caspase-8 under apoptotic stimuli, however, the cleaved RIP1 can not activate NF-B. Consequently, caspase-8 triggers apoptosis by activating the classical caspase cascade [21]. Both apoptosis and necrosis are involved in the neuronal death caused by cerebral ischemia, but the underlying mechanisms remain elusive. It has been reported that Nec-1, a small molecule inhibitor of RIP1 kinase, has shown promise as a neuroprotectant in adult rodent models of myocardial ischemia as well as traumatic
and ischemic brain injury [23]. One report involving myocardial ischemia and reperfusion (I/R) injury showed that enhanced RIP1RIP3 interaction could be inhibited by Necrostatin-1(Nec-1) [24]. To further study the role of RIP3 in cerebral ischemia, we employed the four-vessel occlusion model to develop global cerebral ischemia. Our results illustrated that cerebral I/R promoted the interaction of RIP3 with RIP1 and RIP3 activation, which was inhibited by cerebral ventricle injection of Nec-1. Our study suggested that RIP3 played a vital role in cerebral ischemia neuronal injury; the precise downstream signal pathway is still elusive. Substrates of RIP3, mixed lineage kinase domain-like protein (MLKL) and the mitochondrial phosphatase (PGAM5), are recruited to the pronecrotic RIP1-RIP3 complex [25,26]. However, DAXX, a kind of nucleoprotein, participates in the pronecrotic complex as a novel substrate of RIP3, and is translocated from nucleaus to cytoplasm in response to stress [13]. DAXX expression increases in cells upon exposure to hydrogen peroxide, and is involved in mediating oxidative stress-induced apoptosis [27]. In neuronal cells, dominant negative-DAXX blocks Fas-induced cell death [28]. In addition, FADD/caspase-8 cascade-triggered cell death requires the transcriptional activation of DAXX in normal embryonic motor neurons [29]. Furthermore, DAXX has been identified as a potential compo-
212
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Fig. 4. The effects of Nec-1 on neuronal survival at the fifth day after I/R. A-H. Representative images of HE staining in the hippocampus. I. The surviving neurons were counted and the number of cells was considered to represent the neuron quantity. Data are presented as the mean ± SD (n = 7). Boxed areas in left column are shown at higher magnification in right column. A, C, E, G: ×40; B, D, F, H: ×400. Scale bar in G = 200 m; bar in H = 20 m. ** P < 0.01 vs. sham, ## P < 0.01 vs. I/R group.
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
213
Fig. 5. Nec-1 inhibited the interaction of RIP3 with RIP1, reducing RIP3 increased expression and phosphorylation. (A) The effects of Nec-1 on the interaction of RIP3 with RIP1, the expression of RIP1 and RIP3. Data are presented as the mean ± SD (n = 5). * P < 0.05 vs. sham, # P < 0.05 vs. I/R group. (B) The effects of Nec-1 on the expression and phosphorylation of RIP3. * P < 0.05 vs. sham, # P < 0.05 vs. I/R group.
nent of the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD) [27,30]. Our data suggested that Nec-1 inhibited DAXX cytosol translocation and activation upon ischemia in hippocampus.
impairment of behavior, learning and memory induced by cerebral I/R through suppressing RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction, blocking DAXX translocation from nucleus to cytoplasm and then inactivated DAXX, which provided a new target for further research on cerebral I/R injury.
5. Conclusions Conflict of interests In summary, the present study indicated that Nec-1 increased the survival rate of hippocampal neurons, and attenuated the
The authors declare no competing financial interests.
214
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Fig. 6. Nec-1 inhibited RIP3-DAXX interaction and translocation of DAXX from nucleus to cytoplasm, resulting in inactivation of DAXX. (A) The effects of Nec-1 on the interaction of RIP3 with DAXX and total expression of DAXX. Data are presented as the mean ± SD (n = 5). * P < 0.05 vs. sham, # P < 0.05 vs. I/R group. (B) The effects of Nec-1 on DAXX protein levels in nucleus and cytosol at 6 h of reperfusion. * P < 0.05 vs. sham, # P < 0.05 vs. I/R group. (C) The effects of Nec-1 on DAXX phosphorylation level in cytoplasm. * P < 0.05 vs. sham, # P < 0.05 vs. I/R group.
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Author contributions RGD and RLY conceived and supervised the study; RGD, RLY and KH designed experiments; RLY, KH, JYC, SGZ, LL, PJL and HLL performed experiments; RLY, KH, JYC and SGZ analyzed data; RLY and KH wrote the manuscript and made manuscript revisions. All authors read and approved the manuscript. Acknowledgements The authors thank Prof. Hua Fang, Department of Neurology of Hospital of Xuzhou Medical University, for providing language help and writing assistance. References [1] L. Liu, D. Wang, K.S. Wong, Y. Wang, Stroke and stroke care in China: huge burden, significant workload, and a national priority, Stroke 42 (2011) 3651–3654. [2] C.P. Schuch, R. Diaz, I. Deckmann, J.J. Rojas, B.F. Deniz, L.O. Pereira, Early environmental enrichment affects neurobehavioral development and prevents brain damage in rats submitted to neonatal hypoxia-ischemia, Neurosci. Lett. 617 (2016) 101–107. [3] M.L. Gordan, B. Jungwirth, F. Ohl, K. Kellermann, E.F. Kochs, M. Blobner, Evaluation of neurobehavioral deficits following different severities of cerebral ischemia in rats: a comparison between the modified hole board test and the Morris water maze test, Behav. Brain Res. 235 (2012) 7–20. [4] P.S. Lagali, C.P. Corcoran, D.J. Picketts, Hippocampus development and function: role of epigenetic factors and implications for cognitive disease, Clin. Genet. 78 (2010) 321–333. [5] Y. Li, X. Yang, C. Ma, J. Qiao, C. Zhang, Necroptosis contributes to the NMDA-induced excitotoxicity in rat’s cultured cortical neurons, Neurosci. Lett. 447 (2008) 120–123. [6] I.A. Silver, M. Erecinska, Ion homeostasis in rat brain in vivo: intra- and extracellular [Ca2+] and [H+] in the hippocampus during recovery from short-term, transient ischemia, J. Cereb. Blood Flow Metab. 12 (1992) 759–772. [7] K. Jivan, K. Ranchod, G. Modi, Management of ischaemic stroke in the acute setting: review of the current status, Cardiovasc. J. Afr. 24 (2013) 86–92. [8] D. Zhang, J. Lin, J. Han, Receptor-interacting protein (RIP) kinase family, Cell. Mol. Immunol. 7 (2010) 243–249. [9] E. Meylan, J. Tschopp, The RIP kinases: crucial integrators of cellular stress, Trends Biochem. Sci. 30 (2005) 151–159. [10] S. He, L. Wang, L. Miao, T. Wang, F. Du, L. Zhao, X. Wang, Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha, Cell 137 (2009) 1100–1111. [11] D.W. Zhang, J. Shao, J. Lin, N. Zhang, B.J. Lu, S.C. Lin, M.Q. Dong, J. Han, RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis, Science 325 (2009) 332–336. [12] Y.S. Jung, H.Y. Kim, Y.J. Lee, E. Kim, Subcellular localization of Daxx determines its opposing functions in ischemic cell death, FEBS Lett. 581 (2007) 843–852. [13] Y.-S. Lee, Y. Dayma, M.-Y. Park, K.I. Kim, S.-E. Yoo, E. Kim, Daxx is a key downstream component of receptor interacting protein kinase 3 mediating retinal ischemic cell death, FEBS Lett. 587 (2013) 266–271.
215
[14] Y.L. Niu, C. Li, G.Y. Zhang, Blocking Daxx trafficking attenuates neuronal cell death following ischemia/reperfusion in rat hippocampus CA1 region, Arch. Biochem. Biophys. 515 (2011) 89–98. [15] F.J. Northington, R. Chavez-Valdez, E.M. Graham, S. Razdan, E.B. Gauda, L.J. Martin, Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI, J. Cereb. Blood Flow Metab. 31 (2011) 178–189. [16] W.A. Pulsinelli, J.B. Brierley, A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke 10 (1979) 267–272. [17] X. Zhou, F. Zhang, X. Hu, J. Chen, X. Wen, Y. Sun, Y. Liu, R. Tang, K. Zheng, Y. Song, Inhibition of inflammation by astaxanthin alleviates cognition deficits in diabetic mice, Physiol. Behav. 151 (2015) 412–420. [18] D. Qi, H. Liu, J. Niu, X. Fan, X. Wen, Y. Du, J. Mou, D. Pei, Z. Liu, Z. Zong, X. Wei, Y. Song, Heat shock protein 72 inhibits c-Jun N-terminal kinase 3 signaling pathway via Akt1 during cerebral ischemia, J. Neurol. Sci. 317 (2012) 123–129. [19] N. Talma, W.F. Kok, C.F. de Veij Mestdagh, N.C. Shanbhag, H.R. Bouma, R.H. Henning, Neuroprotective hypothermia − Why keep your head cool during ischemia and reperfusion, Biochim. Biophys. Acta 1860 (2016) 2521–2528. [20] F. Pu, K. Motohashi, T. Kaneko, Y. Tanaka, N. Manome, K. Irie, J. Takata, N. Egashira, R. Oishi, T. Okamoto, Y. Sei, T. Yokozawa, K. Mishima, K. Iwasaki, M. Fujiwara, Neuroprotective effects of kangen-karyu on spatial memory impairment in an 8-Arm radial maze and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia in rats, J. Pharmacol. Sci. 109 (2009) 424–430. [21] Y.S. Cho, S. Challa, D. Moquin, R. Genga, T.D. Ray, M. Guildford, F.K. Chan, Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation, Cell 137 (2009) 1112–1123. [22] X.N. Wu, Z.H. Yang, X.K. Wang, Y. Zhang, H. Wan, Y. Song, X. Chen, J. Shao, J. Han, Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis, Cell Death Differ. 21 (2014) 1709–1720. [23] Z. You, S.I. Savitz, J. Yang, A. Degterev, J. Yuan, G.D. Cuny, M.A. Moskowitz, M.J. Whalen, Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice, J. Cereb. Blood Flow Metab. 28 (2008) 1564–1573. [24] M.I. Oerlemans, J. Liu, F. Arslan, K. den Ouden, B.J. van Middelaar, P.A. Doevendans, J.P. Sluijter, Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo, Basic Res. Cardiol. 107 (2012) 270. [25] L. Sun, H. Wang, Z. Wang, S. He, S. Chen, D. Liao, L. Wang, J. Yan, W. Liu, X. Lei, X. Wang, Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase, Cell 148 (2012) 213–227. [26] Z. Wang, H. Jiang, S. Chen, F. Du, X. Wang, The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways, Cell 148 (2012) 228–243. [27] E. Junn, H. Taniguchi, B.S. Jeong, X. Zhao, H. Ichijo, M.M. Mouradian, Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9691–9696. [28] C. Raoul, C. Barthelemy, A. Couzinet, D. Hancock, B. Pettmann, A.O. Hueber, Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell death, J. Neurobiol. 62 (2005) 178–188. [29] C. Raoul, A.G. Estevez, H. Nishimune, D.W. Cleveland, O. deLapeyriere, C.E. Henderson, G. Haase, B. Pettmann, Motoneuron death triggered by a specific pathway downstream of Fas. potentiation by ALS-linked SOD1 mutations, Neuron 35 (2002) 1067–1083. [30] S. Karunakaran, L. Diwakar, U. Saeed, V. Agarwal, S. Ramakrishnan, S. Iyengar, V. Ravindranath, Activation of apoptosis signal regulating kinase 1 (ASK1) and translocation of death-associated protein, Daxx, in substantia nigra pars compacta in a mouse model of Parkinson’s disease: protection by alpha-lipoic acid, FASEB J. 21 (2007) 2226–2236.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Necrostatin-1 protects hippocampal neurons against ischemia/reperfusion injury via the RIP3/DAXX signaling pathway in rats Rongli Yang a,1 , Kun Hu a,1 , Jieyun Chen a , Shiguang Zhu b , Lei Li a , Hailong Lu a , Pingjing Li a , Ruiguo Dong b,∗ a b
Department of Geriatrics, Affiliated Hospital of Xuzhou Medical University, 99 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China Department of Neurology, Affiliated Hospital of Xuzhou Medical University, 99 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China
h i g h l i g h t s • • • •
Nec-1 attenuates cerebral I/R induced cognitive deficits. Nec-1 preserves CA1 hippocampal neuronal survival under cerebral I/R condition. Nec-1 inhibits RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction. Nec-1 blocks DAXX translocation to cytoplasm, which results in the inactiviation of DAXX.
a r t i c l e
i n f o
Article history: Received 10 February 2017 Received in revised form 16 April 2017 Accepted 8 May 2017 Available online 10 May 2017 Keywords: Global cerebral ischemia/reperfusion Receptor-interacting protein 3 The death-associated protein Necrostatin-1
a b s t r a c t Global cerebral ischemia/reperfusion (I/R) induces selective neuronal injury in CA1 region of hippocampus, leading to severe impairment in behavior, learning and memory functions. However, the molecular mechanism underlying the processes was not elucidated clearly. RIP3 is a key molecular switch connecting apoptosis, necrosis and necroptosis. DAXX, as a novel substrate of RIP3, plays a vital role in ischemia-induced neuronal death. The aim of this study is to investigate the role and mechanism of RIP3/DAXX signaling pathway on neurons in CA1 region of the rat hippocampus after cerebral I/R. Global cerebral ischemia was induced by the method of four-vessel occlusion. RIP1 specific inhibitor Necrostatin1 was administered by intracerebroventricular injection 1 h before ischemia. Open-field, closed-field, and Morris water maze tests were performed respectively to examine the anxiety and cognitive behavior in each group. Hematoxylin and eosinstaining was used to examine the survival of hippocampal CA1 pyramidal neurons. Western blot or immunoprecipitation were carried to detect protein expression, phosphorylation, and interaction. We found that pre-treatment with Nec-1 protected locomotive ability, relieved anxiety behavior, and improved cognitive ability in the rats subjected to cerebral I/R. In addition Moreover, Nec-1 decreased significantly the dead rate of neurons in hippocampal CA1 region after cerebral I/R through suppressing RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction, and then blocked DAXX translocation from nucleaus to cytoplasm, which resulted in the inactiviation of DAXX. We concluded that pre-treatment with Nec-1 can protect neurons in the hippocampal CA1 region against ischemic damage through the RIP3-DAXX signaling pathway. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: I/R, ischemia/reperfusion; RIP3, receptor-interacting protein 3; DAXX, death-associated protein; Nec-1, necrostatin-1; HE, hematoxylin and eosin; PML-NBs, promyelocytic leukemia protein-nuclear bodies; FADD, fas-associated with death domain protein; DMSO, dimethylsulfoxide; RHIM, C-terminal RIP homotypic interaction motif; ANOVA, analysis of variance. ∗ Corresponding author at: Department of Neurology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, PR China. E-mail address: [email protected] (R. Dong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2017.05.016 0304-3940/© 2017 Elsevier B.V. All rights reserved.
Ischemic stroke, is the most common type of stroke and remains the leading cause of death and permanent disability in adults worldwide [1]. The mechanisms underlying cerebral ischemic injury are complicated. This condition often drives neuronal damage in the ischemic tissue [2]. The hippocampus is the cerebral region most vulnerable to ischemia [3]. Damages to this area result in deficits in spatial learning and memory, thereby seriously impacting the quality of life for the patients. There is currently no
208
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
effective approach for prevention and treatment of this memory and cognitive impairment [4]. Ischemia induces hippocampal neurons damage via excitotoxicity of N-methyl-d-aspartate (NMDA), Ca2+ overload, free radical generation, and so on [5,6]. Although current clinical treatment for ischemic stroke is to restore the blood flow to the ischemic area through thrombolytic therapy, the reperfusion injury and the narrow time window and a certain amount of risk for thrombolysis restricts its application for the majority of stroke patients [7]. Receptor-interacting protein 3 (RIP3) is a member of RIP Ser/Thr kinase family, which functions as an essential sensor and regulates cell survival, apoptosis and necrosis to pathogen infection or inflammation [8,9]. RIP3 binds to RIP1 through its C-terminal RIP homotypic interaction motif (RHIM) domain [10]. The kinase activity of RIP3 is required for the caspase-independent cell death [11]. To date, however, the mechanism underlying the RIP3-mediated neuronal death in hippocampal CA1 during global cerebral I/R remains unclear. Death associated protein (DAXX), a kind of nucleoprotein, is a component of promyelocytic leukemia protein-nuclear bodies (PML-NBs), playing a direct role in the transcriptional regulation [12]. Previous study validated that DAXX is a novel substrate of RIP3 [13], and is translocated from the neuronal cytoplasm into the nucleus under stress, which promotes neuronal death [14]. In the present study, we explored whether DAXX could be mediated by RIP3 after cerebral ischemia and its possible influence in this process. Necrostatin-1 (Nec-1), a small molecule inhibitor of RIP1 kinase, can inhibit RIP1-RIP3 interaction and decrease the oxidative injury and programmed necrosis in neonatal hypoxia–ischemia (HI) [15]. We investigated whether Nec-1 can protect rat hippocampal neurons against cerebral I/R injury via the RIP3/DAXX signaling pathway. 2. Experimental procedures 2.1. Materials Antibodies, including rabbit anti-RIP1 (1:200, sc-7881), rabbit anti-p-Ser (1:200, sc-81514) and mouse anti-Actin (1:1000, sc-47778), were purchased from Santa Cruz Biotechnology. Rabbit anti-RIP3 (1:500, ab58828) and Rabbit anti-Histone H3 (1:1000, ab8898) were purchased from Abcam. Rabbit anti-DAXX (1:1000, NB100-56136SS) antibody was obtained from NOVUS Biotechnology. Necrostatin-1 (N9037) was obtained from Sigma Biotechnology. All other chemicals were purchased from Sigma unless indicated otherwise. 2.2. Drug administration Nec-1 was dissolved in 1% DMSO with 0.1 M phosphate buffer solution (PBS). Animals were given Nec-1 (1 ug) or vehicle (0.1 M PBS + 1% DMSO) by means of unilateral intracerebroventricular infusion (anteroposterior, 0.8 mm; lateral, 1.5 mm; depth, 3.5 mm) 1 h before ischemia in a total volume of 5 l at 0.5 l/min. The needle was maintained in place for an additional 5 min before withdrawal to prevent fluid reflux. 2.3. Groups and animal model of ischemia Adult male Sprague-Dawley (SD) rats weighing 250–300 g were used (Shanghai Experimental Animal Center, Chinese Academy of Science). All rats were randomly divided into four groups: sham group, I/R group, DMSO group, Nec-1 group (13 rats in each group). The experimental procedures were approved by local legislation for ethics of experiments on animals. Transient cerebral
ischemia was induced by four-vessel occlusion, as described previously [16]. Briefly, under anesthesia with sodium pentobarbital (30 mg/kg, intraperitoneal), vertebral arteries were electrocauterized, and common carotid arteries were exposed. On the following day, anesthesia was induced with 4% isoflurane and both carotid arteries were occluded with aneurysm clamps to induce cerebral ischemia. After 15 min of occlusion, the aneurysm clamps were removed for reperfusion. Rats that lost their lighting reflex were selected for the experiments. Rectal temperature was maintained at about 37 ◦ C throughout the procedure. Rats with seizures were discarded. Rats were moved to an incubator to keep the proper temperature until they were fully awake. Sham controls were performed using the same surgical procedures, except that the carotid arteries were not occluded. 2.4. Morris water maze testing Evaluation of memory and learning capacity was started on the 5th day after reperfusion following 15 min of ischemia. Morris water maze testing of spatial learning capacity was performed as described previously [17]. The Morris water maze consisted of a large circular black pool (210 cm diameter, 50 cm height, filled to a depth of 30 cm with water at 22 ± 2 ◦ C), which was placed in a darkened room, illuminated by dim light. Within the pool, a submerged platform (black, round, 8 cm diameter, 1 cm below surface) was hidden in a fixed location, 55 cm from the edge of the pool. The rat could climb on the plat-form to escape from the necessity of swimming. The rats were given 3 swimming trials per day on 4 consecutive days with a different starting position in each trial. The rat was given a maximum of 120 s to find the hidden platform and was allowed to stay on it for 20 s. Rats that failed to locate the platform were put onto it by the experimenter for 20 s. After 4 days of training, the platform was removed from the pool and the animal was released from the quadrant opposite where the platform had been located. The length of the trial was 120 s, after which, the rat was taken out of the pool. The proportion of time and swim distance the rat spent searching for the platform in the training quadrant where the previous location of the platform was, were recorded and used as a measure of memory. Morris maze performance was analyzed for latency to find the platform using the ANY-maze video tracking system (Stoelting, Wood Dale, IL, USA) with a CCD camera. 2.5. Open-Field test and closed-Field test Five days after intracerebroventricular injection of Nec-1 or DMSO, the locomotive activity in all the rats were evaluated using an open-field test or a closed-field test. Each rat was placed in the center of an open-field apparatus (W50 × D50 × H30 cm) or a closed-field apparatus (W50 × D50 × H30 cm) and acclimated for 3 min. Afterward, the free-moving behavior was monitored for 5 min and analyzed using the ANY-maze Video Tracking System (Stoelting, Wood Dale, IL, USA) with a CCD camera. The total distance traveled and the crossing lines were analyzed. 2.6. Sample preparation Rats were decapitated immediately 6 h after reperfusion, and then the hippocampal CA1 region was isolated and quickly frozen in liquid nitrogen. Tissues were homogenized in an ice-cold homogenization buffer (50 mM MOPS (pH 7.4), 100 mM KCl, 320 mM sucrose, 50 mM NaF, 0.5 mM MgCl2 , 0.2 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 1 mM Na3 VO4 , 20 mM sodium pyrophosphate, 20 mM -phosphoglycerol, 1 mM p-nitrophenyl phosphate, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 5 g/ml of leupeptin, aprotinin, and pepstatin A), and then centrifuged at 12,000 × g for 15 min at 4 ◦ C. Supernatants were collected and
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
209
protein concentration was determined by the method of Lowry. Samples were stored at −80 ◦ C for future use [18]. 2.7. Immunoprecipitation and immunoblotting Tissue homogenates (400 g of protein) were diluted to the same concentration with RIPA lysis and extraction buffer. Samples were pre-cleared by incubation with protein G beads (20 l; Amersham Biosciences) for 2 h, followed by incubation with 1–2 g of primary antibodies overnight at 4C. Protein G beads were added to the tube for further incubation for 2 h. Samples were then centrifuged at 10,000g for 1 min at 4 ◦ C and the pellets were washed three times with immunoprecipitation buffer. For immunoblotting, bound proteins were eluted by boiling at 100 ◦ C for 10 min in SDS-PAGE loading buffer and then isolated by centrifugation. The supernatants were separated on SDS-PAGE gels and then electrotransferred to a PVDF membrane. After being blocked, membranes were incubated with primary antibodies overnight at 4 ◦ C and incubated with horse-radish peroxidase-conjugated secondary antibodies for 1 h at room temperature, followed by development with ECL Western blotting detection reagents.
Fig. 1. The escape latency of rats in the different groups in the Morris water maze test during 4 consecutive training days. Data are presented as the mean ± SD (n = 8). * P < 0.05 versus respective sham group; # P < 0.05 versus respective I/R group.
Table 1 The escape latency of rats in the different groups in the Morris water maze test during 4 consecutive training days. Latency (sec)
2.8. HE-staining The rats subjected to 5 days of reperfusion were perfusionfixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffin-embedded brain sections (5 m) were prepared and stained with hematoxylin and eosin. Histological evaluations were performed with HE-staining for assessment of neuronal damage in the hippocampus. An initial dissector frame was positioned randomly in hippocampal sectors and cells in every 10th section throughout the entire hippocampus. The number of surviving hippocampal CA1 pyramidal cells per 1 mm of length was counted as the neuronal density. Five independent rats were used in each group and both sides of hippocampi were counted. In brief, cell counts were performed at × 400 magnification with the use of an Olympus BH-2 microscope connected to a Sony charge-coupled device video camera, a motorize stage system, and commercial stereology software. 2.9. Statistical analysis Five animals were randomly selected as samples in all groups for western blotting, immunoprecipitation, and histology assays. Image J (Version 1.30 v) analysis software was used for semiquantitative analysis of the bands. Values were expressed as the means ± SD. Data were analyzed using one-way or two-way ANOVA followed by Tukey’s test, or by t-test. P values of <0.05 were considered significant.
Sham I/R DMSO Nec-1
Day 1
Day 2
Day 3
Day 4
89.25 ± 6.48 107.25 ± 4.53* 108.62 ± 5.24* 92.63 ± 4.24#
68.6 ± 7.07 80.88 ± 6.94* 80.25 ± 5.65* 63.83 ± 4.89#
39.75 ± 6.04 56.25 ± 8.29* 54.38 ± 6.19* 40.88 ± 5.25#
25.75 ± 4.95 37.75 ± 6.54* 36.5 ± 5.10* 31.37 ± 4.53#
Values are mean ± SD (n = 8). * P < 0.05 versus respective sham group. # P < 0.05 versus respective I/R group.
significantly increased the swimming distance and the number of crossings compared with I/R group (P < 0.05, Fig. 2). 3.2. Nec-1 improved locomotor impairment and anxiety-like behavior induced by cerebral I/R The results from open- and closed-field tests showed that the total distance traveled by the rats in the sham group in an open field was significantly longer than that in I/R group or DMSO group (P < 0.05; Fig. 3), and was shorter in I/R group or DMSO group compared with Nec-1 group (P < 0.05; Fig. 3). These changes were consistent with the total distance traveled in the closed field, in which the total distance traveled by the Nec-1 group was longer than that in I/R group or DMSO group with regard to crossing the lines in the closed field. In the closed-field test, the rats in I/R group showed a shorter travelling distance and fewer crossings (P < 0.05). Treatment with Nec-1 increased the travelling distance and number of crossings compared with I/R group (P < 0.05).
3. Results 3.1. Nec-1 improved memory deficit and cognitive impairment induced by cerebral I/R In Morris water maze, during the training period, the escape latency in each group gradually decreased (Fig. 1 and Table 1). The latencies in I/R or DMSO group were significantly higher than that in sham groups, but decreased significantly in the Nec-1 group compared with I/R group or DMSO group (P < 0.05; Fig. 1 and Table 1). Spatial memory was measured on the 5th day. During this test, the platform was removed, the swimming distance and the number of crossings in I/R group or DMSO group were greatly decreased compared with sham group (P < 0.05, Fig. 2). Pre-treatment with Nec-1
3.3. Nec-1 protected hippocampal CA1 neurons against global cerebral I/R injury In HE-staining slides, normal pyramidal cells in the sham group (Fig. 4A and B) showed round and pale stained nuclei, whereas dead cells in the I/R group and DMSO group (Fig. 4C and F) displayed pyknotic nuclei. In Nec-1 group (Fig. 4G and H), the neuronal degeneration significantly decreased compared with I/R group. In the four groups, the surviving pyramidal cells counted within a 1-mm length of the CA1 region were 207.67 ± 18.06 in sham group, 31.50 ± 2.88 in I/R group, 31.67 ± 5.54 in DMSO group, 121.67 ± 6.74 in Nec-1 group, corresponding to Fig. 4B, D, F, and H, respectively. Taken together, the results indicated that treat-
210
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Fig. 2. Nec-1 improved memory deficit and cognitive impairment induced by cerebral I/R. (A) The track maps of rats in the different groups on Day 5 without the platform. (B) Total swimming distance traveled in 120 s for each group. (C) Number of crossings in 120 s for each group. Data are presented as the mean ± SD (n = 8). * P < 0.05 versus respective sham group; # P < 0.05 versus respective I/R group.
ment with Nec-1 had a neuroprotective effect against cerebral I/R-induced neuronal cell death in vivo.
3.4. Nec-1 inhibited the interaction of RIP3 with RIP1, reducing RIP3 expression and phosphorylation To explore the role of Nec-1 on RIP3-medicated pathway, interaction of RIP3 with RIP1, expression of RIP1 and RIP3, phosphorylation of RIP3 were measured by immunoblotting and Immunoprecipitation. As shown in Fig. 5, interaction of RIP3 with RIP1, expression of RIP1 and RIP3, phosphorylation of RIP3 in I/R group were increased significantly when compared with that of sham group (P < 0.05). Nec-1 blocked significantly RIP1-RIP3 interaction, RIP1 and RIP3 expression, RIP3 phosphorylation compared with that of I/R group (P < 0.05).
Besides, DAXX subdivision in the cells was measured by immunoblotting (Fig. 6B). As illustrated in Fig. 6B, in I/R group, nuclear DAXX protein level was increased obviously, while cytoplasmic DAXX protein level was decreased distinctly, which indicated that cerebral ischemia reperfusion could induce DAXX translocation to cytoplasm from nucleus. Nec-1 reversed significantly DAXX translocation; this was corresponding to decreased DAXX protein level in the nucleus and its increased protein level in the cytoplasm, which suggested that Nec-1 could block DAXX translocation from nucleaus to cytoplasm during cerebral ischemia stress. In addition, phosphorylation of DAXX in cytoplasm was measured by immunoprecipitation. As shown in Fig. 6C, DAXX phosphorylation in I/R group were increased significantly compared with that of sham group (P < 0.05), and Nec-1 reduced DAXX phosphorylation compared with that of I/R group (P < 0.05). 4. Discussion
3.5. Nec-1 inhibited RIP3-DAXX interaction and translocation of DAXX from nucleus to cytoplasm, resulting in inactivation of DAXX In current study, we used immunoprecipitation to detect interaction of RIP3 with DAXX in each group. As shown in Fig. 6A, interaction of RIP3 with DAXX in I/R group were increased significantly compared with that of sham group (P < 0.05). Nec-1 could block RIP3-DAXX interaction compared with that of I/R group (P < 0.05).
Hippocampus is vulnerable to global cerebral I/R injury. The pattern of behavior, learning, and memory impairments attribute to hippocampal damage [3,19]. Previous studies indicated that most extensive neuronal damage in the CA1 layer of the hippocampus was caused by cerebral ischemia, further resulting in impairment of cognitive ability and other neurological deficits [20]. Our findings suggested that Nec-1 enhanced the survival rate of hippocampal neurons and reduced the impairment of behavior, learning, and
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
211
Fig. 3. Nec-1 improved locomotor impairment and anxiety-like behavior induced by cerebral I/R. (A) The track maps of different groups. (B) Total distance traveled during 5 min. (C) Crossing lines during 5 min. (B, C) Data are presented as the mean ± SD (n = 8). * P < 0.05 versus respective sham group; # P < 0.05 versus respective I/R group.
memory induced by cerebral ischemia, which involved the RIP3DAXX signaling pathway. The family members of RIP are critical for cell necrosis and apoptosis, in which RIP1 and RIP3 are involved in the signaling pathways for cell death induced by FasL and TNF␣. RIP3 binds to RIP1 through its C-terminal RIP homotypic interaction motif (RHIM) domain in response to TNF␣ [10]. TNF␣ stimulation leads to the formation of TNF receptor 1 (TNFR1) signaling complex I, and complex II containing RIP1, RIP3, FADD (Fas-associated with death domain protein) and caspase-8, of which the activation initiates ischemic neuron death [21]. Although the other complex II components such as FADD and caspase-8 contribute to the size of complex II, the amyloid complex of RIP1 and RIP3 are the core of complex II [22]. Both RIP1 and RIP3 are cleaved by caspase-8 under apoptotic stimuli, however, the cleaved RIP1 can not activate NF-B. Consequently, caspase-8 triggers apoptosis by activating the classical caspase cascade [21]. Both apoptosis and necrosis are involved in the neuronal death caused by cerebral ischemia, but the underlying mechanisms remain elusive. It has been reported that Nec-1, a small molecule inhibitor of RIP1 kinase, has shown promise as a neuroprotectant in adult rodent models of myocardial ischemia as well as traumatic
and ischemic brain injury [23]. One report involving myocardial ischemia and reperfusion (I/R) injury showed that enhanced RIP1RIP3 interaction could be inhibited by Necrostatin-1(Nec-1) [24]. To further study the role of RIP3 in cerebral ischemia, we employed the four-vessel occlusion model to develop global cerebral ischemia. Our results illustrated that cerebral I/R promoted the interaction of RIP3 with RIP1 and RIP3 activation, which was inhibited by cerebral ventricle injection of Nec-1. Our study suggested that RIP3 played a vital role in cerebral ischemia neuronal injury; the precise downstream signal pathway is still elusive. Substrates of RIP3, mixed lineage kinase domain-like protein (MLKL) and the mitochondrial phosphatase (PGAM5), are recruited to the pronecrotic RIP1-RIP3 complex [25,26]. However, DAXX, a kind of nucleoprotein, participates in the pronecrotic complex as a novel substrate of RIP3, and is translocated from nucleaus to cytoplasm in response to stress [13]. DAXX expression increases in cells upon exposure to hydrogen peroxide, and is involved in mediating oxidative stress-induced apoptosis [27]. In neuronal cells, dominant negative-DAXX blocks Fas-induced cell death [28]. In addition, FADD/caspase-8 cascade-triggered cell death requires the transcriptional activation of DAXX in normal embryonic motor neurons [29]. Furthermore, DAXX has been identified as a potential compo-
212
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Fig. 4. The effects of Nec-1 on neuronal survival at the fifth day after I/R. A-H. Representative images of HE staining in the hippocampus. I. The surviving neurons were counted and the number of cells was considered to represent the neuron quantity. Data are presented as the mean ± SD (n = 7). Boxed areas in left column are shown at higher magnification in right column. A, C, E, G: ×40; B, D, F, H: ×400. Scale bar in G = 200 m; bar in H = 20 m. ** P < 0.01 vs. sham, ## P < 0.01 vs. I/R group.
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
213
Fig. 5. Nec-1 inhibited the interaction of RIP3 with RIP1, reducing RIP3 increased expression and phosphorylation. (A) The effects of Nec-1 on the interaction of RIP3 with RIP1, the expression of RIP1 and RIP3. Data are presented as the mean ± SD (n = 5). * P < 0.05 vs. sham, # P < 0.05 vs. I/R group. (B) The effects of Nec-1 on the expression and phosphorylation of RIP3. * P < 0.05 vs. sham, # P < 0.05 vs. I/R group.
nent of the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD) [27,30]. Our data suggested that Nec-1 inhibited DAXX cytosol translocation and activation upon ischemia in hippocampus.
impairment of behavior, learning and memory induced by cerebral I/R through suppressing RIP1-RIP3 interaction and RIP3 activation along with RIP3-DAXX interaction, blocking DAXX translocation from nucleus to cytoplasm and then inactivated DAXX, which provided a new target for further research on cerebral I/R injury.
5. Conclusions Conflict of interests In summary, the present study indicated that Nec-1 increased the survival rate of hippocampal neurons, and attenuated the
The authors declare no competing financial interests.
214
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Fig. 6. Nec-1 inhibited RIP3-DAXX interaction and translocation of DAXX from nucleus to cytoplasm, resulting in inactivation of DAXX. (A) The effects of Nec-1 on the interaction of RIP3 with DAXX and total expression of DAXX. Data are presented as the mean ± SD (n = 5). * P < 0.05 vs. sham, # P < 0.05 vs. I/R group. (B) The effects of Nec-1 on DAXX protein levels in nucleus and cytosol at 6 h of reperfusion. * P < 0.05 vs. sham, # P < 0.05 vs. I/R group. (C) The effects of Nec-1 on DAXX phosphorylation level in cytoplasm. * P < 0.05 vs. sham, # P < 0.05 vs. I/R group.
R. Yang et al. / Neuroscience Letters 651 (2017) 207–215
Author contributions RGD and RLY conceived and supervised the study; RGD, RLY and KH designed experiments; RLY, KH, JYC, SGZ, LL, PJL and HLL performed experiments; RLY, KH, JYC and SGZ analyzed data; RLY and KH wrote the manuscript and made manuscript revisions. All authors read and approved the manuscript. Acknowledgements The authors thank Prof. Hua Fang, Department of Neurology of Hospital of Xuzhou Medical University, for providing language help and writing assistance. References [1] L. Liu, D. Wang, K.S. Wong, Y. Wang, Stroke and stroke care in China: huge burden, significant workload, and a national priority, Stroke 42 (2011) 3651–3654. [2] C.P. Schuch, R. Diaz, I. Deckmann, J.J. Rojas, B.F. Deniz, L.O. Pereira, Early environmental enrichment affects neurobehavioral development and prevents brain damage in rats submitted to neonatal hypoxia-ischemia, Neurosci. Lett. 617 (2016) 101–107. [3] M.L. Gordan, B. Jungwirth, F. Ohl, K. Kellermann, E.F. Kochs, M. Blobner, Evaluation of neurobehavioral deficits following different severities of cerebral ischemia in rats: a comparison between the modified hole board test and the Morris water maze test, Behav. Brain Res. 235 (2012) 7–20. [4] P.S. Lagali, C.P. Corcoran, D.J. Picketts, Hippocampus development and function: role of epigenetic factors and implications for cognitive disease, Clin. Genet. 78 (2010) 321–333. [5] Y. Li, X. Yang, C. Ma, J. Qiao, C. Zhang, Necroptosis contributes to the NMDA-induced excitotoxicity in rat’s cultured cortical neurons, Neurosci. Lett. 447 (2008) 120–123. [6] I.A. Silver, M. Erecinska, Ion homeostasis in rat brain in vivo: intra- and extracellular [Ca2+] and [H+] in the hippocampus during recovery from short-term, transient ischemia, J. Cereb. Blood Flow Metab. 12 (1992) 759–772. [7] K. Jivan, K. Ranchod, G. Modi, Management of ischaemic stroke in the acute setting: review of the current status, Cardiovasc. J. Afr. 24 (2013) 86–92. [8] D. Zhang, J. Lin, J. Han, Receptor-interacting protein (RIP) kinase family, Cell. Mol. Immunol. 7 (2010) 243–249. [9] E. Meylan, J. Tschopp, The RIP kinases: crucial integrators of cellular stress, Trends Biochem. Sci. 30 (2005) 151–159. [10] S. He, L. Wang, L. Miao, T. Wang, F. Du, L. Zhao, X. Wang, Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha, Cell 137 (2009) 1100–1111. [11] D.W. Zhang, J. Shao, J. Lin, N. Zhang, B.J. Lu, S.C. Lin, M.Q. Dong, J. Han, RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis, Science 325 (2009) 332–336. [12] Y.S. Jung, H.Y. Kim, Y.J. Lee, E. Kim, Subcellular localization of Daxx determines its opposing functions in ischemic cell death, FEBS Lett. 581 (2007) 843–852. [13] Y.-S. Lee, Y. Dayma, M.-Y. Park, K.I. Kim, S.-E. Yoo, E. Kim, Daxx is a key downstream component of receptor interacting protein kinase 3 mediating retinal ischemic cell death, FEBS Lett. 587 (2013) 266–271.
215
[14] Y.L. Niu, C. Li, G.Y. Zhang, Blocking Daxx trafficking attenuates neuronal cell death following ischemia/reperfusion in rat hippocampus CA1 region, Arch. Biochem. Biophys. 515 (2011) 89–98. [15] F.J. Northington, R. Chavez-Valdez, E.M. Graham, S. Razdan, E.B. Gauda, L.J. Martin, Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI, J. Cereb. Blood Flow Metab. 31 (2011) 178–189. [16] W.A. Pulsinelli, J.B. Brierley, A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke 10 (1979) 267–272. [17] X. Zhou, F. Zhang, X. Hu, J. Chen, X. Wen, Y. Sun, Y. Liu, R. Tang, K. Zheng, Y. Song, Inhibition of inflammation by astaxanthin alleviates cognition deficits in diabetic mice, Physiol. Behav. 151 (2015) 412–420. [18] D. Qi, H. Liu, J. Niu, X. Fan, X. Wen, Y. Du, J. Mou, D. Pei, Z. Liu, Z. Zong, X. Wei, Y. Song, Heat shock protein 72 inhibits c-Jun N-terminal kinase 3 signaling pathway via Akt1 during cerebral ischemia, J. Neurol. Sci. 317 (2012) 123–129. [19] N. Talma, W.F. Kok, C.F. de Veij Mestdagh, N.C. Shanbhag, H.R. Bouma, R.H. Henning, Neuroprotective hypothermia − Why keep your head cool during ischemia and reperfusion, Biochim. Biophys. Acta 1860 (2016) 2521–2528. [20] F. Pu, K. Motohashi, T. Kaneko, Y. Tanaka, N. Manome, K. Irie, J. Takata, N. Egashira, R. Oishi, T. Okamoto, Y. Sei, T. Yokozawa, K. Mishima, K. Iwasaki, M. Fujiwara, Neuroprotective effects of kangen-karyu on spatial memory impairment in an 8-Arm radial maze and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia in rats, J. Pharmacol. Sci. 109 (2009) 424–430. [21] Y.S. Cho, S. Challa, D. Moquin, R. Genga, T.D. Ray, M. Guildford, F.K. Chan, Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation, Cell 137 (2009) 1112–1123. [22] X.N. Wu, Z.H. Yang, X.K. Wang, Y. Zhang, H. Wan, Y. Song, X. Chen, J. Shao, J. Han, Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis, Cell Death Differ. 21 (2014) 1709–1720. [23] Z. You, S.I. Savitz, J. Yang, A. Degterev, J. Yuan, G.D. Cuny, M.A. Moskowitz, M.J. Whalen, Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice, J. Cereb. Blood Flow Metab. 28 (2008) 1564–1573. [24] M.I. Oerlemans, J. Liu, F. Arslan, K. den Ouden, B.J. van Middelaar, P.A. Doevendans, J.P. Sluijter, Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo, Basic Res. Cardiol. 107 (2012) 270. [25] L. Sun, H. Wang, Z. Wang, S. He, S. Chen, D. Liao, L. Wang, J. Yan, W. Liu, X. Lei, X. Wang, Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase, Cell 148 (2012) 213–227. [26] Z. Wang, H. Jiang, S. Chen, F. Du, X. Wang, The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways, Cell 148 (2012) 228–243. [27] E. Junn, H. Taniguchi, B.S. Jeong, X. Zhao, H. Ichijo, M.M. Mouradian, Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9691–9696. [28] C. Raoul, C. Barthelemy, A. Couzinet, D. Hancock, B. Pettmann, A.O. Hueber, Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell death, J. Neurobiol. 62 (2005) 178–188. [29] C. Raoul, A.G. Estevez, H. Nishimune, D.W. Cleveland, O. deLapeyriere, C.E. Henderson, G. Haase, B. Pettmann, Motoneuron death triggered by a specific pathway downstream of Fas. potentiation by ALS-linked SOD1 mutations, Neuron 35 (2002) 1067–1083. [30] S. Karunakaran, L. Diwakar, U. Saeed, V. Agarwal, S. Ramakrishnan, S. Iyengar, V. Ravindranath, Activation of apoptosis signal regulating kinase 1 (ASK1) and translocation of death-associated protein, Daxx, in substantia nigra pars compacta in a mouse model of Parkinson’s disease: protection by alpha-lipoic acid, FASEB J. 21 (2007) 2226–2236.