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. 2014 Nov 26;35(3):407–416. doi: 10.1007/s10571-014-0136-8

Expression and Cell Distribution of SENP3 in the Cerebral Cortex After Experimental Subarachnoid Hemorrhage in Rats: A Pilot Study

Yi-qing Yang 1, Hua Li 1, Xiangsheng Zhang 2, Chun-xi Wang 1, Qing Sun 1, Song Li 3, Weide Li 3, Wei Li 1, Ke Ding 1, Ming Liu 4, Zhuang Yu 1, Chun-hua Hang 1,✉
PMCID: PMC11486192  PMID: 25423917

Abstract

Subarachnoid hemorrhage (SAH) is one of the life-threatening diseases with high morbidity and mortality rates. Small ubiquitin-like modifier (SUMO)-specific proteases 3 (SENP3), a member of the SUMO-specific protease family, was identified as an isopeptidase that deconjugates SUMOylation (The covalent modification by SUMO) of modified protein substrates. It is reported that SUMO-2/3 conjugation, a member of SUMOylation, presented neuroprotection. The study aimed to evaluate the expression of SENP3 and to explore its role potential role in SAH. A total of 95 Sprague–Dawley rats were randomly divided into sham group and SAH groups at 6, 12, 24, 48 h, day 3, day 5, and day 7. SAH groups suffered experimental SAH by injection with 0.3 ml nonheparinized autoblood into the prechiasmatic cistern. SENP3 expression is surveyed by western blot analysis, real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence. The levels of cleavage caspase-3 were determined by western blot and immunohistochemistry. SENP3 protein expression was significantly up-regulated after SAH which peaked at 24 h; however, the mRNA expression of SENP3 remained unchanged. Meanwhile, the level of cleaved caspase-3 was also increased after SAH. There is a highly positive correlation between cleavage caspase-3 and SENP3 in protein level. Immunofluorescent results showed that the expression of SENP3 was increased in neurons, rather than astrocytes nor microglia. Our findings indicated a possible role of SENP3 in the pathogenesis of early brain injury mediated by apoptosis following SAH.

Keywords: Subarachnoid hemorrhage, SENP3, Cleavage caspase-3

Introduction

Subarachnoid hemorrhage (SAH), especially aneurysm subarachnoid hemorrhage (aSAH), is a fatal disease with high morbidity and disability rates (Cahill 2006). Although cerebral vasospasm (CVS) was regarded as a unique and paramount factor of poor outcome following SAH for several decades, current series of studies and largish reviews illustrate that early brain injury (EBI) is another factor influencing outcome. Therefore, more and more researches convert from CVS to EBI (Sehba and Pluta 2011). The EBI was considered widely to be the injury occurred in 72 h after SAH.

Small ubiquitin-like modifier (SUMO) is covalently linked to a variety of proteins and is deconjugated by SUMO-specific proteases (SENPs) (Hay 2005). It modifies protein at post-translation and functions in various kinds of cell signaling pathways (Yeh and Gong 2000). SENPs are an isopeptidase in guaranteeing SUMO homeostasis between modified protein substrates (SUMOylation) and not modified protein substrates (deSUMOylation) (Sun et al. 2013; Yeh 2009). This balance influences differentiation, growth, targeting, and communication between cells which are highly complex and tightly regulated (Wilkinson and Nakamura 2010). For example, disrupting it contributed to cancer development and progression (Han et al. 2010; Sun et al. 2013). And it is also important in the central nervous system (CNS). General increase of SUMO-2/3 conjugation is a neuroprotective response to severe stress (Guo et al. 2013). Extra SENP3 could reverse this neuroprotection in a ischemia model (Yeh 2009). SNEP3 is prevented from proteasomal degradation-related ubiquitin when calculated ROS stimulate cells (Yan et al. 2010).

Few studies examining SENP3 in the CNS indicated that this enzyme is related to apoptosis, which is one of the most important mechanisms of EBI following SAH (Cahill 2006). In a vitro model of ischemia, declined SENP3 leaded to a suppression of caspase-mediated neuronal apoptosis (Guo et al. 2013). In spinal cord injury (SCI) model, SENP3 was up-regulation that Implicating for neuronal apoptosis (Wei et al. 2012). However, there are no studies concentrating on expression of SENP3 in brain tissue after SAH. In order to acquire more knowledge of SENP3 after SAH and coming up with any possibility of a relationship between SENP3 and apoptosis in SAH rats, this study was designed to identify the time course of SENP3 expression in cerebral cortex and to clarify the correlation between SENP3 and apoptosis in early brain injury following SAH.

Materials and Methods

Animal Preparation

Male Sprague–Dawley rats (280–320 g) were acquired from Animal Center of Jinling Hospital. The rats were raised on a 12-h dark–light cycle circumstance. They were given free access to food and water. All procedures were approved by the Animal Care and Use Committee of Nanjing University and accorded to Guide for the Care and Use of Laboratory Animals by National Institutes of Health.

Animal Model of SAH

The prechiasmatic injection model was used to imitate SAH (Jeon et al. 2010). Specifically, after intraperitoneal anesthesia with pentobarbital sodium (50 mg/kg) (Sigma, St. Louis, MO, USA), they were located prone in a stereotactic frame. After careful disinfection treatment, a midline scalp incision was taken and a 1-mm hole was drilled at 7.5 mm anterior to the bregma in the midline, at an angle of 30E caudally. Then they were turned into supine position. Carefully disinfecting again, then we use insulin injection needle (BD Science) to obtain 300 μl of their own blood from femoral artery. The needle (BD Science) was advanced 11 mm into the prechiasmatic cistern through this hole, and the blood was injected into the prechiasmatic cistern over 20 s. Sham animals were operated the same surgery process but not injection with blood into the prechiasmatic cistern. After finishing these procedures, 1 ml of 0.9 % NaCl solution was injected subcutaneously to prevent dehydration. And the rats were returned to their cages. Rats with SAH were randomly divided into seven sub-groups and killed by ventricle perfusion at 6, 12, 24, 48 h, and on day 3, day 5, day 7 post-SAH (n = 6/subgroup). Another 18 rats were chosen randomly for immunohistochemical (IHC) study of sham group, 24 and 72 h group (n = 6/subgroup). Our study found that there was no statistical difference of all items detected among sham groups at each time point. Therefore, sham group animals were killed at 24 h after sham operation.

Perfusion-Fixation and Tissue Preparation

The rats were anesthetized as above, and perfused through the left cardiac ventricle with 0.9 % NaCl solutions until the effluent from the right atrium was clear. Animals which had obvious clots in the prechiasmatic cistern were selected for further analysis. Then blood clots on the brain tissue were cleared, the temporal lobe tissue (Fig. 1) near the hematoma was obtained on ice and stored in −80 °C for western blot and realtime-PCR analysis. For immunohistochemistry and immunofluorescence study, the rats were perfused with 0.9 % NaCl solutions, then perfused 4 % buffered paraformaldehyde; the brain was immersed in 4 % buffered paraformaldehyde 8 h then embedded in paraffin for immunohistochemistry study and frozen in OCT for immunofluorescence study.

Fig. 1.

Fig. 1

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Left one sham group rat brain. Right one SAH rat brain that was collected in 24 h following SAH induced

Western Blot Analysis

Completely homogenizing and centrifuging (at 14,000g for 15 min at 4 °C) of just enough brain tissues were prepared for western blot analysis. Sequently, the supernate was collected. The samples were added SDS sample buffer, followed by boiling for 5 min at 100 °C. Samples (10 μl/lane) were separated by electrophoresis on 10 % SDS-polyacrylamide gels for 30 min at 80 V, then shift to 100 min at 110 V. When electrophoresis was done, it was transferred onto PVDF for 2 h at 200 mA. The membrane was blocked with 5 % defatted milk for 2 h at room temperature, after that incubated with anti-SENP3 antibody (diluted 1:1,000 in 5 % defatted milk, Cell Signaling Technology), active caspase-3 (anti-rabbit, 1:500; Cell Signaling, #9664), and β-actin (diluted 1:5,000 in 5 % defatted milk, Bioworld, USA) at 4 °C with mild shaking for 8 h. Following washing for 10 min each for four times in TBS + Tween 20 (TBST), the membrane was incubated in the appropriate secondary antibody (diluted 1:5,000 in TBST) for 2 h at room temperature. The chemiluminescence (ECL, Thermo Scientific, USA) visualizes the blotted bands. And the bands were exposed to X-ray photographic film. Relative changes in protein expression were evaluated from the mean pixel density by using UN-SCAN-IT, compared with β-actin, and calculated as target protein expression/β-actin expression ratios.

RNA Isolation and Quantitative Real-Time PCR

Rat cerebral tissues were isolated by TRIzol Reagent (TAKARA Biotechnology). The concentration of the RNA was confirmed by spectrophotometric analysis (OD260/280). The quantity of RNA was measured by OD260. The isolated RNA was stored at −80 °C until it being analyzed. RNA was reverse-transcribed to cDNA by Reverse Transcriptase Reagent (TAKARA Biotechnology) and oligo dT primers. Quantitative real-time PCR analysis was conducted by the Agilent Technologies Strata gene Mx3000P real-time PCR system (Gene times Technology, Inc.) and real-time SYBRGreen PCR technology. A reaction unit contained 1 μl of each forward and reverse primer (10 μM), 1 μl cDNA, 12.5 μl SYBRGreen (TAKARA Biotechnology), and nuclease-free water to a final 25 μl. And the primers were synthesized by Life Technologies (Invitrogen, Shanghai, China). The sequences applied were from the database at NCBI for rat SENP3 and β-actin. SENP3 forward and reverse primers were 5′-GGGCTTCCGGGTATCCTA-3′ and 5′-TGCGTCGCCTTACATCAA-3′; β-actin forward and reverse primers were 5′-AGGGAAATCGTGCGTGAC-3′ and 5′-CGCTCATTGCCGATAGTG-3′. After 95 °C for 30 s, 40 PCR cycles were performed, each cycle including a denaturation step (95 °C, 5 s) and an annealing step (60 °C, 30 s). Total RNA concentrations from each sample were normalized by quantity of β-actin messenger RNA (mRNA), and the expression levels of target genes were calculated by the 2−ΔΔCq method. All samples were analyzed in triplicate.

Immunohistochemical Staining

The tissue was fixed with the 4 % paraformaldehyde and embedded in paraffin. IHC staining was performed as our previous study (Bawa-Khalfe 2010). Detailedly, the tissue sections (4 μm) were used for IHC staining; the sections were deparaffinized as usual and incubated with 3 % H2O2 in phosphate-buffered saline (PBS) for 10 min. Sections were blocked with 5 % normal fetal bovine serum in PBS for 2 h followed by incubation with anti-SENP3 antibody (diluted 1:200, Cell Signaling Technology) and active caspase-3 (anti-rabbit, 1:100; Cell Signaling, #9664), respectively. After washing carefully for half an hour, each of the sections was incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (diluted 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at room temperature. After washing for half an hour again, diaminobenzidine was used as a chromogen and counter staining was done with hematoxylin. The negative control was also performed without adding SENP3 antibody and active caspase-3 antibody, respectively. Meanwhile, the other steps were the same between the experiment sections and negative control. In detail, three coronary sections of temporal lobe tissue in each rat brain sample, with a minimum of 150 μm from the adjoining section, were used for cell counting in each sample. Six randomly non-overlapping high power areas (×400) per section were selected and observed. Then mean percentage of robust SENP3-positive cells in the six views was regarded as the data for each section. The final average percentage of the three sections was regarded as the data for the sample. Six samples in each group were employed for the statistical analysis. The percentage of robust SENP3-positive cells was identified, calculated, and analyzed under a light microscope by an investigator blinded to the grouping. Robust expression of SENP3 cells was defined as presenting dark buffy grains in cells shown by arrows in Fig. 5. The robust SENP3-positive expression percentage was defined as which the number of robust SENP3-positive cells accounted for in total number of SENP3-positive cells of each visual field.

Fig. 5.

Fig. 5

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SENP3 expression indentified by immunohistochemistry in brain cortex. SENP3 expression could be observed in the sham and 24 h post-SAH group (a) SENP3 expression in the sham group: robust expression of SENP3 could be observed seldom in the sham group; b SENP3 expression in the 24 h post-SAH group: robust expression of SENP3 obviously enhanced in the SAH group at 24 h after SAH. c and d Enlarged images of a and b, respectively; robust expression of SENP3 cells was defined as presenting dark buffy grains in cells shown by arrows. Scalar bars in a and b present 50 μm, while 20 μm in c and d. e Quantification of robust-positive cells for SENP3. Values were acquired from averaging three sections in every rat. SAH induced up-regulation of SENP3 expression in cortex near subarachnoid space. Bars represent the mean ± SE (n = 6). # P < 0.01 compared with sham group

Immunofluorescence Staining

Immunofluorescence staining was performed according to our previous study in our laboratory (Li et al. 2013). Brain tissue was fixed with 4 % araformaldehyde overnight and dipped in 20 % saccharose PBS for 2 days and then in 30 % saccharose PBS for another 2 days to remove water in the tissue. Sections 6 μm in thickness were sliced and blocked with 5 % normal fetal bovine serum in PBS containing 0.1 % Triton X-100 for 2 h at room temperature prior to incubation with anti-neuron-specific nuclear protein (NeuN) antibody (Millipore, USA, 1:200) and anti-SENP3 antibody (Cell Signaling Technology 1:100) or anti-ionized calcium binding adapter molecule 1 (Iba1) antibody (abcam, 1:200) and anti-SENP antibody (Cell Signaling Technology 1:100) or anti-glial fibrillary acidic protein (GFAP) antibody (Millipore, USA, 1:200) and anti-SENP3 antibody (Cell Signaling Technology 1:100) over night at 4 °C. After sections were washed three times with PBS for 45 min, they were incubated with proper secondary antibodies (AlexaFluor488 and AlexaFluor594, 1:200) for 1 h at room temperature. The slides were washed with PBS again three times for 45 min prior to be counter stained by DAPI for 2 min. After three washes again, the slides were covered by microscopic glass with anti-fade mounting medium for further study. Negative controls were prepared by omitting the primary antibodies. Fluorescence microscopy imaging was performed using ZEISS HB050 inverted microscope system and handled by Image-Pro Plus 6.0 software (Media Cybernetics, USA) and Adobe Photoshop CS5 (Adobe Systems, San Jose, USA). In the merged photomicrographs, yellow color indicates co-localization-positive expression. And the co-localization-positive expression proportion was defined as which the number of co-localization-positive expression accounted for in total number of SENP3-positive expression of each type-specific marker group. Six randomly non-overlapping high power areas (×400) per section were selected and observed. Then mean percentage of co-localization cells in the six views was regarded as the data for each section. The final average percentage of the three sections was regarded as the data for the sample. Six samples in each group were employed for the statistical analysis. The co-localization-positive expression proportion was identified, calculated, and analyzed under a light microscope by an investigator blinded to the grouping.

Statistical Analysis

All data were presented as mean ± SE. SPSS 21.0 (SPSS Inc., Chicago) was applied for statistical analysis of the data. The measurements were analyzed to ANOVA followed by Tukey’s post-hoc test. The relationship between SENP3 protein and cleaved caspase-3 expression was evaluated by the linear regression model. A value of P < 0.05 was considered as statistical significance.

Results

The Mortality After SAH

None of the sham group rats died. And they had not any evidence of SAH. The mortality of SAH group was 28 % (23/83).

The Level of SENP3 Protein Expression in Brain Cortex Following SAH

Low level of SENP3 was identified in the sham group while the level of SENP3 protein increased significantly by 12 h after experimental SAH, peaked at 24 h and declined back to baseline level after day 3 post-SAH. There was a statistically significant difference between the sham group and 12, 24, 48, day 3 group (P < 0.01, respectively) (Figs. 1, 2). The above results demonstrated that SAH could cause significant up-regulation of SENP3 in the temporal lobe tissue in the early part of 12 h post-injury, and keep it at a high level for 3 days. The peak appeared at 24 h post-SAH.

Fig. 2.

Fig. 2

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Top representative autoradiogram of SENP3 expression in the cortex of the brain after SAH. It shows that the expression of SENP3 protein was increased in 12, 24, 48, 3 days post-SAH group and peaked at 24 h. Bottom quantitative analysis of the western blot results for SENP3. It shows that SENP3 levels in SAH groups are significantly higher than the counterpart of sham group. Bars represent the mean ± SE (n = 6, each group). # P < 0.01 compared with sham group. * P < 0.05 compared with sham group

SENP3 mRNA Level in the Brain Cortex After SAH

The expression of SENP3 mRNA was detected by quantitative real-time PCR. This part of the study showed that the SENP3 mRNA expression was not changed in the SAH group which was in a time-dependent manner contrast to the counterpart in sham group (Fig. 3).

Fig. 3.

Fig. 3

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States of SENP3 expression at mRNA level following SAH in the brain cortex. The expression of SENP3 mRNA was detected by quantitative real-time PCR. The level of SENP3 expression was not statistically changed after SAH

Expression and Distribution of SENP3 in Brain Cortex After SAH

The expression and distribution of SENP3 were identified by IHC staining and immunofluorescence staining. SENP3 expression could be observed in both the sham and 24 h post-SAH group. However, robust expression of SENP3 could seldom be observed in the sham group while it obviously enhanced in 24 h post-SAH group (Fig. 4). Semi-quantitative analysis proved that there was a statistical signification between the sham group and the 24 h post-SAH group (P < 0.01) (Fig. 5). To identify in which kind of brain cells SENP3 was increased in 24 h post-SAH group, double immunofluorescence staining was detected for SENP3 and different phenotype-specific markers including neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), and ionized calcium binding adapter molecule 1 (Iba1). As shown in Fig. 6, SENP3 was expressed in both sham group and 24 h post-SAH group. However, further quantitative analysis of the co-localization-positive expression proportion showed that the ratio of NeuN in 24 h post-SAH group significantly increased, when compared with the counterpart of sham group. However, SAH did not change the ratio of GFAP-positive expression proportion and Iba-1 (Fig. 6). These results revealed that expression of SENP3 was increased in neurons, but not in astrocytes or microglia in the injured brain cortex following SAH.

Fig. 4.

Fig. 4

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Top representative autoradiogram of cleaved caspase-3 expression in the cortex of the brain after SAH. It shows that the expression of cleaved caspase-3 protein was enhanced in the 6, 12, 24, 48 h, and 3 days post-SAH group and peaked at 24 h. Bottom quantitative analysis of the western blot results for cleaved caspase-3. It shows that cleaved caspase-3 levels in these groups are significantly higher than counterpart of sham group. Bars represent the mean ± SE (n = 6, each group). # P < 0.01 compared with sham group

Fig. 6.

Fig. 6

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Representative photomicrographs showed brain cortex double immunofluorescent staining for SENP3 (red) (A1–F1) and different phenotype-specific markers (green) (A2–F2) in sham or 24 h post-SAH group. Nucleus was counterstained with DAPI (blue) (A3–F3) in the same view in each section. A4–F4 present merged images of SENP3 (red) and each phenotype-specific marker (green). There were three kinds of cell type-specific markers including NeuN which is a neuron marker (A2, B2), GFAP which is an astrocytes marker (C2, D2) and Iba1 which is a microglia marker (E2, F2). In these merged photomicrographs, yellow color indicates co-localization-positive expression, both SENP3-positive expression (red) and each type of markers-positive expression (green). Scale bars 10 μm G the quantitative analysis of the co-localization-positive expression proportion. The proportion is defined as which the number of co-localization-positive expression accounts for in total number of SENP3-positive expression of each type-specific marker group. Values of both the sham group and 24 h post-SAH group were collected from averaging three sections in every rat. The figure showed that cell distribution of SENP3 after SAH was statistical difference in the neurons, but was not changed in the other phenotypes. Bars represent the mean ± SE (n = 6). # P < 0.01 compared with sham group (Color figure online)

Expression of Cleavage Caspase-3 Increased In Brain Cortex Following SAH

Our data showed that the total quantity of cleaved caspase-3 is low in the sham group in western blot (Fig. 4). Compared with that of the sham group, cleaved caspase-3 in the injured brain tissue was statistically increased as early as 6 h after SAH and peaked at 24 h post-SAH in this study. There is obviously positive relationship between cleaved caspase-3 and SENP3 in protein level (r = 0.8529, P < 0.01). Moreover, cleaved caspase-3 IHC staining demonstrated that positive cells could hardly be found in sham group. In contrast to post-SAH group, both 24 h group and 3 days group, positive cells could easily be found (Fig. 7). There were statistical significances between sham group and each SAH group (P < 0.05). In addition, positive cells in 3 day group statistically decreased compared with counterpart in 24 h group.

Fig. 7.

Fig. 7

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Cleaved caspase-3 expression indentified by immunohistochemistry in brain cortex. Cleaved caspase-3 expression could be observed in 24 h post-SAH group and 3 day post-SAH group, could hardly be found in sham group (a) Cleaved caspase-3 expression in the sham group: positive cells could be observed seldom in the sham group; (b, c) cleaved caspase-3 expression in the 24 h post-SAH group and the 3 days post-SAH group, respectively: expression of cleaved caspase-3 obviously enhanced in the SAH group at 24 h and 3 day after SAH. Scalar bars in a, b and c present 50 μm (d) quantification of positive cells for cleaved caspase-3. Values were acquired from averaging three sections in every rat. SAH induced up-regulation of cleaved caspase-3 expression in cortex near subarachnoid space. Bars represent the mean ± SE (n = 6). * P < 0.05 SAH group (both 24 h and 3 day group) compared with sham group. # P < 0.05 24 h group compared with 3 day group

Discussion

In this study, we reported that the protein expression of SENP3 was up-regulated while gene transcription did not change in the brain cortex in an experimental rat SAH model. These results suggest that SENP3 regulation is a procedure after transcription. IHC and immunofluorescence staining demonstrated that the SENP3-positive cells were obviously increased in the brain cortex. Moreover, expression of SENP3 was increased in neurons, rather than in astrocytes or microglia in the injured brain cortex following SAH. Meanwhile, the protein level of cleaved caspase-3 was also dramatically increased after SAH. A positive correlation between SENP3 and cleaved caspase-3 could be found through statistical analysis, suggesting that SENP3 might play a role in inducing apoptosis in EBI following SAH, which may conduce to the poor outcome of SAH.

Over Expression of SENP3 in Protein Level

Conjugation of SUMO to specific substrates which are certain cellular proteins mediates numerous cell signaling pathways including protein stabilization, apoptosis, intracellular transport, and regulation of transcription (Tan et al. 2010). The covalent modification by SUMO, a isopeptide bond, is invertible and regulated by a family of sentrin/SUMO-specific proteases (SENPs) (Yeh and Gong 2000). SENP3 (SMT3IP1), a member in this family, has been proved to function as a SUMO-specific protease (Hay 2005). It is demonstrated that SENP3, as an isopeptidase, cleaves the isopeptide bond between lysine side chain of a substrate and the glycine residue of SUMO (Yeh and Gong 2000). Normal level of SENP3 which deconjugates modified proteins is considerable for keeping balance between SUMOylation and deSUMOylation. The balance is essential for guarantying normal physiology (Bawa-Khalfe 2010). For example, elevated SENP3 can be detected in both prostate cancer and additional carcinomas including ovarian, lung, rectum, and colon (Han et al. 2010). It is reported that overexpression of SENP3 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein (Nishida and Yamada 2011). SENP3 reverses SUMOylation of nucleophosmin and is indispensable for rRNA processing (Haindl et al. 2008). Moreover, in CNS, proof of up-regulated SENP3 in neurons could be found, especially in a SCI model and an ischemia model. It is reported that up-regulated SENP3 was mostly located in neurons in SCI rats (Guo et al. 2013; Wei et al. 2012). In this study, overexpression of SENP3 was also found in the cerebral cortex after experimental SAH in rats, which matched the previous findings in CNS. What is more, it peaked at 24 h post-SAH and was presented mostly in neurons rather than in astrocytes or microglia.

No Change of SENP3 Gene Transcription

It is clear that SENP3 protein levels can be mediated via reactive oxygen species (ROS); a binding of Hsp90 to SENP3 leads to SENP3 stabilization under oxidative stress (accumulation of ROS), and that this is achieved by blockage of the CHIP-mediated ubiquitination which is constantly under non-stress (Yan et al. 2010). EBI after SAH immediately appears after insult. It is widely accepted that EBI is associated with ischemia following SAH, destruction of blood brain barrier, simulation of hemolytic products and apoptosis of neural cells (Gaetani et al. 1990). All factors above can contribute to oxidative stress, especially to the accumulation of ROS. It is proved that accumulated ROS after SAH contributes to brain edema in rats. When this change had been recovered, neurological impairment avoided consequently (Lu et al. 2009). In a in vitro experiment, H2O2 induced a high level expression of SENP3 in neurons (Wei et al. 2012). In this study, protein levels of SENP3 increased and mRNA levels of SENP3 did not change after SAH, which proved that SENP3 regulation following SAH is a cellular procedure after transcription. Furthermore, considering increased ROS after SAH, induced high expression of SENP3 by H2O2 and the mechanism of SENP3 stabilization, as mentioned above, a hypothesis should be concluded that overexpression of SENP3 is a post-translation procedure, which is induced by accumulated ROS following SAH.

Increased Expression of SENP3 and Apoptosis

Lots of studies have indicated that apoptosis is involved in the pathogenesis of secondary brain injury including EBI after SAH (Cahill 2006). Following SAH, apoptosis has been detected in the cortex, hippocampus, vasculature, and blood brain barrier (Yuksel 2012; Zubkov et al. 2001; Park et al. 2004; Ostrowski and Colohan 2005; Zhou et al. 2005). Different kinds of apoptotic pathways including the death receptor pathway, caspase-dependent pathway, caspase-independent pathway, and the mitochondrial pathway are activated after SAH (Cahill 2006). In addition, relationship between SENP3 and apoptosis was discussed in several studies. It is demonstrated that increased expression of SENP3 restrained Mdm2-mediated p53 ubiquitination and consequential degradation in proteasome (Nishida and Yamada 2011). The expression of SENP3 rose in rat SCI model. Moreover, the co-localization of SENP3 and cleaved-caspase-3 was checked in the spinal cord (Wei et al. 2012). It is proved that depletion of SENP3 prolongs Drp1 SUMOylation, which suppressed Drp1-mediated cytochrome c release and caspase-mediated neurons death in an ischemia model (Guo et al. 2013). In this study, the protein level of cleaved caspase-3 was dramatically elevated and reached the maximum in its protein level after 24 h post-SAH and histological method confirmed this result. What is more, there was a positive relationship between SENP3 and cleaved caspase-3. The correlation implied that SENP3 might play a role in inducing apoptosis in EBI following SAH, which may conduce to the poor outcome of SAH. To demonstrate this assumption, further studies with intervention of SENP3 are still needed.

Summary

To our knowledge, this is the first study to reveal the expression of SENP3 in the brain after SAH. It is found that SAH caused an obvious up-regulation of SENP3 protein expression in rat brain, especially in neurons. However, mRNA expressions of SENP3 remained unchanged. In addition, it could be postulated that SENP3 might play an important role in the EBI after SAH. However, the full role of SENP3 in EBI needs further investigation.

Acknowledgments

The authors acknowledge support for this study by grants from National Natural Science Foundation, China (No. 81171170 to C.-H.H. and No. 81371294 to C.-H.H.) and Nature Science Foundation of Jiangsu Province, China (BK2010459).

Conflict of interest

The authors declare that they have no conflict of interest.

Abbreviations

SAH

Subarachnoid hemorrhage

EBI

Early brain injury

CNS

Central nerve system

CVS

Cerebral vasospasm

ICP

Intracranial pressure

SUMO

Small ubiquitin-like modifier

SENPs

SUMO-specific proteases

SCI

Spinal cord injury

NeuN

Neuron-specific nuclear protein

Iba1

Ionized calcium binding adapter molecule 1

GFAP

Glial fibrillary acidic protein

ROS

Reactive oxygen species

References

  1. Bawa-Khalfe T, Yeh ET (2010) SUMO losing balance: SUMO proteases disrupt SUMO homeostasis to facilitate cancer development and progression. Genes Cancer 1:748–752 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cahill J (2006) Calvert JW, Zhang JH Mechanisms of early brain injury after subarachnoid hemorrhage. J Cereb Blood Flow Metab 26:1341–1353 [DOI] [PubMed] [Google Scholar]
  3. Gaetani P, Marzatico F, Baena RRY, Pacchiarini L, Vigano T et al (1990) Arachidonic-acid metabolism and pathophysiologic aspects of subarachnoid hemorrhage in rats. Stroke 21:328–332 [DOI] [PubMed] [Google Scholar]
  4. Guo C, Hildick KL, Luo J, Dearden L, Wilkinson KA et al (2013) SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia. EMBO J 32:1514–1528 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Haindl M, Harasim T, Eick D, Muller S (2008) The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing. EMBO Rep 9:273–279 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Han Y, Huang C, Sun XX, Xiang BG, Wang M et al (2010) SENP3-mediated de-conjugation of SUMO2/3 from promyelocytic leukemia is correlated with accelerated cell proliferation under mild oxidative stress. J Biol Chem 285:12906–12915 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hay RT (2005) SUMO: a history of modification. Mol Cell 18:1–12 [DOI] [PubMed] [Google Scholar]
  8. Jeon H, Ai J, Sabri M, Tariq A, Macdonald RL (2010) Learning deficits after hemorrhage in rats. Neuroscience 169:1805–1814 [DOI] [PubMed] [Google Scholar]
  9. Li W, Ling HP, You WC, Ji XJ, Tang Y et al (2013) Recombinant high-mobility group box 1 protein (HMGB-1) promotes myeloid differentiation primary response protein 88 (Myd88) upregulation in mouse primary cortical neurons. Neurol Sci 34:847–853 [DOI] [PubMed] [Google Scholar]
  10. Lu H, Zhang DM, Chen HL, Lin YX, Hang CH et al (2009) N-acetylcysteine suppresses oxidative stress in experimental rats with subarachnoid hemorrhage. J Clin Neurosci 16:684–688 [DOI] [PubMed] [Google Scholar]
  11. Nishida T, Yamada Y (2011) The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation. Biochem Biophys Res Commun 406:285–291 [DOI] [PubMed] [Google Scholar]
  12. Ostrowski RP, Colohan ART, Zhang JH (2005) Mechanisms of hyperbaric oxygen-induced neuroprotection in a rat model of subarachnoid hemorrhage. J Cereb Blood Flow Metab 25:554–571 [DOI] [PubMed] [Google Scholar]
  13. Park S, Yamaguchi M, Zhou C, Calvert JW, Tang J et al (2004) Neurovascular protection reduces early brain injury after subarachnoid hemorrhage. Stroke 35:2412–2417 [DOI] [PubMed] [Google Scholar]
  14. Sehba FA, Pluta RM, Zhang JH (2011) Metamorphosis of subarachnoid hemorrhage research: from delayed vasospasm to early brain injury. Mol Neurobiol 43:27–40 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sun ZJ, Hu SQ, Luo QQ, Ye DX, Hu D et al (2013) Overexpression of SENP3 in oral squamous cell carcinoma and its association with differentiation. Oncol Rep 29:1701–1706 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tan JAT, Song J, Chen Y, Durrin LK (2010) Phosphorylation-dependent interaction of SATB1 and PIAS1 directs SUMO-regulated caspase cleavage of SATB1. Mol Cell Biol 30:2823–2836 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wei HX, Teng HL, Huan WP, Zhang SW, Fu HR et al (2012) An upregulation of SENP3 after spinal cord injury: implications for neuronal apoptosis. Neurochem Res 37:2758–2766 [DOI] [PubMed] [Google Scholar]
  18. Wilkinson KA, Nakamura Y, Henley JM (2010) Targets and consequences of protein SUMOylation in neurons. Brain Res Rev 64:195–212 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yeh ETH (2009) SUMOylation and de-SUMOylation: wrestling with life’s processes. J Biol Chem 284:8223–8227 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yan S, Sun XX, Xiang BG, Cang H, Kang XL et al (2010) Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90. EMBO J 29:3773–3786 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yeh ETH, Gong LM, Kamitani T (2000) Ubiquitin-like proteins: New wines in new bottles. Gene 248:1–14 [DOI] [PubMed] [Google Scholar]
  22. Yuksel S, Tosun YB, Cahill J, Solaroglu I (2012) Early brain injury following aneurysmal. Subarachnoid hemorrhage: emphasis on cellular apoptosis. Turk Neurosurg 22:529–533 [DOI] [PubMed] [Google Scholar]
  23. Zhou CM, Yamaguchi M, Colohan ART, Zhang JH (2005) Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab 25:572–582 [DOI] [PubMed] [Google Scholar]
  24. Zubkov AY, Tibbs RE, Clower B, Ogihara K, Aoki K et al (2001) Apoptosis in basilar endothelial cells in a canine double hemorrhage model. Acta Neurochir Suppl 77:29–31 [DOI] [PubMed] [Google Scholar]

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