Transmembrane protein 166 regulates autophagic and apoptotic activities following focal cerebral ischemic injury in rats

Experimental Neurology 234 (2012) 181–190

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Transmembrane protein 166 regulates autophagic and apoptotic activities following focal cerebral ischemic injury in rats Li Li a, Nikan H. Khatibi b, Qin Hu a, c, Junhao Yan a, c, Chunhua Chen a, c, Jingyan Han d, Dalong Ma e, Yinyu Chen e,⁎⁎, 1, Changman Zhou a, 1,⁎ a

Department of Anatomy and Histology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Department of Anesthesiology, Loma Linda Medical Center, Loma Linda, CA, USA Department of Neurosurgery, Loma Linda Medical Center, Loma Linda, CA, USA d Microcirculation Research Center, Peking University Health Science Center, Beijing, China e Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Science Center, Beijing, China b c

a r t i c l e

i n f o

Article history: Received 4 November 2011 Revised 13 December 2011 Accepted 17 December 2011 Available online 29 December 2011 Keywords: Autophagy Apoptosis TMEM166 Cerebral ischemia MCAO

a b s t r a c t Transmembrane protein 166 (TMEM166) is a lysosomal/endoplasmic reticulum-associated protein found in various species where it acts as a regulator of programmed cell death, mediating both autophagy and apoptosis. In the present study, we investigated the role of TMEM166 following MCAO injury in rats to determine whether the structural damages following injury were orchestrated in part by TMEM166. One hundred and fifty six male Sprague–Dawley rats were randomly divided into 4 groups: Sham, MCAO, MCAO + control siRNA, MCAO + TMEM166 siRNA. Outcomes were measured including mortality rate, brain edema, BBB disruption, and neurobehavioral testing. Western blotting techniques measured the expression of key proautophagic and apoptotic proteins such as TMEM166, Beclin-1, cleaved casepase-3 and Bcl-2/Bax. The study found that TMEM166 siRNA treatment significantly reduced the mortality rate, cerebral edema, neurobehavioral deficits, and BBB disruption as measured by Evan's blue assay following MCAO injury. Immunohistochemical staining and western blotting analysis demonstrated an increased expressions of TMEM166, Beclin-1, LC3, cleaved casepase-3 and Bcl-2/Bax in the infarcted areas. This study suggests that TMEM166 induces autophagy and apoptosis may in fact play a significant role in cell death following MCAO injury and its mediation may be through the crosstalk of Bcl-2. By blocking the activity of TMEM166 using siRNA, we were able to prevent the cell loss that occured following cerebral ischemia injury. This translated into a preservation of functional integrity and an improvement in mortality. © 2012 Elsevier Inc. All rights reserved.

Introduction Cerebral stroke is the leading cause of adult disability and the second overall cause of mortality worldwide. With close to 80% of all stroke subtypes being ischemic, research has focused extensively on means of reducing consequences following ischemic stroke episodes; this includes reducing both neuronal and non-neuronal cell death. At least three distinct modes of cell death have been identified and implicated with the ischemic stroke process — they include necrosis, apoptosis, and autophagy. While the first two have been extensively

⁎ Correspondence to: C. Zhou, Department of Anatomy and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xueyuan Road, Haidian Qu, Beijing 100191, China. Fax: + 86 10 8280 1164. ⁎⁎ Correspondence to: Y. Chen, Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Science Center, Beijing 100191, China. E-mail addresses: [email protected] (Y. Chen), [email protected] (C. Zhou). 1 Contributed equally to this work. 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2011.12.038

researched and supported, the discussion around autophagy remains inconclusive. This is in part because of the mixed research suggesting autophagy is an essential process for cellular metabolism and survival (Komatsu et al., 2005; Kuma et al., 2004; Shintani and Klionsky, 2004), while on the other hand, it may also be involved in cell death (Ohsawa et al., 1998; Uchiyama, 2001; Yu et al., 2004; Zhu et al., 2007), specifically orchestrating the effects in cerebral ischemia (Chu, 2008; Park et al., 2009; Uchiyama et al., 2008b). Transmembrane protein 166 (TMEM166) is a lysosomal/endoplasmic reticulum-associated protein found in various species including humans, chimpanzees, rats, mice, and dogs where it acts as a regulator of programmed cell death, mediating both autophagy and apoptosis. Recently, studies have suggested that over-expression of TMEM166 induces the autophagy process and may contribute to further cellular dysfunction and activation of programmed cell death (He et al., 2009). However, other studies have suggested a different role for TMEM166, suggesting more neuroprotective properties. Thus, further understanding of the TMEM166 protein may provide new information about signaling pathways between the apoptosis

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and autophagy processes and clarify whether it provides cellular protection or propagates damage (Wang et al., 2007). Beclin-1 (the homologue of the yeast Apg6/Vps30) participates in the early stages of autophagy, promoting the nucleation of the autophagic vesicle and recruiting proteins from the cytosol (Ferraro and Cecconi, 2007), which is now used as a marker of final autophagosome formation. In contrast to Beclin-1, LC3 (a mammalian homologue of yeast Apg 8/Aut7) was the first protein identified from the autophagosome membrane (Kabeya et al., 2000). There are three isoforms of LC3 mRNA: LC3A, LC3B, and LC3C. After synthesis, LC3 is subsequently processed to LC3-I (cytosolic form) and modified to the active LC3-II form (membrane bound form) by posttranslational modification. LC3-II, after conjugation and lipidation with phosphatidylethanolamine, binds to the outer membrane of autophagosomes. LC3 is another reliable marker of autophagosome formation in mammalian cells, and the relative amount of LC3-II reflects autophagic activity. Accordingly, in the present study we investigated the role of the TMEM166 protein following a middle cerebral artery occlusion (MCAO) induced injury in rats — specifically evaluating whether TMEM166 could orchestrate the induction of neuronal autophagy and apoptosis following focal cerebral ischemia. In order to test these aims, we used a TMEM166 siRNA protein and corresponding results of mortality rate, brain edema accumulation, blood–brain-barrier (BBB) disruption, and neurobehavioral deficits, and compared them to those animals treated with the control siRNA. Additionally, western blotting techniques were utilized to evaluate the expression of key pro-autophagy and apoptotic proteins such as TMEM166, Beclin-1, cleaved casepase-3 and Bcl-2/Bax.

96 h to normoxic atmosphere, at 37 °C, whereas controls were constantly maintained under standard conditions. TMEM166 siRNAs synthesis Specific siRNAs targeting against TMEM166 (targeting sequence 5-TGATAAGGATCTCTTGCCA-3; si-TMEM166) were designed and chemically synthesized and PAGE purified according to the manufacturer's instructions (Genechem Corporation, Shanghai, China). Nonsilencing siRNA that had no sequence homology to any known human genes was used as the control. All siRNAs were dissolved at a concentration of 20 μm in buffer containing 20 mM KCl, 6 mM HEPES, pH 7.5, 0.2 mM MgCl2. Hypoxia induced forebrain neurons expressing TMEM166-GFP were cultured on the coverslips, stained with either MAP-2 or GFAP fluorescence immunohistochemical staining with Tracker Red for 15 min at 37 °C, and observed by fluorescence microscopy (detail description see double and triple fluorescence labeling). Real-time quantitative PCR A quantitative real-time PCR assay was carried out in an ABI Sequence Detection System (Applied Biosystems, USA) with specific primers for TMEM166 and GAPDH. Preliminary reactions were run to optimize the concentration and ratio of each primer set. All the cDNA templates were diluted 100 times and 8 ll of each diluted cDNA template was used in a 20 ll real-time PCR amplification system of SYBR Green PCR Master Mix Kit as the manufacturer directed. The expression level of normal oxygen (no hypoxia) as control was treated as the baseline.

Materials and methods Infection efficiency of siRNA in rat brain All protocols for this study were evaluated and approved by the Animal Care and Use Committee at Peking University Health Sciences Center and with the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience (Beijing, certificate no. SCXK 2002-0001). To verify the effect of TMEM166 siRNA, quantitative real-time PCR assay was carried out in an ABI Sequence Detection System (Applied Biosystems, USA) with specific primers for TMEM166 and GAPDH on hypoxia-induced forebrain cultured neurons from Sprague–Dawley rat embryos.

To test if the siRNA could be successfully transfected into the neuronal cells following intracerebroventricular injection, a fluorescence conjugated siRNA (Santa Cruz; sc-36869) was used immediately after MCAO in the pilot experiments (n = 2). At 24 h after injection, the animals were anesthetized and sacrificed by intracardiac perfusion of ice-cold 4% paraformaldehyde in PBS. The brain was removed, postfixed, cryoprotected in 30% sucrose in PBS and embedded in Tissue Tek. Cryostat sections (20 μm) were collected on slides and observed under a fluorescence microscope (Olympus, BX51) (Chen et al., 2009; Hu et al., 2009).

Neuronal cell culture and exposure to hypoxia Experimental groups and MCAO model Primary forebrain cultured neurons were obtained from fourteen day old Sprague–Dawley rat embryos as previously described (Lievre et al., 2000). Briefly, living embryos were excised by cesarean section performed under anesthesia with halothane. Whole embryos were placed in culture medium previously equilibrated at 37 °C and consisting of a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium (50:50, ICN Pharmaceuticals, Costa Mesa, CA, USA) supplemented with 5% inactivated fetal calf serum (Valbiotech, Paris, France). Forebrains were carefully collected, meninges were dissected and gently dispersed in culture medium. After centrifugation at 700 ×g for 10 min, the pellet was re-dispersed in the same medium and passed through a 46 mm-pore size nylon mesh. The cell suspension was transferred into 35 mm petri dishes (Falcon, Becton Dickinson, Le Pont-de-Claix, France) pre-coated with poly-L-lysine. The final density was 106 cells per dish. Cultures were then placed at 37 °C in a humidified atmosphere of 95% air/5% CO2. Six day old neuronal cell cultures were committed to hypoxia for 6 h by transferring the culture dishes to a humidified incubation chamber thermoregulated at 37 °C and flushed by a gas mixture consisting of 95% N2/5% CO2. Cultures were then returned for the next

One hundred and fifty six male Sprague–Dawley rats weighing 300–350 g were housed in a 12-hour light/dark cycle at a controlled temperature and humidity with free access to food and water. A total of 120 rats (36 died within 24 h) were randomly assigned to one of four groups: Sham (n = 30), MCAO (n= 30), MCAO + control siRNA (n= 30) and MCAO + TMEM166 siRNA groups (n= 30). Among the groups, 6 animals were sacrificed for brain water content, 8 for western blotting, 8 for histological examination, and 8 for Evan's blue content examination. MCAO was induced by the endovascular suture rat model as previously described by Chen et al. (2009) with some added modifications. Briefly, animals were anesthetized using 4% isoflurane with a mixture of 60% medical air and 40% oxygen and anesthesia was maintained with 2% isoflurane. The right common carotid, internal carotid and external carotid arteries were surgically exposed. The external carotid artery was then isolated and coagulated. A 40 nylon suture with silicon (Doccol Co.) was inserted into the internal carotid artery through the external carotid artery stump and gently advanced to occlude the MCA. The mean arterial blood pressure, heart rate, arterial blood gases, and blood glucose levels before, during, and after ischemia were analyzed.

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Following 2 h of MCAO, the suture was carefully removed to restore blood flow, the neck incision was closed and the rats were allowed to recover. The body (rectal) temperature at 37.0±0.58 °C was carefully monitored by a feedback-controlled infrared heating pad during the postoperative period until the animal had completely recovered from the anesthesia. After the experiment, animals were housed individually with free access to food and water until they were sacrificed.

left hemispheres. Brain samples were homogenized in 600 μL of PBS and centrifuged (30 min, 15,000 rcf, 4 °C). The supernatant was collected and equal amounts of 50% trichloroacetic acid were added followed by another centrifuge (30 min, 15,000 rcf, 4 °C). The amount of Evan's blue dye was measured by the spectrophotometer and quantified according to a standard curve (Genesis 10uv; Thermo Fisher Scientific, Waltham, MA).

Drug administration

Brain water content

TMEM166 siRNA (5 μL; Santa Cruz; sc-94711) was diluted with the same volume of transfection reagent. The mixture (10 μL) was injected intracerebroventricularly immediately after MCAO according to the established protocol (Hu et al., 2009). In the control siRNA group, rats were treated with the same volume of control siRNA (Santa Cruz; sc-37007). The anesthetized animals were placed in a stereotaxic apparatus and then the siRNA was injected into the right lateral ventricle over 3 min using a Hamilton microsyringe (coordinates 0.8 mm posterior to the bregma, 1.5 mm lateral to the midline, 4.5 mm ventral to the surface of the skull).

Brain water content was measured as previously described (Xi et al., 2002). Briefly, animals were decapitated under deep anesthesia. Brains were immediately removed and cut into four parts: both ipsilateral and contralateral basal ganglia and cortex, cerebellum and brain stem were collected as an internal control. Tissue samples were weighed on an electronic analytical balance (APX-60, Denver Instrument) to the nearest 0.1 mg to obtain the wet weight (WW). The tissue was then dried at 100 °C for 24 h to determine the dry weight (DW). Brain water content (%) was calculated as [(WW − DW)/WW] × 100.

Neurobehavioral deficits

TTC staining

Neurological outcomes were assessed by a blinded observer at 24 h post-MCAO using the Modified Garcia Score (Garcia et al., 1995). The Modified Garcia Score is an 18-point sensorimotor assessment system consisting of six tests with scores of 0–3 for each test (max score = 18). These seven tests included: (i) spontaneous activity, (ii) side stroking, (iii) vibris touch, (iv) limb symmetry, (v) climbing, (vi) forelimb walking.

2,3,7-Triphenyltetrazolium chloride (TTC) staining for infarction volume was conducted as previously described (Chen et al., 2009; Hu et al., 2009). Briefly, at 24 h post-MCAO rats (sham group, n = 8; MCAO group, n = 8; TMEM166 siRNA group, n = 5; control siRNA, n = 5) were deeply anesthetized with ketamine and then decapitated. Brains were then rapidly removed and sliced into 2-mm-thick coronal sections in an adult rat brain matrix (Kent Scientific Corporation). The slices were stained in 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) for 30 min at 37 °C in the dark (Chen et al., 2009). The infarction area and hemisphere area of each section were traced and measured using an image analysis system [Imaging-Pro-Plus (OLYMPUS)]. The possible interference of brain edema to infarction volume was corrected by the following standard method: the non-infarcted area of the ipsilateral hemisphere/total non-infarcted area (from both the ipsilateral and contralateral hemispheres).

Blood–brain-barrier permeability BBB permeability was measured as previously reported (Uyama et al., 1988). Briefly, Evan's blue dye (4%; 2.5 mL/kg) was injected intravenously into the jugular vein and allowed to circulate for 1 h. This was followed by perfusion with PBS (20 mL) via the aorta. The rats were then decapitated, brains harvested and divided into right and

Control

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Fig. 1. Silencing expression of TMEM166 in hypoxia-induced autophagy in forebrain neurons by siRNA. A. RT-PCR results for TMEM166 mRNA expression. B. Quantification of the expression level of TMEM166 detected by real time PCR. TMEM166 siRNA showed strong inhibitory effects for TMEM166 mRNA expression, whereas TMEM166 mRNA expression was not inhibited by non-silencing siRNA. The expression levels in the control group were treated as 1.0 #p b 0.05 si-TMEM166 vs non-silencing. C. Effects of TMEM166 siRNA on the expression of TMEM166-GFP fusion protein. Cultured neurons were transfected with TMEM166-GFP alone (C1) or co-transfected with non-silencing siRNA (C2) and TMEM166 siRNA (C3) respectively. Twenty four hours following transfection, cells were observed with fluorescence microscopy. TMEM166 siRNA showed much fainter fluorescence than cells transfected with non-silencing.

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antibody, on another section of the same brain provided a negative control for each staining.

Histology Animals were sacrificed with deep anesthesia and perfused through the left ventricles with 200 mL of ice-cold 0.1 mol/L PBS followed by 400 mL of 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4). Brains were post-fixed in the same fixative overnight. Following fixation, brains were cryoprotected in 30% sucrose in PBS for over 48 h at 4 °C. Coronal brain sections (20 μm thick) were cut on a cryostat (Leica CM3050 S). A series of sections was obtained from all animal groups. Sections from each animal were divided into several groups for immunohistochemistry and immunofluorescence staining, respectively. The results were observed under an Olympus BX51 microscope. TUNELpositive cell staining was also conducted as previously described and was counted under microscopy (Chen et al., 2009; Hu et al., 2009).

Double and triple fluorescence labeling Double and triple fluorescence labeling were conducted as previously described (Zhou et al., 2005). For double labeling, brain coronal sections from MCAO group rats were incubated by primary antibodies rabbit anti-TMEM166 and goat anti-Beclin-1 (1:200) overnight at 4 °C. The sections were treated with second antibodies conjugated with fluorescence dyes: goat anti-rabbit IgG Texas Red 1:200 (Santa Cruz Inc.), and donkey anti-goat IgG-AMCA (blue aminomethylcoumarin acetate) 1:200 (Jackson Immuno Research Inc., Pennsylvania, USA) in the dark for 2 h at room temperature respectively. For triple labeling, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) fluorescence staining (green, fluorescein dUTP and dNTP Kit, Roche Inc., protocol) was added to double fluorescence labeling. Sections were covered with 30% glycerin and observed under fluorescent microscope operating with a digital camera (Olympus, BX51). Digitalized microphotographs of immunofluorescent sections were saved. The merged images were generated by the Image ProPlus software.

Immunohistochemistry staining Immunohistochemical staining was conducted as previously described (Chen et al., 2009; Hu et al., 2009). Briefly, five series of sections from each group animals were used for the following primary polyclonal antibodies: (1) rabbit anti-TMEM166 (Santa Cruz Inc.); (2) goat antiBeclin-1; (3) rabbit anti-LC3 (Santa Cruz Inc.). Sections were treated with a different species ABC Kit according to the species of antibodies (Santa Cruz Inc.). Peroxidase activity was revealed by dipping the sections for 5 min in a mixture containing 3-diaminobenzidine (DAB) and H2O2 at room temperature. The sections were dehydrated and cover-slipped. Application of control serum, instead of the primary

A1

Western blotting Two series brain infarction samples were prepared for western blotting. One series contained 6 time groups of focal ischemia

A2 Control siRNA

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A3

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β-actin

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80

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60

40

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0 Sham

6h

12h

24h

48h

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Fig. 2. Infection efficiency of siRNA in rat brain and TMEM166 expression post ischemia at 6, 12, 24, 48,96 h and 7 d. A1–A3. siRNA conjugated with fluorescence was administered to the rat brain. Images were obtained from 20-μm cryosections of rat brain using an OLYMPUS BX51 microscope with fluorescence light. A2 and A3 showed the merging image of GFP with MAP-2 (red) and GFAP (red) in the cortex, which indicated the injected siRNA infected the neurons and astrocytes. B. Western blotting analysis of TMEM166 expression post ischemia at 6, 12, 24, 48,96 h and 7 d. TMEM166 significantly increased at 12 h, the peak of expression was at 24 h. *p b 0.05 MCAO vs Sham.

L. Li et al. / Experimental Neurology 234 (2012) 181–190

A 40 33.33% (15/45)

20

# 14.28% (5/35)

10 0% (0/30)

0 Sham

MCAO

Control siRNA

TMEM166 siRNA

B 20 #

18

Neurological scores

Data analysis The statistical differences between two groups were analyzed using the established t-test. Multiple comparisons were statistically analyzed with one-way analysis of variance (ANOVA) followed by Tukey multiple comparison post-hoc analysis or Student–Newman– Keuls test on ranks. A p-value of less than 0.05 was considered statistically significant.

34.78% (16/46)

30

Mortality

(sacrificed at 6, 12, 24, 48, 96 h, and 7 days); another series contained 4 experimental groups (Sham, MCAO, control siRNA and TMEM166siRNA). Samples (50 μg protein; Bradford dye-binding procedure, BioRad Laboratories) were separated onto SDS-polyacrylamide membrane as previously described (Chen et al., 2009; Hu et al., 2009), and probed with the anti-TMEM166 antibody (1:1000, Santa Cruz Inc.); anti-Beclin-1 antibody (1:1000, monoclonal; BD Transduction Laboratories, Lexington, KY), anti-cleaved casepase-3 (1:500 monoclonal, 1:1000, polyclonal, respectively; BD Transduction Laboratories, Lexington, KY), anti-Bax (1:1000, mouse polyclonal, Santa Cruz Inc.) and anti-Bcl-2 (1:1000, rabbit polyclonal, Santa Cruz Inc.). A monoclonal antibody against β-actin (1:4000, Sigma) was used as a control for protein gel loading. Immunoblots were then probed with an ECL Plus chemiluminescence reagent kit (Amersham Biosciences, Arlington Heights, IL) and visualized with the imagine system (Bio-Rad, Versa Doc, model 4000). The data were analyzed by the software Image J.

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16 14 12

*

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8 6 4

Results TMEM166 siRNA reduced TMEM166 mRNA levels In order to effectively determine the specificity of the TMEM166 siRNA translation, siRNA was used to silence the expression of TMEM166 in hypoxic cultured rat forebrain neurons. Both nonsilencing siRNA and TMEM166 siRNA (si-TMEM166) were transfected into neurons combined with the TMEM166-GFP vector. At 48 h after transfection, TMEM166 mRNA and protein levels were significantly reduced in cells transfected with si-TMEM166, as assessed by quantitative real-time RT-PCR (Figs. 1A, B) and fluorescence microscopy (Fig. 1C). Twenty four hours following intracerebroventricular injection of control siRNA, the fluorescence material was observed in the brain parenchyma (Fig. 2A1). The double fluorescence immunohistochemical staining showed the siRNA-GFP was co-labeled with MAP-2 (Fig. 2A2) and GFAP (Fig. 2A3). It indicated that injected siRNA successfully transfected into the neurons (marked by MAP-2) and astrocytes (marked by GFAP). Western blotting analysis revealed decreased expression of TMEM166 following injury The expression of TMEM166 from brain infarction in different times was shown by western blot analysis of TMEM166 expression at 6, 12, 24, 48,96 h, and 7 days following MCAO (Fig. 2B). The expression of the TMEM166 protein slightly increased as early as 6 h following MCAO, augmented significantly at 12 h, and increased to the highest level at 24 h, then reduced continuously at both 48 and 96 h (p b 0.05) until no significant difference was seen by 7 days (p b 0.05; Fig. 2B). TMEM166 siRNA reduced mortality rates and neurobehavioral deficits following MCAO injury Twenty four hours following MCAO injury, approximately 36 rats (total number started with 156 rats) died at 24 h (Fig. 3A). With regard to mortality, they were found to be: MCAO group 34.78% (16/46 rats), control siRNA 33.33% (15/45 rats), TMEM166 siRNA

2 0 Sham

MCAO

Control siRNA

TMEM166 siRNA

Fig. 3. Mortality rate and neurobehavioral scores. A. There was a significant difference among the MCAO, control siRNA, and TMEM166 siRNA groups with regard to mortality (p = 0.030, p = 0.020, respectively) by the Fisher exact test. A probability value of p b 0.05 was considered statistically significant. B. All animals developed neurobehavioral deficits following MCAO injury. Treatment with TMEM166 siRNA could markedly improve the neurobehavioral deficits (i.e. increase scores) at 24 h. *p b 0.05 MCAO vs Sham; #TMEM166 vs MCAO and control siRNA.

14.28% (5/35 rats), and 0% (0/30 rats) in the sham group. Statistical analysis revealed that there was a significant difference in mortality between the MCAO, control siRNA groups and TMEM166 siRNA group (p = 0.030, p = 0.020, respectively). However, no statistical significance in mortality rate was observed between the MCAO and MCAO + control siRNA group. The mean neurobehavioral scores were shown in Fig. 3B. No deficits were observed among sham operated animals; however, there was a marked decline in the MCAO and control siRNA groups. The TMEM166 siRNA showed a statistically significant improvement (i.e. reduction) in neurobehavioral deficits when compared to MCAO and control siRNA groups at 24 h (p b 0.05). TMEM166 siRNA reduced infarction volume following focal brain ischemia Twenty four hours following MCAO injury, rat brains were evaluated for infarction volume using TTC staining and specific imaging software. Representative samples of TTC-stained brain sections were shown in Fig. 4A with corresponding infarction volumes shown in Fig. 4B. We observed a significant increase in infarction volume in the MCAO group compared to the non-operated sham group (28 ± 5% vs 48 ± 6%, p b 0.05) and the control siRNA group (26.0 ± 3%). This was in contrast to the TMEM166 siRNA group which showed a marked reduction in infarction volume compared to both the MCAO and control siRNA groups (41 ± 5%, p b 0.05 vs MCAO and control siRNA respectively).

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A Sham

Control siRNA

MCAO

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B

TMEM166 siRNA

60 * p<0.05vs Sham # p<0.05vs MCAO

50 40

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30

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Fig. 4. TTC staining and infarction ratios of rat brains. A. Representative samples of TTC-stained brain sections from rats sacrificed at 24 h following ischemia. Severe infarction was shown in MCAO and MCAO + control siRNA rats. The white areas represented the infarction regions in these sections. TMEM166 siRNA treatment reduced the infarction volume sharply, especially in the cortex. No ischemic lesion was found in the sham group. B. Statistical analysis of the infarction ratio which was calculated by taking the non-infarcted area of the ipsilateral hemisphere divided by the total non-infarcted area (from both the ipsilateral and contralateral hemispheres). TMEM166 siRNA treatment significantly reduced the infarction area. *p b 0.05 MCAO vs Sham; #TMEM166 vs MCAO and control siRNA.

TMEM166 siRNA reduced BBB disruption and brain edema BBB permeability was measured using the Evan's blue assay technique (Figs. 5A, B). Compared with the sham operated animal group, MCAO and control siRNA groups demonstrated a marked extravasation of Evan's blue dye in both hemispheres at 24 h (pb 0.05) — especially in the brain stem and cerebellum (pb 0.01). Treatment with TMEM166 siRNA significantly reduced the amount of Evan's blue extravasations in both hemispheres (p b 0.05 vs MCAO and control siRNA).

Brain water content was shown in Fig. 5C. There were no significant differences observed among the groups for the contralateral hemisphere, brain stem and cerebellum water content for TMEM166 siRNA vs MCAO and control siRNA. Compared to sham operated animals, there was a significant increase in brain water content in ipsilateral hemisphere in both the MCAO and control siRNA groups at 24 h post-MCAO injury (p b 0.05). Subsequent treatment with TMEM166 siRNA showed a statistically significant reduction in brain water content (p b 0.05).

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Evans blue (µg/g of brain tissue)

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84 82 80

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78 76 74 72 70

Ipsi- Hetero- Brain lateral lateral stem

Cerebellum

Fig. 5. Evan's blue extravasation and brain water content. A. Representative samples of Evan's blue-stained brain sections from rats sacrificed at 24 h following MCAO brain injury. Negative dye leakage was observed in the sham group while notable staining was seen in the ischemic areas in the MCAO group and control siRNA group. TMEM166 siRNA treated rats displayed weak Evan's blue staining in the respected infarcted areas. B. Vascular leakage was determined by measuring the amount of brain-extracted Evan's blue by spectrophotometry at 620 nm and expressed as μg/g of brain tissue. TMEM166 siRNA group demonstrated a reduced brain EB content compared with the MCAO and MCAO + control siRNA groups (n= 8, pb 0.05). Values were expressed as means±SEM. Evan's blue content analysis. C. The brain water content of the ipsilateral ischemic hemisphere, the contralateral hemisphere, and brain stem/cerebellum. The ipsilateral hemisphere of TMEM166 siRNA group had a significantly decreased water content compared with the MCAO and MCAO+ control siRNA groups (n= 8, p b 0.05, ANOVA). The results of the contralateral hemisphere, brain stem and cerebellum from all groups showed no difference. *pb 0.05 MCAO vs Sham; #TMEM166 vs MCAO and control siRNA.

TMEM166 siRNA significantly reduced the autophagic activity Immunohistochemical staining showed increased expressions of TMEM166, Beclin-1 and LC3 in infarcted regions at 24 h following MCAO (Figs. 6A1–A4) as well as in control siRNA group (Figs. 6B1–B4) — they were negative in sham animals (images are not presented). The TMEM166 siRNA reduced the expressions of TMEM166, Beclin-1 and LC3 in the penumbral areas of infarcted brain in both the MCAO and control siRNA animal groups (Figs. 6C1–C4). Western blotting analysis of brain tissue sample from infarcted areas demonstrated an increased expressions of TMEM166 and Beclin-1 in the MCAO and control siRNA groups compared with those of sham operated animals

(Figs. 7A,B. p b 0.05). Following administration of TMEM166 siRNA, the levels of TMEM166 and Beclin-1 were significantly decreased (p b 0.05). TMEM166 siRNA reduced apoptotic activity Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. Strong TUNEL positive cellular nuclei were observed in the penumbra of brain infarction in MCAO and control siRNA animals (Figs. 6A5, B5). In rats treated with TMEM166 siRNA, the number and density of TUNEL staining were also decreased (Fig. 6C5). Following western blotting analysis,

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Fig. 6. Immunohistochemistry staining in the infarction area. The expressions of TMEM166, Beclin-1, and LC3 in the infarcted areas were significantly increased in the MCAO group (A1–A4) and MCAO + control siRNA group (B1–B4). Markedly increased TUNEL positive cells (A5, B5). TMEM166 siRNA could significantly decrease the levels of TMEM166, Beclin1, and LC3 (C1–C4). The intensity and positive cells of TUNEL staining were markedly declined (C5). The double (D1–D3) and triple (D1–D5) immunofluorescence staining showed the positive TMEM166 (D1, Texas Red) and Beclin-1 (D2, blue aminomethylcoumarin acetate) co-localized in the neuronal cells (D3), which also expressed the TUNEL (D4, D5 green, fluorescein dUTP and dNTP). Figs A1, B1 and C1, bar = 100 μm; Figs A2–D5, bar = 20 μm. The “arrow” showed the co-localized cell.

the levels of cleaved casepase-3 expression were significantly increased in both the MCAO and control-siRNA groups compared to sham animals (p b 0.05; Fig. 7C). The increased expression was then significantly reduced following TMEM166 siRNA administration (p b 0.05 vs MCAO and control siRNA). Double fluorescence staining revealed a relationship expression between autophagy and apoptosis Double fluorescence immunohistochemistry labeling showed both TMEM166 (positive in red; Fig. 6D1) and Beclin-1 (positive in blue; Fig. 6D2) were found within the same neurons (Fig. 6D3). Further staining with TUNEL (green, Fig. 6D4), demonstrated that the signals of TMEM166, Beclin-1 and TUNEL were also in the same neurons (Fig. 6D5). In Fig. 7, western blotting analysis revealed the levels of Bax expression in the MCAO and control siRNA groups were significantly higher than that of the sham animals. Additionally, Bcl-2 expression in the MCAO and control siRNA groups was significantly lower than that of the sham group (p b 0.05, Fig. 7D); moreover, TMEM166 siRNA not only reduced the expression of Bax, but also increased the expression of Bcl-2 (p b 0.05, Fig. 7D). Discussion The present study was designed to investigate the role of transmembrane protein 166 (TMEM166) following MCAO injury in rats — specifically investigating whether TMEM166 could orchestrate the induction of cell autophagy and apoptosis in this type of injury. Our experiments found that following brain injury there was a significant increase in mortality rate, brain edema accumulation, and neurobehavioral

deficits in our rat population; and that following administration of TMEM166 siRNA, there was remarkable reduction. Additionally, further investigation revealed a significant reduction in brain infarction volume following TMEM166 siRNA administration, while both immunohistochemical and western blotting analysis demonstrated the involvement of key pro-autophagic and apoptotic related proteins. Accordingly, these results suggest that TMEM166 is a key orchestrator of autophagic and apoptotic functions following MCAO injury in rats and that through its modulation, we could preserve the structural integrity of the BBB, translating into functional preservation of our rat population. All three modes of cellular death – necrosis, apoptosis and autophagy – have been seen following cerebral ischemic injury (Lipton, 1999). Whereas the role of necrosis and apoptosis has been clearly defined, the precise role of autophagy still remains to be elucidated. This is in part because of the mixed literature that has published both its harmful and protective potentials. On one hand researchers have shown that autophagy plays a key role in cellular survival pathways in response to nutrient deprivation as well as various disease processes including Huntington's, Parkinson's, and Alzheimer's disease (Levine and Yuan, 2005). Yet on the other hand, studies investigating cellular death following neonatal hypoxia–ischemia have shown a more neuroprotective role (Carloni et al., 2008). As a result, it was important for us to investigate what role and in what capacity the autophagic process had among subjects injured by focal brain ischemia. This would allow us to better understand potential upstream targets that can be blocked to reduce downstream consequences following brain injury, i.e. brain edema, BBB disruption, neurobehavioral deficits. Autophagy, also known as cellular self-digestion, is a dynamic intracellular process responsible for degrading cytoplasmic proteins

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Fig. 7. Western blotting analysis of the infarcted brain tissue in each respective group. The expression levels of TMEM166 (A) Beclin-1 (B), cleaved casepase-3 (C) and Bcl-2/Bax (D) in the different groups were significantly increased in the MCAO and MCAO + control siRNA groups compared to those of the sham group (p b 0.05). However, administration of TMEM166 siRNA reduced the levels of TMEM166, Beclin-1, cleaved casepase-3 and Bcl-2/Bax proteins. *p b 0.05 MCAO vs Sham; #TMEM166 vs MCAO and control siRNA.

and organelles in response to external stressors (Kondo et al., 2005; Maiuri et al., 2007b). To date, a number of complicated signaling pathways have been implicated in the regulation of autophagy — one of which is TMEM166, also known as FLJ13391. TMEM166 is a novel lysosomal and endoplasmic reticulum associated membrane protein that contains a putative transmembrane domain. As a novel regulator involved in both autophagy and apoptosis (Wang et al., 2007), our results suggest for the first time that TMEM166 can also be a regulator for both autophagy and apoptosis following MCAO injury. This is important because cellular death leads to a variety of complications including the accumulation of brain edema which causes a rise in intracranial pressure, a reduction in cerebral blood flow causing further ischemic damage — including neuronal cell death and long term neurobehavioral impairment. Accordingly, in the current study, we found that by blocking the TMEM166 with siRNA, we could subsequently reduce brain edema and improve BBB disruption following MCAO injury. This preservation of structural integrity translated into improvements in neurobehavioral capabilities as well. The initial step of the autophagy pathway is the elongation of isolated membranes surrounding cytoplasmic constituents called autophagosomes. Subsequently, the autophagosomes fuse with lysosomes, generating the single membraned autophagolysosome whose contents are degraded by lysosomal hydrolases (Yoshimori, 2004). Autophagy that occurs in ischemic stroke involves the rearrangement

of subcellular membranes into autophagosomes and autophagic vacuoles (Klionsky and Emr, 2000). Similarly, in the current study we found that high levels of TMEM166 lead to massive lysosomal activation and ultimately cell demise following MCAO injury (Figs. 6 and 7). Previously it was reported that hypoxic–ischemic neuronal death was largely prevented by Atg7 deficiency (Uchiyama et al., 2008a) and Beclin-1siRNA (Zheng et al., 2009) — we found that once TMEM166 was inhibited by siRNA, the expressions of Beclin-1 and LC3 were attenuated, suggesting that TMEM166 mediated the autophagic cell death pathway through Beclin-1 and LC3 following MCAO injury. The crosstalk between ‘self-eating’ (autophagy) and ‘self-killing’ (apoptosis) remains unclear, even though the involvement of a functional and physical Bec1/Bcl-2 interaction has been suggested (Maiuri et al., 2007a). A novel protein, NAF-1 (nutrient-deprivation autophagy factor-1), that binds Bcl-2 at the ER. NAF-1 is a component of the inositol-1,4,5 trisphosphate (IP3) receptor complex, which contributes to the interaction of Bcl-2 with Bec1and is required for Bcl-2 to functionally antagonize Bec1-mediated autophagy (Chang et al., 2010). Apoptosis and autophagy can occur concurrently within damaged neurons, leading to cell death. In the current study, in order to confirm whether TMEM166 mediated apoptosis and autophagy following MCAO injury, TMEM166, Beclin-1 and LC3 immunohistochemistry and TUNEL labeling were performed. The positive results (Fig. 6) suggested that autophagy and apoptosis occurred somehow as a

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consequence of TMEM166 activation. This was also confirmed by western blotting which showed a significant reduction in Beclin-1 and cleaved casepase-3 following TMEM166 siRNA administration compared to MCAO or MCAO + control siRNA groups (Figs. 7B,C). Further evaluation using TTC infarction volume assay and neurobehavioral deficits observation also confirmed the TMEM166 functions. Thus to this point we can conclude that autophagy and apoptosis cell death may be orchestrated through TMEM166 and that its inhibition can reduce the cell loss following ischemia. TMEM166 is an essential mediator of cell death and plays a key functional role in the process of autophagy and apoptosis (Wang et al., 2007). Like Beclin-1 and Virtual Mono Poly Analogue Synthesizer (VMP1, TMEM49) proteins, TMEM166 binds to and neutralizes prosurvival members of the Bcl-2 family to promote apoptosis (Ferraro and Cecconi, 2007). Proteins from Bcl-2 family not only participate in the regulation of apoptosis, but are also important inducers or inhibitors of autophagy (Levine et al., 2008). Along with Beclin-1, TMEM166 may also be another avenue of cross-talk between the apoptotic and autophagic pathway (Liu et al., 2010; Maiuri et al., 2007b; Wang et al., 2007). Generally, B-cell lymphoma 2 (Bcl-2) or the Bcl-2 homologue B-cell lymphoma-extra large (Bcl-XL) complex is formed by the combination of a the Bcl-2 homology domain 3 (BH3) domain in Beclin-1 and the BH3 binding groove of Bcl-2/Bcl-XL (Liu et al., 2010). Under specific stimuli, BH3-only proteins like Bad can competitively antagonize the interaction between Beclin-1 and Bcl-2/Bcl-XL. As a result, BH3-only proteins may differentially induce autophagy and apoptosis by acting on (at least) two distinct subcellular compartments (Maiuri et al., 2007a; Oltersdorf et al., 2005). In our study, we found that the level of Bcl-2/ Bax significantly changed in MCAO and TMEM166 siRNA groups (Fig. 7D). It is our belief that TMEM166 could also mediate cell autophagy and apoptosis through the Bcl-2 family. In conclusion, this study suggests that TMEM166 induced autophagy and apoptosis may in fact play a significant role in the cell death process following MCAO injury and its mediation may be through the Bcl-2. By blocking the activity of TMEM166 using siRNA, we were able to improve outcomes that occured following MCAO injury. This preservation of structural integrity translated into a preservation of functional capabilities and an improvement in mortality. That being said, more studies evaluating the exact mechanistic relationship between autophagy and apoptosis and its molecular pathway are warranted. Acknowledgments This work was partially supported by: The National Key Project for Basic Research of China (2011 CB910103) and The National Natural Science Foundation of China (30971527). The authors extend special thanks to Ms. Yuan Zhou, a PhD student in the department of biologic statistics, Louisianan State University in New Orleans for our data statistic analysis. References Carloni, S., Buonocore, G., Balduini, W., 2008. Protective role of autophagy in neonatal hypoxia–ischemia induced brain injury. Neurobiol. Dis. 32, 329–339. Chang, N.C., Nguyen, M., Germain, M., Shore, G.C., 2010. Antagonism of Beclin 1dependent autophagy by Bcl-2 at the endoplasmic reticulum requires NAF-1. EMBO J. 29, 606–618. Chen, C., Hu, Q., Yan, J., Yang, X., Shi, X., Lei, J., Chen, L., Huang, H., Han, J., Zhang, J.H., Zhou, C., 2009. Early inhibition of HIF-1alpha with small interfering RNA reduces ischemic-reperfused brain injury in rats. Neurobiol. Dis. 33, 509–517. Chu, C.T., 2008. Eaten alive: autophagy and neuronal cell death after hypoxia–ischemia. Am. J. Pathol. 172, 284–287. Ferraro, E., Cecconi, F., 2007. Autophagic and apoptotic response to stress signals in mammalian cells. Arch. Biochem. Biophys. 462, 210–219. Garcia, J.H., Wagner, S., Liu, K.F., Hu, X.J., 1995. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 26, 627–634.

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