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Neuroprotective DAMPs member prothymosin alpha has additional beneficial actions against cerebral ischemia-induced vascular damages
Journal of Pharmacological Sciences 132 (2016) 100e104
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short communication
Neuroprotective DAMPs member prothymosin alpha has additional beneficial actions against cerebral ischemia-induced vascular damages Shiori Maeda a, Keita Sasaki a, Sebok Kumar Halder a, Wakako Fujita b, Hiroshi Ueda a, * a b
Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan Department of Frontier Life Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2016 Received in revised form 25 April 2016 Accepted 18 May 2016 Available online 27 May 2016
Prothymosin alpha (ProTa) suppresses stress-induced necrosis of cultured cortical neurons. As neuroprotection alone could not explain the long-lasting protective actions against cerebral ischemia by ProTa, we further examined whether ProTa, in addition to neuroprotective effects, has other anti-ischemic activities. When recombinant mouse ProTa (rmProTa) at 0.3 mg/kg was intravenously (i.v.) given 2 h after the start of tMCAO, all mice survived for more than 14 days. In evaluation of CD31- and tomato lectin-labeling as well as IgG and Evans blue leakage, rmProTa treatment (0.1 mg/kg) largely blocked ischemia-induced vascular damages. Therefore, rmProTa has novel beneficial effects against ischemiainduced brain damage through vascular mechanisms. © 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Cerebral ischemia Prothymosin alpha Vascular and neuronal protection
Nuclear protein prothymosin alpha (ProTa) is extracellularly released from neuronal cells upon ischemic or starving stress and protects neurons in an autocrinic or paracrinic manner (1e4). Accordingly, ProTa is supposed to possess a nature of damageassociated molecular patterns (DAMPs), but it has robustness actions to keep the cellular survival against different aspects of stress (3e5). ProTa has been identified as an anti-necrosis factor from the conditioned medium of serum- and supplement-free primary culture of rat embryonic cortex (1,3,6). In the culture of cortical neurons, one of underlying molecular mechanisms was evidenced by ProTa-mediated externalization of glucose transporter 1 and 4, which had been endocytosed by ischemic or starving stress (1e3). This mechanism prevents the necrotic energy crisis due to cellular ATP loss (1,2). Additional anti-necrotic mechanism by ProTa is related to the activation of caspase 3, which cleaves poly (ADPribose) polymerase, resulting in the suppression of necrotic cell death by inhibiting a rapid decrease in mitochondrial ATP
Abbreviations: CBB, Coomassie Brilliant Blue; IgG, immunoglobulin G; i.p., intraperitoneal; i.v., intravenous; ProTa, prothymosin alpha; TEV, tobacco etch virus; tMCAO, transient middle cerebral artery occlusion. * Corresponding author. Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyomachi, Nagasaki 852-8521, Japan. Tel.: þ81 95 819 2421; fax: þ81 95 819 2420. E-mail address: [email protected] (H. Ueda). Peer review under responsibility of Japanese Pharmacological Society.
production (3,7,8). However, ProTa remarkably protects the ischemic brain (9). Detailed analyses revealed that ProTa in vivo inhibits not only necrosis, but also apoptosis by a help of endogenous brain-derived neurotrophic factor or erythropoietin (2,3,10). Similar mechanisms of necrosis inhibition and neurotrophic factormediated apoptosis inhibition were also observed in retinal ischemiaereperfusion model (11). Furthermore, in vivo protective roles of ProTa were evidenced in experiments using treatments with neutralizing ProTa antibody or ProTa antisense oligonucleotide (2,11). In cerebral ischemia or stroke, the blockade of neuronal death would be the most important endpoint for medicinal treatments. However, neuroprotection alone is not enough for successful treatments, since neuroinflammation due to microvascular occlusion may persistently causes neuronal damages (12). As systemic administration of ProTa remarkably protects the ischemic brain for longer periods (9), but neuroprotective function alone could not explain the long-lasting protective actions against cerebral ischemia by ProTa, we hypothesize that ProTa might have additional beneficial effects against cerebral ischemia. In the present study, we attempted to examine the effects of ProTa on vascular damages by cerebral ischemia. Male C57BL/6J 6-week-old mice weighing 20e24 g were purchased from TEXAM Corporation (Nagasaki, Japan) and used for all experiments. Mice were kept in a room maintained at temperature of 21 ± 2 C and relative humidity of 55 ± 5% with a 12eh light/dark
http://dx.doi.org/10.1016/j.jphs.2016.05.006 1347-8613/© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Maeda et al. / Journal of Pharmacological Sciences 132 (2016) 100e104
cycle. Mice had free access to standard laboratory diet and tap water. All experiments were approved by the Nagasaki University Animal Care Committee (Nagasaki, Japan). Mouse recombinant ProTa (rmProTa), V8 serine protease (V8)digested rmProTa and V8 were diluted in phosphate-buffered saline (PBS). Mice were intravenously (i.v.) treated with rmProTa (0.03, 0.1, or 0.3 mg/kg), V8-digested rmProTa (0.1 or 0.3 mg/kg), V8 (2.5 or 7.5 mg/kg) or PBS (vehicle) 1, 2, 3, or 4 h after the start of cerebral ischemia induced by transient middle cerebral artery occlusion (1 h tMCAO). The details of tMCAO, ProTa preparation and its proteolysis by V8 protease, western blotting, tissue preparation, histological analysis, labeling of blood vessels with Lycopersicon esculentum (tomato) lectin and cluster of differentiation 31 (CD31), detection of endogenous immunoglobulin G (IgG) leakage, Evans blue extravasation, and behavioral assessments were provided in Supplementary materials and methods. Statistical analyses were performed using TukeyeKramer multiple comparison post-hoc analysis after a one-way analysis of variance (ANOVA). Survival ratio was analyzed by Log-rank test after KaplaneMeier survival plots using GraphPad Prism 6 software. Results are shown as means ± standard error of the mean (S.E.M). p < 0.05 was considered significant. As shown in Supplementary Fig. 1, rmProTa was prepared by sequential methodological steps including PCR amplification of tobacco etch virus (TEV)-ProTa, insertion of TEV-ProTa into the pGEX4T1 expression vector, transformation of pGEX4T1-TEV-ProTa into BL21 Escherichia coli (E. coli), lysis of BL21 E. coli cells, and purification of rmProTa through glutathione sepharose column, Mono Q ion-exchange column and Centriprep YM-3 filter. Following removal of endotoxin contamination by EndTrap affinity system, rmProTa was finally dialyzed against PBS. We obtained 0.9 mg rmProTa in 3 ml of PBS from 4 l of Luria broth culture medium as the initial volume. The endotoxin level in purified rmProTa was 0.09 EU/ml (0.9 EU/kg). When sample fractions at different purification steps were run on sodium dodecyl sulphate (SDS)polyacrylamide gel, a band for rmProTa (12.5 kDa) was detected by staining the SDS-polyacrylamide gel with Coomassie Brilliant Blue (CBB) (Fig. 1A). A band for rmProTa was disappeared in CBB-stained SDS-polyacrylamide gel when rmProTa was digested with V8 (Fig. 1B). The purity of rmProTa was also confirmed by western blotting using antibodies against N- and C-terminal sequences in ProTa (Fig. 1C and 1D, respectively). To determine the better condition for rmProTa administration against cerebral ischemia, mice were treated with rmProTa (0.3 mg/ kg, i.v.) 1, 2, 3, or 4 h after the start of tMCAO. Vehicle (PBS)-treated mice died within 7 days after tMCAO (Fig. 2A). When rmProTa was given 2 h after the start of tMCAO, all mice survived (significant, p < 0.01, Log-rank test) for more than 14 days (Fig. 2A). Significant effects in survival were also obtained by rmProTa administration at 1 h (significant, p < 0.01), but not at 3 or 4 h (not significant, p > 0.05) after the start of tMCAO (Fig. 2A). Previous studies reported that tMCAO-type cerebral ischemia caused motor dysfunction, which was evaluated by increase in neurological scores (13,14). We also observed that neurological scores were significantly increased 4 and 7 days after the start of tMCAO, whereas administration of rmProTa 2 h after the start of tMCAO markedly decreased neurological scores 4 and 7 days after tMCAO (Fig. 2B). Neurological scores were also significantly decreased 7 days after tMCAO when mice were treated with rmProTa 1 h, but not 3 or 4 h after the start of tMCAO (Fig. 2B). ProTa-caused increase in survival ratio and decrease in neurological scores were abolished in mice treated with V8-digested rmProTa (0.3 mg/kg, i.v.) 2 h after the start of tMCAO (Fig. 2C and 2D, respectively). To examine the dose-dependent protective activity of rmProTa against cerebral ischemia, mice were treated (i.v.) with 0.03, 0.1, or
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0.3 mg/kg rmProTa 2 h after the start of tMCAO. Administration of rmProTa at a dose of 0.3 mg/kg markedly reversed tMCAO-induced increase in neurological scores 4 and 7 days after tMCAO (Fig. 2E). Neurological scores were also significantly decreased 7 days after tMCAO when mice were treated with 0.1 mg/kg rmProTa, but not with 0.03 mg/kg (Fig. 2E). Administration of V8-digested rmProTa (0.3 mg/kg, i.v.) abolished ProTa-caused decrease in neurological scores 4 and 7 days after tMCAO (Fig. 2E). However, V8 alone did not show any effects against tMCAO-induced decrease in survival ratio (Fig. 2C) and increase in neurological scores (Fig. 2D, E). To confirm whether rmProTa treatment 2 h after the start of tMCAO protects the brain from ischemic damages, histological analysis using Nissl staining of brain sections was performed 24 h after tMCAO. We observed that the ischemic core was clearly demarcated from the surrounding brain region (penumbra), and Nissl-stained neuronal cells were shrunken in the regions of ipsilateral striatum and somatosensory cortex 24 h after tMCAO in vehicle (PBS)-treated tMCAO mice (Supplementary Fig. 2A), consistent with previous studies with brain ischemia (15,16). In contrast, administration (i.v.) of 0.3 mg/kg rmProTa 2 h after the start of tMCAO partially, but significantly reduced infarct volume from 49.9 ± 2.2 to 35.6 ± 4.8% (n ¼ 3) and inhibited shrunken of neuronal cells 24 h after tMCAO, as shown in Supplementary Fig. 2A and 2B. Next, we examined whether ProTa has protective actions against ischemia-induced vascular damages. Mice were treated (i.v.) with rmProTa (0.1 mg/kg), or V8-digested rmProTa (0.1 mg/kg) 2 h after the start of tMCAO, and coronal brain sections (þ0.98 mm from Bregma) were labeled with CD31 and tomato lectin, which are considered as endothelial markers of blood vessels (17,18) as described in Supplementary materials and methods. The damage of blood vessels in terms of disrupted labeling of CD31 and tomato lectin was observed in the region of somatosensory cortex 24 h after tMCAO (Fig. 3A and Supplementary Fig. 3A, B). Moreover, the length of blood vessels was significantly decreased 24 h after tMCAO (Fig. 3B). Administration of rmProTa significantly inhibited ischemia-induced damage of blood vessels 24 h after tMCAO (Fig. 3A, B and Supplementary Fig. 3A, B). ProTa-caused inhibition of cerebral vascular damages was abolished when mice were treated with V8-digested rmProTa 2 h after the start of tMCAO (Fig. 3A, B and Supplementary Fig. 3A, B). Several recent studies reported that cerebral ischemia caused vascular disruption/permeability in the evaluation of endogenous immunoglobulin G (IgG) or Evans blue leakage in the brain of mice and rats (19e23). Based on previous reports and our present findings using CD31 and tomato lectin labeling, we further conducted quantitative analysis of endogenous IgG and Evans blue leakage following tMCAO to explore whether ProTa blocks cerebral ischemia-induced vascular disruption/permeability. Mice were treated (i.v.) with rmProTa (0.1 mg/kg), V8-digested rmProTa (0.1 mg/kg), or V8 alone 2 h after the start of tMCAO, and endogenous IgG or Evans blue leakage was assessed 24 h after tMCAO. Immunostaining revealed that IgG was markedly leaked after tMCAO, whereas administration of rmProTa significantly reduced the leakage of IgG (Fig. 3C, D). ProTa-caused inhibition of IgG leakage following tMCAO was abolished when mice were treated with V8-digested rmProTa (Fig. 3C, D). Similarly, administration of rmProTa significantly reduced tMCAO-induced leakage of Evans blue 24 h after tMCAO (Fig. 3C, E). Administration of V8-digested rmProTa abolished ProTa-caused reduction of Evans blue leakage 24 h after tMCAO (Fig. 3C, E). However, V8 alone had no effects on tMCAO-induced leakage of IgG or Evans blue 24 h after tMCAO (Fig. 3CeE). Recombinant mouse ProTa (rmProTa) at 0.3 mg/kg (i.v.) showed potent survival effects in mice with tMCAO (1 h)-type cerebral
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Fig. 1. Preparation of recombinant mouse ProTa (rmProTa). (A) SDS-polyacrylamide gel electrophoresis analysis of the rmProTa purity at different purification steps. Samples were run on a 15% polyacrylamide gel and subsequently stained with CBB. M, protein marker; lane 1, supernatants of disrupted BL21 E. coli by Nonidet-P40 lysis buffer and sonication; lane 2, the flow-through of sample 1 through glutathione sepharose column; lane 3, the washed fraction of sample 2; lane 4, the eluates by glutathione elution buffer; lane 5, TEV protease digests of sample 4; lane 6, the flow-through of sample 5 with glutathione sepharose column; lane 7, rmProTa purified with a Mono Q column purified of sample 6; lane 8, the concentrated solution of sample 7 with a Centriprep YM-3; lane 9, the flow-through of sample in the Centriprep YM-3; lane 10, treatment of sample 8 with EndTrap blue kit to remove endotoxin; lane 11, dialysis of sample 10 with PBS. (B) SDS-polyacrylamide gel was loaded with rmProTa (1 mg/lane) and V8-digested rmProTa (1 mg/lane). The band of rmProTa (12.5 kDa) was detected by CBB staining of SDS-polyacrylamide gel and using a protein marker (M). ProTa band was not observed when rmProTa was digested with V8. (C, D) For western blotting, SDS-polyacrylamide gel was loaded with rmProTa (1 mg/lane) and V8-digested rmProTa (1 mg/lane). Detection of rmProTa was performed using antibodies against N-terminus ProTa (C) and C-terminus ProTa (D).
ischemia, as previously reported using rat model (9). Most beneficial administration schedule of rmProTa was 2 h after the start of cerebral ischemia, and all mice survived for more than 14 days. Similar results of critical periods for rmProTa effects were also observed in the evaluation of neurological scores. Moreover, histological analysis using Nissl staining confirmed that rmProTa administration 2 h after the start of tMCAO significantly reduced infarct volume and inhibited neuronal damage. We previously reported that significant amount of Myc-tagged recombinant rat
ProTa (i.v.) was observed only in ischemic hemisphere in the brain (9), suggesting the efficient brain penetration of ProTa requires ischemia-induced bloodebrain barrier damage. This view may explain the fact that rmProTa treatment at 1 h after the start of ischemia shows weaker effects than that at 2 h. The fact that rmProTa-induced complete survival effects after ischemic stroke suggests that ProTa may have additional beneficial effects against cerebral vascular damages in addition to direct survival effects against neuronal cell death (1,3,9), since blood
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Fig. 2. Therapeutic period of ProTa treatment against cerebral ischemia. (A, B) Recombinant mouse ProTa (rmProTa) was administered (i.v.) 1, 2, 3, or 4 h after the start of cerebral ischemia (tMCAO) in mice. Vehicle (Veh) represents PBS injection (i.v.) 1 h after the start of tMCAO. Evaluation of survival ratio (A) and neurological scores (B) after tMCAO. n ¼ 8. (C, D) PBS (vehicle), rmProTa, V8-digested rmProTa, or V8 alone was injected (i.v.) 2 h after the start of tMCAO. Evaluation of survival ratio (C) and neurological scores (D) following tMCAO. n ¼ 8. (E) Following PBS (vehicle), rmProTa, V8-digested rmProTa, or V8 administration (i.v.) 2 h after the start of tMCAO, the neurological scores were assessed 4 and 7 days after tMCAO. n ¼ 8. Data are means ± S.E.M. *, #p < 0.05, **, ##p < 0.01.
Fig. 3. ProTa blocks cerebral ischemia-induced vascular damages. (A, B) Mice were treated (i.v.) with PBS (vehicle), rmProTa (0.1 mg/kg) or V8-digested rmProTa (0.1 mg/kg) 2 h the start of after cerebral ischemia (1 h tMCAO). (A) Representative merged images showing somatosensory cortex 1 (S1) in the brain (þ0.98 mm from Bregma) labeled with CD31 and tomato lectin 24 h after tMCAO (CD31, red; tomato lectin, green). (B) Quantitative analysis of the length of blood vessels with merged images of tomato lectin and CD31 at 24 h after tMCAO. Data were obtained from 3 independent mice for each group. (CeE) Mice were treated (i.v.) with PBS (vehicle), rmProTa (0.1 mg/kg), V8-digested rmProTa (0.1 mg/kg) or V8 alone 2 h after the start of cerebral ischemia (1 h tMCAO). (C) Representative images showing detection of endogenous IgG leakage (left panels) and Evans blue leakage (right panels) 24 h after tMCAO. (C) Quantitative analysis of endogenous IgG leakage 24 h after tMCAO. n ¼ 3. (D) Quantitative analysis of Evans blue leakage 24 h after tMCAO. n ¼ 5. Data are means ± S.E.M. *, #, yp < 0.05.
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vessel damages evaluated by CD31- and tomato lectin-labeling were still observed 24 h after tMCAO. Surprisingly, systemic administration of rmProTa at 0.1 mg/kg 2 h after the start of tMCAO caused a potent inhibition of blood vessel damages 24 h after tMCAO. The beneficial effects of rmProTa were also observed in the evaluation of IgG or Evans blue leakage after tMCAO. Although rmProTa-caused neuronal protection was partial in histological assessment, strong vascular protection against tMCAO was obtained by rmProTa treatment. In the present study, IgG leakage indicates the total vascular damages till 24 h after tMCAO, while Evans blue leakage does the weakness of blood brain barrier during the period between 22 and 24 h after tMCAO. Therefore, these results suggest that rmProTa inhibits unidentified molecular mechanisms causing at least 24-h long vascular damages as well as neuroprotective effects at early phase after ischemia. Further studies to examine the direct effects of rmProTa on cultured endothelial cells, and brain and peripheral inflammatory cells, would clarify the molecular mechanisms of rmProTa-induced beneficial effects against stroke. Conflict of interest The authors indicated no potential conflicts of interest. Acknowledgments This work was partially supported by the Platform project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Agency for Medical Research and Development (AMED), and from Grants-in-Aid for Scientific Research on Challenging Exploratory Research (to H. U.: 25670061) from MEXT. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jphs.2016.05.006. References (1) Ueda H, Fujita R, Yoshida A, Matsunaga H, Ueda M. Identification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in cortical neuronal cultures. J Cell Biol. 2007;176:853e862. (2) Ueda H, Matsunaga H, Uchida H, Ueda M. Prothymosin alpha as robustness molecule against ischemic stress to brain and retina. Ann N Y Acad Sci. 2010;1194:20e26. (3) Ueda H. Prothymosin alpha and cell death mode switch, a novel target for the prevention of cerebral ischemia-induced damage. Pharmacol Ther. 2009;123: 323e333.
(4) Matsunaga H, Ueda H. Stress-induced non-vesicular release of prothymosinalpha initiated by an interaction with S100A13, and its blockade by caspase-3 cleavage. Cell Death Differ. 2010;17:1760e1772. (5) Halder SK, Matsunaga H, Ueda H. Neuron-specific non-classical release of prothymosin alpha: a novel neuroprotective damage-associated molecular patterns. J Neurochem. 2012;123:262e275. (6) Ueda H. Prothymosin alpha plays a key role in cell death mode-switch, a new concept for neuroprotective mechanisms in stroke. Naunyn Schmiedebergs Arch Pharmacol. 2008;377:315e323. (7) Chaitanya GV, Steven AJ, Babu PP. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal. 2010;8:31. (8) Fatokun AA, Dawson VL, Dawson TM. Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities. Br J Pharmacol. 2014;171: 2000e2016. (9) Fujita R, Ueda H. Prothymosin-alpha1 prevents necrosis and apoptosis following stroke. Cell Death Differ. 2007;14:1839e1842. (10) Sasaki Y, Fukushima N, Yoshida A, Ueda H. Low-density induced apoptosis of cortical neurons is inhibited by serum factors. Cell Mol Neurobiol. 1998;18: 487e496. (11) Fujita R, Ueda M, Fujiwara K, Ueda H. Prothymosin-alpha plays a defensive role in retinal ischemia through necrosis and apoptosis inhibition. Cell Death Differ. 2009;16:349e358. (12) Deddens LH, Van Tilborg GA, Mulder WJ, De Vries HE, Dijkhuizen RM. Imaging neuroinflammation after stroke: current status of cellular and molecular MRI strategies. Cerebrovasc Dis. 2012;33:392e402. (13) Wang J, Dotson AL, Murphy SJ, Offner H, Saugstad JA. Adoptive transfer of immune subsets prior to MCAO does not exacerbate stroke outcome in splenectomized mice. J Syst Integr Neurosci. 2015;1:20e28. (14) Ma Y, Li Y, Zhang C, Zhou X, Wu Y. Neuroprotective effect of 4methylcyclopentadecanone on focal cerebral ischemia/reperfusion injury in rats. J Pharmacol Sci. 2014;125:320e328. (15) Li H, Zhang N, Lin HY, Yu Y, Cai QY, Ma L, et al. Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice. BMC Neurosci. 2014;15:58. (16) Tajiri S, Oyadomari S, Yano S, Morioka M, Gotoh T, Hamada JI, et al. Ischemiainduced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ. 2004;11:403e415. (17) Li P, Mao L, Liu X, Gan Y, Zheng J, Thomson AW, et al. Essential role of program death 1-ligand 1 in regulatory T-cell-afforded protection against blood-brain barrier damage after stroke. Stroke. 2014;45:857e864. (18) Mazzetti S, Frigerio S, Gelati M, Salmaggi A, Vitellaro-Zuccarello L. Lycopersicon esculentum lectin: an effective and versatile endothelial marker of normal and tumoral blood vessels in the central nervous system. Eur J Histochem. 2004;48:423e428. (19) Huang J, Li Y, Tang Y, Tang G, Yang GY, Wang Y. CXCR4 antagonist AMD3100 protects blood-brain barrier integrity and reduces inflammatory response after focal ischemia in mice. Stroke. 2013;44:190e197. (20) Liu Y, Tang G, Li Y, Wang Y, Chen X, Gu X, et al. Metformin attenuates bloodbrain barrier disruption in mice following middle cerebral artery occlusion. J Neuroinflammation. 2014;11:177. (21) Mao XW, Pan CS, Huang P, Liu YY, Wang CS, Yan L, et al. Levo-tetrahydropalmatine attenuates mouse bloodebrain barrier injury induced by focal cerebral ischemia and reperfusion: involvement of Src kinase. Sci Rep. 2015;5: 11155. (22) Fernandez-Lopez D, Faustino J, Daneman R, Zhou L, Lee SY, Derugin N, et al. Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat. J Neurosci. 2012;32:9588e9600. (23) Hou ST, Nilchi L, Li X, Gangaraju S, Jiang SX, Aylsworth A, et al. Semaphorin3A elevates vascular permeability and contributes to cerebral ischemia-induced brain damage. Sci Rep. 2015;5:7890.
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short communication
Neuroprotective DAMPs member prothymosin alpha has additional beneficial actions against cerebral ischemia-induced vascular damages Shiori Maeda a, Keita Sasaki a, Sebok Kumar Halder a, Wakako Fujita b, Hiroshi Ueda a, * a b
Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan Department of Frontier Life Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2016 Received in revised form 25 April 2016 Accepted 18 May 2016 Available online 27 May 2016
Prothymosin alpha (ProTa) suppresses stress-induced necrosis of cultured cortical neurons. As neuroprotection alone could not explain the long-lasting protective actions against cerebral ischemia by ProTa, we further examined whether ProTa, in addition to neuroprotective effects, has other anti-ischemic activities. When recombinant mouse ProTa (rmProTa) at 0.3 mg/kg was intravenously (i.v.) given 2 h after the start of tMCAO, all mice survived for more than 14 days. In evaluation of CD31- and tomato lectin-labeling as well as IgG and Evans blue leakage, rmProTa treatment (0.1 mg/kg) largely blocked ischemia-induced vascular damages. Therefore, rmProTa has novel beneficial effects against ischemiainduced brain damage through vascular mechanisms. © 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Cerebral ischemia Prothymosin alpha Vascular and neuronal protection
Nuclear protein prothymosin alpha (ProTa) is extracellularly released from neuronal cells upon ischemic or starving stress and protects neurons in an autocrinic or paracrinic manner (1e4). Accordingly, ProTa is supposed to possess a nature of damageassociated molecular patterns (DAMPs), but it has robustness actions to keep the cellular survival against different aspects of stress (3e5). ProTa has been identified as an anti-necrosis factor from the conditioned medium of serum- and supplement-free primary culture of rat embryonic cortex (1,3,6). In the culture of cortical neurons, one of underlying molecular mechanisms was evidenced by ProTa-mediated externalization of glucose transporter 1 and 4, which had been endocytosed by ischemic or starving stress (1e3). This mechanism prevents the necrotic energy crisis due to cellular ATP loss (1,2). Additional anti-necrotic mechanism by ProTa is related to the activation of caspase 3, which cleaves poly (ADPribose) polymerase, resulting in the suppression of necrotic cell death by inhibiting a rapid decrease in mitochondrial ATP
Abbreviations: CBB, Coomassie Brilliant Blue; IgG, immunoglobulin G; i.p., intraperitoneal; i.v., intravenous; ProTa, prothymosin alpha; TEV, tobacco etch virus; tMCAO, transient middle cerebral artery occlusion. * Corresponding author. Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyomachi, Nagasaki 852-8521, Japan. Tel.: þ81 95 819 2421; fax: þ81 95 819 2420. E-mail address: [email protected] (H. Ueda). Peer review under responsibility of Japanese Pharmacological Society.
production (3,7,8). However, ProTa remarkably protects the ischemic brain (9). Detailed analyses revealed that ProTa in vivo inhibits not only necrosis, but also apoptosis by a help of endogenous brain-derived neurotrophic factor or erythropoietin (2,3,10). Similar mechanisms of necrosis inhibition and neurotrophic factormediated apoptosis inhibition were also observed in retinal ischemiaereperfusion model (11). Furthermore, in vivo protective roles of ProTa were evidenced in experiments using treatments with neutralizing ProTa antibody or ProTa antisense oligonucleotide (2,11). In cerebral ischemia or stroke, the blockade of neuronal death would be the most important endpoint for medicinal treatments. However, neuroprotection alone is not enough for successful treatments, since neuroinflammation due to microvascular occlusion may persistently causes neuronal damages (12). As systemic administration of ProTa remarkably protects the ischemic brain for longer periods (9), but neuroprotective function alone could not explain the long-lasting protective actions against cerebral ischemia by ProTa, we hypothesize that ProTa might have additional beneficial effects against cerebral ischemia. In the present study, we attempted to examine the effects of ProTa on vascular damages by cerebral ischemia. Male C57BL/6J 6-week-old mice weighing 20e24 g were purchased from TEXAM Corporation (Nagasaki, Japan) and used for all experiments. Mice were kept in a room maintained at temperature of 21 ± 2 C and relative humidity of 55 ± 5% with a 12eh light/dark
http://dx.doi.org/10.1016/j.jphs.2016.05.006 1347-8613/© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Maeda et al. / Journal of Pharmacological Sciences 132 (2016) 100e104
cycle. Mice had free access to standard laboratory diet and tap water. All experiments were approved by the Nagasaki University Animal Care Committee (Nagasaki, Japan). Mouse recombinant ProTa (rmProTa), V8 serine protease (V8)digested rmProTa and V8 were diluted in phosphate-buffered saline (PBS). Mice were intravenously (i.v.) treated with rmProTa (0.03, 0.1, or 0.3 mg/kg), V8-digested rmProTa (0.1 or 0.3 mg/kg), V8 (2.5 or 7.5 mg/kg) or PBS (vehicle) 1, 2, 3, or 4 h after the start of cerebral ischemia induced by transient middle cerebral artery occlusion (1 h tMCAO). The details of tMCAO, ProTa preparation and its proteolysis by V8 protease, western blotting, tissue preparation, histological analysis, labeling of blood vessels with Lycopersicon esculentum (tomato) lectin and cluster of differentiation 31 (CD31), detection of endogenous immunoglobulin G (IgG) leakage, Evans blue extravasation, and behavioral assessments were provided in Supplementary materials and methods. Statistical analyses were performed using TukeyeKramer multiple comparison post-hoc analysis after a one-way analysis of variance (ANOVA). Survival ratio was analyzed by Log-rank test after KaplaneMeier survival plots using GraphPad Prism 6 software. Results are shown as means ± standard error of the mean (S.E.M). p < 0.05 was considered significant. As shown in Supplementary Fig. 1, rmProTa was prepared by sequential methodological steps including PCR amplification of tobacco etch virus (TEV)-ProTa, insertion of TEV-ProTa into the pGEX4T1 expression vector, transformation of pGEX4T1-TEV-ProTa into BL21 Escherichia coli (E. coli), lysis of BL21 E. coli cells, and purification of rmProTa through glutathione sepharose column, Mono Q ion-exchange column and Centriprep YM-3 filter. Following removal of endotoxin contamination by EndTrap affinity system, rmProTa was finally dialyzed against PBS. We obtained 0.9 mg rmProTa in 3 ml of PBS from 4 l of Luria broth culture medium as the initial volume. The endotoxin level in purified rmProTa was 0.09 EU/ml (0.9 EU/kg). When sample fractions at different purification steps were run on sodium dodecyl sulphate (SDS)polyacrylamide gel, a band for rmProTa (12.5 kDa) was detected by staining the SDS-polyacrylamide gel with Coomassie Brilliant Blue (CBB) (Fig. 1A). A band for rmProTa was disappeared in CBB-stained SDS-polyacrylamide gel when rmProTa was digested with V8 (Fig. 1B). The purity of rmProTa was also confirmed by western blotting using antibodies against N- and C-terminal sequences in ProTa (Fig. 1C and 1D, respectively). To determine the better condition for rmProTa administration against cerebral ischemia, mice were treated with rmProTa (0.3 mg/ kg, i.v.) 1, 2, 3, or 4 h after the start of tMCAO. Vehicle (PBS)-treated mice died within 7 days after tMCAO (Fig. 2A). When rmProTa was given 2 h after the start of tMCAO, all mice survived (significant, p < 0.01, Log-rank test) for more than 14 days (Fig. 2A). Significant effects in survival were also obtained by rmProTa administration at 1 h (significant, p < 0.01), but not at 3 or 4 h (not significant, p > 0.05) after the start of tMCAO (Fig. 2A). Previous studies reported that tMCAO-type cerebral ischemia caused motor dysfunction, which was evaluated by increase in neurological scores (13,14). We also observed that neurological scores were significantly increased 4 and 7 days after the start of tMCAO, whereas administration of rmProTa 2 h after the start of tMCAO markedly decreased neurological scores 4 and 7 days after tMCAO (Fig. 2B). Neurological scores were also significantly decreased 7 days after tMCAO when mice were treated with rmProTa 1 h, but not 3 or 4 h after the start of tMCAO (Fig. 2B). ProTa-caused increase in survival ratio and decrease in neurological scores were abolished in mice treated with V8-digested rmProTa (0.3 mg/kg, i.v.) 2 h after the start of tMCAO (Fig. 2C and 2D, respectively). To examine the dose-dependent protective activity of rmProTa against cerebral ischemia, mice were treated (i.v.) with 0.03, 0.1, or
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0.3 mg/kg rmProTa 2 h after the start of tMCAO. Administration of rmProTa at a dose of 0.3 mg/kg markedly reversed tMCAO-induced increase in neurological scores 4 and 7 days after tMCAO (Fig. 2E). Neurological scores were also significantly decreased 7 days after tMCAO when mice were treated with 0.1 mg/kg rmProTa, but not with 0.03 mg/kg (Fig. 2E). Administration of V8-digested rmProTa (0.3 mg/kg, i.v.) abolished ProTa-caused decrease in neurological scores 4 and 7 days after tMCAO (Fig. 2E). However, V8 alone did not show any effects against tMCAO-induced decrease in survival ratio (Fig. 2C) and increase in neurological scores (Fig. 2D, E). To confirm whether rmProTa treatment 2 h after the start of tMCAO protects the brain from ischemic damages, histological analysis using Nissl staining of brain sections was performed 24 h after tMCAO. We observed that the ischemic core was clearly demarcated from the surrounding brain region (penumbra), and Nissl-stained neuronal cells were shrunken in the regions of ipsilateral striatum and somatosensory cortex 24 h after tMCAO in vehicle (PBS)-treated tMCAO mice (Supplementary Fig. 2A), consistent with previous studies with brain ischemia (15,16). In contrast, administration (i.v.) of 0.3 mg/kg rmProTa 2 h after the start of tMCAO partially, but significantly reduced infarct volume from 49.9 ± 2.2 to 35.6 ± 4.8% (n ¼ 3) and inhibited shrunken of neuronal cells 24 h after tMCAO, as shown in Supplementary Fig. 2A and 2B. Next, we examined whether ProTa has protective actions against ischemia-induced vascular damages. Mice were treated (i.v.) with rmProTa (0.1 mg/kg), or V8-digested rmProTa (0.1 mg/kg) 2 h after the start of tMCAO, and coronal brain sections (þ0.98 mm from Bregma) were labeled with CD31 and tomato lectin, which are considered as endothelial markers of blood vessels (17,18) as described in Supplementary materials and methods. The damage of blood vessels in terms of disrupted labeling of CD31 and tomato lectin was observed in the region of somatosensory cortex 24 h after tMCAO (Fig. 3A and Supplementary Fig. 3A, B). Moreover, the length of blood vessels was significantly decreased 24 h after tMCAO (Fig. 3B). Administration of rmProTa significantly inhibited ischemia-induced damage of blood vessels 24 h after tMCAO (Fig. 3A, B and Supplementary Fig. 3A, B). ProTa-caused inhibition of cerebral vascular damages was abolished when mice were treated with V8-digested rmProTa 2 h after the start of tMCAO (Fig. 3A, B and Supplementary Fig. 3A, B). Several recent studies reported that cerebral ischemia caused vascular disruption/permeability in the evaluation of endogenous immunoglobulin G (IgG) or Evans blue leakage in the brain of mice and rats (19e23). Based on previous reports and our present findings using CD31 and tomato lectin labeling, we further conducted quantitative analysis of endogenous IgG and Evans blue leakage following tMCAO to explore whether ProTa blocks cerebral ischemia-induced vascular disruption/permeability. Mice were treated (i.v.) with rmProTa (0.1 mg/kg), V8-digested rmProTa (0.1 mg/kg), or V8 alone 2 h after the start of tMCAO, and endogenous IgG or Evans blue leakage was assessed 24 h after tMCAO. Immunostaining revealed that IgG was markedly leaked after tMCAO, whereas administration of rmProTa significantly reduced the leakage of IgG (Fig. 3C, D). ProTa-caused inhibition of IgG leakage following tMCAO was abolished when mice were treated with V8-digested rmProTa (Fig. 3C, D). Similarly, administration of rmProTa significantly reduced tMCAO-induced leakage of Evans blue 24 h after tMCAO (Fig. 3C, E). Administration of V8-digested rmProTa abolished ProTa-caused reduction of Evans blue leakage 24 h after tMCAO (Fig. 3C, E). However, V8 alone had no effects on tMCAO-induced leakage of IgG or Evans blue 24 h after tMCAO (Fig. 3CeE). Recombinant mouse ProTa (rmProTa) at 0.3 mg/kg (i.v.) showed potent survival effects in mice with tMCAO (1 h)-type cerebral
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Fig. 1. Preparation of recombinant mouse ProTa (rmProTa). (A) SDS-polyacrylamide gel electrophoresis analysis of the rmProTa purity at different purification steps. Samples were run on a 15% polyacrylamide gel and subsequently stained with CBB. M, protein marker; lane 1, supernatants of disrupted BL21 E. coli by Nonidet-P40 lysis buffer and sonication; lane 2, the flow-through of sample 1 through glutathione sepharose column; lane 3, the washed fraction of sample 2; lane 4, the eluates by glutathione elution buffer; lane 5, TEV protease digests of sample 4; lane 6, the flow-through of sample 5 with glutathione sepharose column; lane 7, rmProTa purified with a Mono Q column purified of sample 6; lane 8, the concentrated solution of sample 7 with a Centriprep YM-3; lane 9, the flow-through of sample in the Centriprep YM-3; lane 10, treatment of sample 8 with EndTrap blue kit to remove endotoxin; lane 11, dialysis of sample 10 with PBS. (B) SDS-polyacrylamide gel was loaded with rmProTa (1 mg/lane) and V8-digested rmProTa (1 mg/lane). The band of rmProTa (12.5 kDa) was detected by CBB staining of SDS-polyacrylamide gel and using a protein marker (M). ProTa band was not observed when rmProTa was digested with V8. (C, D) For western blotting, SDS-polyacrylamide gel was loaded with rmProTa (1 mg/lane) and V8-digested rmProTa (1 mg/lane). Detection of rmProTa was performed using antibodies against N-terminus ProTa (C) and C-terminus ProTa (D).
ischemia, as previously reported using rat model (9). Most beneficial administration schedule of rmProTa was 2 h after the start of cerebral ischemia, and all mice survived for more than 14 days. Similar results of critical periods for rmProTa effects were also observed in the evaluation of neurological scores. Moreover, histological analysis using Nissl staining confirmed that rmProTa administration 2 h after the start of tMCAO significantly reduced infarct volume and inhibited neuronal damage. We previously reported that significant amount of Myc-tagged recombinant rat
ProTa (i.v.) was observed only in ischemic hemisphere in the brain (9), suggesting the efficient brain penetration of ProTa requires ischemia-induced bloodebrain barrier damage. This view may explain the fact that rmProTa treatment at 1 h after the start of ischemia shows weaker effects than that at 2 h. The fact that rmProTa-induced complete survival effects after ischemic stroke suggests that ProTa may have additional beneficial effects against cerebral vascular damages in addition to direct survival effects against neuronal cell death (1,3,9), since blood
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Fig. 2. Therapeutic period of ProTa treatment against cerebral ischemia. (A, B) Recombinant mouse ProTa (rmProTa) was administered (i.v.) 1, 2, 3, or 4 h after the start of cerebral ischemia (tMCAO) in mice. Vehicle (Veh) represents PBS injection (i.v.) 1 h after the start of tMCAO. Evaluation of survival ratio (A) and neurological scores (B) after tMCAO. n ¼ 8. (C, D) PBS (vehicle), rmProTa, V8-digested rmProTa, or V8 alone was injected (i.v.) 2 h after the start of tMCAO. Evaluation of survival ratio (C) and neurological scores (D) following tMCAO. n ¼ 8. (E) Following PBS (vehicle), rmProTa, V8-digested rmProTa, or V8 administration (i.v.) 2 h after the start of tMCAO, the neurological scores were assessed 4 and 7 days after tMCAO. n ¼ 8. Data are means ± S.E.M. *, #p < 0.05, **, ##p < 0.01.
Fig. 3. ProTa blocks cerebral ischemia-induced vascular damages. (A, B) Mice were treated (i.v.) with PBS (vehicle), rmProTa (0.1 mg/kg) or V8-digested rmProTa (0.1 mg/kg) 2 h the start of after cerebral ischemia (1 h tMCAO). (A) Representative merged images showing somatosensory cortex 1 (S1) in the brain (þ0.98 mm from Bregma) labeled with CD31 and tomato lectin 24 h after tMCAO (CD31, red; tomato lectin, green). (B) Quantitative analysis of the length of blood vessels with merged images of tomato lectin and CD31 at 24 h after tMCAO. Data were obtained from 3 independent mice for each group. (CeE) Mice were treated (i.v.) with PBS (vehicle), rmProTa (0.1 mg/kg), V8-digested rmProTa (0.1 mg/kg) or V8 alone 2 h after the start of cerebral ischemia (1 h tMCAO). (C) Representative images showing detection of endogenous IgG leakage (left panels) and Evans blue leakage (right panels) 24 h after tMCAO. (C) Quantitative analysis of endogenous IgG leakage 24 h after tMCAO. n ¼ 3. (D) Quantitative analysis of Evans blue leakage 24 h after tMCAO. n ¼ 5. Data are means ± S.E.M. *, #, yp < 0.05.
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vessel damages evaluated by CD31- and tomato lectin-labeling were still observed 24 h after tMCAO. Surprisingly, systemic administration of rmProTa at 0.1 mg/kg 2 h after the start of tMCAO caused a potent inhibition of blood vessel damages 24 h after tMCAO. The beneficial effects of rmProTa were also observed in the evaluation of IgG or Evans blue leakage after tMCAO. Although rmProTa-caused neuronal protection was partial in histological assessment, strong vascular protection against tMCAO was obtained by rmProTa treatment. In the present study, IgG leakage indicates the total vascular damages till 24 h after tMCAO, while Evans blue leakage does the weakness of blood brain barrier during the period between 22 and 24 h after tMCAO. Therefore, these results suggest that rmProTa inhibits unidentified molecular mechanisms causing at least 24-h long vascular damages as well as neuroprotective effects at early phase after ischemia. Further studies to examine the direct effects of rmProTa on cultured endothelial cells, and brain and peripheral inflammatory cells, would clarify the molecular mechanisms of rmProTa-induced beneficial effects against stroke. Conflict of interest The authors indicated no potential conflicts of interest. Acknowledgments This work was partially supported by the Platform project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Agency for Medical Research and Development (AMED), and from Grants-in-Aid for Scientific Research on Challenging Exploratory Research (to H. U.: 25670061) from MEXT. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jphs.2016.05.006. References (1) Ueda H, Fujita R, Yoshida A, Matsunaga H, Ueda M. Identification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in cortical neuronal cultures. J Cell Biol. 2007;176:853e862. (2) Ueda H, Matsunaga H, Uchida H, Ueda M. Prothymosin alpha as robustness molecule against ischemic stress to brain and retina. Ann N Y Acad Sci. 2010;1194:20e26. (3) Ueda H. Prothymosin alpha and cell death mode switch, a novel target for the prevention of cerebral ischemia-induced damage. Pharmacol Ther. 2009;123: 323e333.
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