Peptides 43 (2013) 68–75 Contents lists available at SciVerse ScienceDirect Peptides j ourna l ho me pa g e: www.elsevier.com/locate/peptides Therapeutic benefits of 9-amino acid peptide derived from prothymosin alpha against ischemic damages Sebok Kumar Halder, Junya Sugimoto, Hayato Matsunaga, Hiroshi Ueda ∗ Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan a r t i c l e i n f o Article history: Received 19 January 2013 Received in revised form 27 February 2013 Accepted 27 February 2013 Available online 7 March 2013 Keywords: Blood vessel Ischemia Neuroprotective peptide Prothymosin alpha a b s t r a c t Prothymosin alpha (ProT), a nuclear protein, plays multiple functions including cell survival. Most recently, we demonstrated that the active 30-amino acid peptide sequence/P 30 (amino acids 49–78) in ProT retains its substantial activity in neuroprotection in vitro and in vivo as well as in the inhibition of cerebral blood vessel damages by the ischemic stress in retina and brain. But, it has remained to identify the minimum peptide sequence in ProT that retains neuroprotective activity. The present study using the experiments of alanine scanning suggested that any amino acid in 9-amino acid peptide sequence/P 9 (amino acids 52–60) of P 30 peptide is necessary for its survival activity of cultured rat cortical neurons against the ischemic stress. In the retinal ischemia-perfusion model, intravitreous injection of P 9 24 h after ischemia significantly inhibited the cellular and functional damages at day 7. On the other hand, 2,3,5- triphenyltetrazolium chloride (TTC) staining and electroretinogram assessment showed that systemic delivery with P 9 1 h after the cerebral ischemia (1 h tMCAO) significantly blocks the ischemia-induced brain damages. In addition, systemic P 9 delivery markedly inhibited the cerebral ischemia (tMCAO)- induced disruption of blood vessels in brain. Taken together, the present study provides a therapeutic importance of 9-amino acid peptide sequence against ischemic damages. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Ischemic stress in brain and retina causes common expression of cellular and functional damages, which include diverse injury- related cascades underlying necrosis and apoptosis, along with subsequent production and secretion of different cytotoxic medi- ators [7–10,21,31,34,40,44,51–53]. In addition to the release of cell-damaging mediators, some neuroprotective molecules, such as brain-derived neurotrophic factor, fibroblast growth factor and erythropoietin are simultaneously elevated after the onset of ischemia, and cause limited amelioration of ischemic injury through an inhibition of apoptosis, but not necrosis, a key mecha- nism of cell death [3,4,13,25,28,29,41,44,48]. Hence, it is essential to develop neuroprotective agents that target the mechanism of necrosis under ischemic condition. We previously identified prothymosin alpha (ProT) as a necrosis-inhibitory factor in the conditioned medium of Abbreviations: ERG, electroretinogram; GCL, ganglion cell layer; H&E, hema- toxylin and eosin; INL, inner nuclear layer; IPL, inner plexiform layer; i.v., intravenously; i.vt., intravitreously; ONL, outer nuclear layer; OPL, outer plexiform layer; ProT, prothymosin alpha; tMCAO, transient middle cerebral artery occlu- sion; TTC, 2,3,5-triphenyltetrazolium chloride. ∗ Corresponding author. Tel.: +81 95 819 2421; fax: +81 95 819 2420. E-mail address: ueda@nagasaki-u.ac.jp (H. Ueda). serum-free primary culture of cortical neurons [11,45]. It has been clarified that ProT inhibits ischemia-induced damages in brain and retina through blockade of necrosis and apoptosis [12,13,46,48]. Several studies established a relationship between ProT and cell survival [1,23,27,30,47,49,50], and distinct amino acid sequences in ProT are separately involved in this survival phenomenon [6,24,43]. Among them, the peptide sequence (amino acids 32–52) of the central domain in ProT participates in the cell defensive mechanisms against oxidative stress through an interaction with Nrf2–Keap1 inhibitory complex [18,24,33]. The N- terminal sequence in ProT (amino acids 2–29), corresponding to thymosin alpha 1, shows anti-cancer activity and induces immun- odefensive action against viral infections [5,14,15,36], whereas C-terminal sequence (amino acids 89–109, 99–109 and 100–109) of human ProT is involved in the induction of pro-inflammatory activity through toll-like receptor signaling and dendritic cell matu- ration [42,43]. Recently, the cell survival action of mid part (amino acids 41–83) in human ProT against mutant huntingtin-caused cytotoxicity has been discussed [6]. Most recently, we reported that active core peptide sequence comprised of 30 amino acids (P 30 : amino acids 49–78) in ProT exerts its full survival effect in cultured cortical neurons against the ischemic stress and potently blocks the ischemia-induced cellular and functional damages in brain and retina and reverses the damage of cerebral blood vessels in the in vivo studies using various ischemic models [17]. However, it is interesting to be investigated which peptide sequence with 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.02.022