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- Materials and methods
Prothymosin alpha (ProTα), a nuclear protein, is implicated in the inhibition of ischemia-induced necrosis as well as apoptosis in the brain and retina. Although ProTα has multiple biological functions through distinct regions in its sequence, it has remained which region is involved in this neuroprotection. This study reported that the active core peptide sequence P30 (amino acids 49–78) of ProTα exerts its full survival effect in cultured cortical neurons against ischemic stress. Our in vivo study revealed that intravitreous administration of P30 at 24 h after retinal ischemia significantly blocks the ischemia-induced functional damages of retina at day 7. In addition, P30 completely rescued the retinal ischemia-induced ganglion cell damages at day 7 after the ischemic stress, along with partial blockade of the loss of bipolar, amacrine, and photoreceptor cells. On the other hand, intracerebroventricular (3 nmol) or systemic (1 mg/kg; i.v.) injection of P30 at 1 h after cerebral ischemia (1 h tMCAO) significantly blocked the ischemia-induced brain damages and disruption of blood vessels. Systemic P30 delivery (1 mg/kg; i.v.) also significantly ameliorated the ischemic brain caused by photochemically induced thrombosis. Taken together, this study confers a precise demonstration about the novel protective activity of ProTα-derived small peptide P30 against the ischemic damages in vitro and in vivo.
Ischemic damages in the central nervous system including brain and retina are associated with the rapid and severe loss of functional and cellular responses through the mechanisms of necrosis as well as apoptosis by several types of cytotoxic mediators (White et al. 2000; Paolucci et al. 2003; Ueda and Fujita 2004; Feigin 2005; Flynn et al. 2008; Fornage 2009; Dvoriantchikova et al. 2010; Neroev et al. 2010; Sims and Muyderman 2010; Yin et al. 2010; Iadecola and Anrather 2011; Witmer et al. 2011). At the same time, several neuroprotective molecules such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), and erythropoietin (EPO) are produced upon ischemia to play limited attenuation of ischemic damages through the anti-apoptosis mechanisms, without exerting protective activity against necrosis (Siren et al. 2001; Korada et al. 2002; Maiese et al. 2004; Blanco et al. 2008; Fujita et al. 2009; Madinier et al. 2009; Ueda et al. 2010; Bejot et al. 2011).
Prothymosin alpha (ProTα) has been identified in the conditioned medium of serum-free primary culture of cortical neurons, as an anti-necrosis factor (Ueda et al. 2007). In addition, ProTα potently inhibits the ischemia-induced damages in brain and retina (Fujita and Ueda 2007; Fujita et al. 2009; Ueda et al. 2010). It is interesting that ProTα has distinct actions, which are all related to the cell survival (Jiang et al. 2003; Ueda 2008; Mosoian et al. 2010; Ueda et al. 2012). Some studies revealed that different peptide sequences in ProTα are implicated with these survival actions. The peptide sequence in the central domain of ProTα (amino acids; a.a. 32–52) is related to the interaction with Kelch-like ECH-associated protein 1 (Keap1), which play roles in the induction of oxidative stress-protecting genes expression by liberating Nrf2 from the Nrf2-Keap1 inhibitory complex (Karapetian et al. 2005). The N-terminal sequence in ProTα (a.a. 2–29), corresponding to thymosin alpha 1, which has an ability to induce anti-cancer effects (Garaci et al. 2007; Danielli et al. 2012). In addition, thymosin alpha 1 has been approved in 35 countries for the treatment of hepatitis B and C, and as an immune stimulant and adjuvant (Goldstein and Goldstein 2009; Pierluigi et al. 2010). Previous reports suggested that C-terminal region (a.a. 89–109, 99–109 and 100–109) of human ProTα exerts immunoenhancing effects including pro-inflammatory activity through the stimulation of monocytes via toll-like receptor (TLR) signaling, induces dendritic cell maturation and adopts β-sheet conformation (Skopeliti et al. 2009). Most recently, there is a report about the survival activity of the middle part (a.a. 41–83) of human ProTα against mutant huntington-caused cytotoxicity in the cultured cells (Dong et al. 2012). However, it remains to be elucidated which region is responsible for the neuroprotection against ischemia-induced neuronal damages. In this study, we have attempted to see the neuroprotective activity of ProTα-derived small peptide against ischemic damages in vitro and in vivo.
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- Materials and methods
This study demonstrates three major findings. First, active core peptide domain P30 (a.a. 49–78) derived from ProTα retains the original survival activity in cultured neuronal cells against ischemic (serum-free) stress. Second, characterizations of P30 actions reveal that it potently inhibits the ischemia-induced damages in retina and brain. Third, P30 induces protective action against ischemia-induced disruption of cerebral blood vessels.
Several in vitro studies reported about the different sequence-specific functions of ProTα, which is also involved in the mechanisms of cell survival (Jiang et al. 2003; Karapetian et al. 2005; Skopeliti et al. 2007; Ueda et al. 2007; Ueda 2009; Mosoian et al. 2010; Danielli et al. 2012; Dong et al. 2012). On the basis of previous information, we firstly designed in vitro experiments to find out the sequence-specific neuroprotective actions of ProTα using various deletion mutants of GST-ProTα in neuronal cells culture under ischemic stress. The peptides lacking sequence (a.a. 1–29), which belongs to thymosin alpha 1 (a.a. 2–29), sequence (a.a. 1–48), which mostly covers the binding region for Keap1, or C-terminal sequences (a.a. 79–112 and 102–112) completely retained the original survival activity as like ProTα. However, the significant decrease in survival effect was observed by the deficiency of parts of the central core peptide sequence comprised of 30 amino acids in ProTα (a.a. 49–78). Interestingly, this central active core peptide of ProTα referred as P30 (a.a. 49–78) itself exerts full survival action in neuronal cells against ischemia. Retinal ischemia causes the functional and cellular damages in different layers of retina through several destructive cascade of mechanisms, as consequence of visual impairment and blindness (Osborne et al. 2004). Our recent in vivo studies suggested that ProTα potently inhibits this ischemia-induced functional and cellular damages of retina (Fujita et al. 2009; Ueda et al. 2010). To evaluate the in vivo protective effect of P30 against ischemic damages, ischemic retina was post-treated with P30. The findings using H&E staining and ERG study revealed that P30 significantly blocks the retinal ischemia-induced decrease in cells number of different layers and retinal thickness. In addition, immunohistochemical analysis clarified that P30 completely rescues the retinal ischemia-induced ganglion cell damages, along with the partial but significant blockade of the loss of bipolar, amacrine, and photoreceptor cells. Stroke following cerebral ischemia (tMCAO) or photothrombotic brain ischemia causes the neuronal damages, along with adequate disruption of cerebral blood vessels (Beck and Plate 2009; Hofmeijer and van Putten 2012; Krysl et al. 2012). We previously explained the protective role of ProTα against cerebral ischemia-induced brain damages (Fujita and Ueda 2007; Ueda 2009; Ueda et al. 2010). The present findings of TTC staining and neurological assessment suggested that intracerebroventicular (3 nmol, i.c.v.) or systemic (1 mg/kg, i.v.) treatment with P30 at 1 h after cerebral ischemia (1 h tMCAO) significantly blocks ischemia-induced brain damages. Following immunostaining with tomato lectin in P30-treated (1 mg/kg, i.v.) ischemic mice, the complete recovery of ischemia-induced (tMCAO) cerebral blood vessels damages was observed through day 1, a consideration of P30 as a new angiogenic factor. In addition, systemic administration with P30 (1 mg/kg, i.v.) significantly ameliorated the ischemic brain caused by photochemically induced thrombosis (PIT), a representative clinical model of cerebral ischemia.
The present investigations were performed following several routes of the administration of P30. According to the fact that retinal ischemia possesses high reproducibility and quantitation to understand the pathophysiological changes and signaling pathways under ischemic condition (Prasad et al. 2010), we used this ischemic injury as a simple model for screening of survival activity by i.vt. administration of P30. We already reported that i.v. administration with full-length ProTα induces protective effect against retinal ischemia (Fujita et al. 2009). In brain ischemia, we firstly decided to perform i.c.v. administration of P30 to evaluate the improvement of ischemic injury, and successfully confirmed against ischemic brain damages. Our recent studies revealed that myc-tagged ProTα (1 mg/kg) is penetrated to the damaged area of brain at least 3 h after brain ischemia by intraperitoneal (i.p.) administration, and that systemic administration (i.p. and i.v.) of ProTα ameliorates brain ischemia-induced functional and cellular damages (Fujita and Ueda 2007). It is well known that brain ischemic stress disrupts the blood–brain barrier (BBB) (Paul et al. 2001; Fujita and Ueda 2007). Thus, we presume that like ProTα, systemic administrated P30 would penetrate to the damaged brain through the disrupted BBB. Although relationship between route of administration and penetrated amounts of P30 to the brain are not clear, isotope and/or fluorescence labeling might be useful method for the calculation of penetration. In the systemic administration, ProTα and P30 exercise the maximum improvement effect against brain ischemia in 100 μg/kg (equivalent 8.08 nmoles/kg) and 1 mg/kg (equivalent 0.30 μmoles/kg), respectively. This difference of efficacy between ProTα and P30 might be because of the stability of P30 in vivo, though GST-ProTα and GST-P30 (a.a. 49–78) showed similar survival activity in this in vitro study. However, the modification of amino acid and/or mutation in sequence of P30 may provide a better solution to improve the stability and survival activity of P30. This should be the next issue to address.
Cortical neurons in serum-free primary culture rapidly die by necrosis, which is completely inhibited by ProTα (Fujita and Ueda 2003; Ueda et al. 2007). As ProTα also protects the retinal ischemia-induced necrosis and apoptosis through the up-regulation of BDNF and EPO, and this retinal protection is completely abolished by antisense oligodeoxynucleotide or antibody treatment against ProTα (Fujita et al. 2009; Ueda et al. 2010), it should be an interesting next subject to investigate whether the same mechanisms are involved in the P30-induced functional and cellular protection against ischemic damages. Despite of being neuroprotective activity of several proteins, peptides have been detected as a new class of attractable therapeutic molecule owing to their diversity, synthesis, and higher capability to penetrate the challenging targets (Archakov et al. 2003; Watt 2006; Gozes 2007; Patel et al. 2007; Meade et al. 2009). Taken together, this study confers a precise demonstration about the broad-spectrum protective activity of ProTα-derived small peptide P30 against ischemic damages in vitro and in vivo. Thus, it is evident that P30 mimics the in vitro and in vivo neuroprotective actions of ProTα. The sequence homology of P30 domain in ProTα among all species is highly conserved; furthermore, this sequence is completely equal in human, rat, and mouse. From these facts, it is speculated that P30 domain may plays important roles in robustness of ProTα against neuronal damages.
In conclusion, ProTα-derived peptide P30 exerted its survival actions in cultured neurons against ischemic stress. P30 significantly blocked the ischemia-induced functional and cellular damages in retina as well as in brain, along with inhibition of the cerebral blood vessels disruption. Therefore, detailed mechanisms underlying neuroprotection by ProTα-derived small peptide may provide a novel therapeutic approach for the treatment of ischemic damages in the central nervous system.