Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Address correspondence and reprint requests to Dr. Hiroshi Ueda, Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. E-mail: firstname.lastname@example.org
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.
Materials and methods
Male C57/BLJ mice weighing 20–25 g were purchased from Tagawa Experimental Animals (Nagasaki, Japan) and used for all the experiments. Mice were kept in a room maintained at constant temperature (21 ± 2°C) and relative humidity (55 ± 5%) with an automatic 12 h light/dark cycle with free access to standard laboratory diet and tap water. Animal care and all experimental procedures were formally approved by Nagasaki University Animal Care and Use Committee (Animal Experiments Approval Number: 1104190914).
Expression constructs and purification procedures for GST-fusion rat ProTα deletion mutants
The rat ProTα gene was amplified from cDNA derived from rat embryonic brain. The gene constructions for expression of recombinant GST-ProTα deletion mutants (Full-length, ∆1-29, ∆1-48, ∆1-68, ∆1-86, ∆30-112, ∆58-112, ∆79-112, and ∆102-112) were previously described (Ueda et al. 2007; Matsunaga and Ueda 2010). Here, we newly made a GST-ProTα-49-78. The amplified genes blunted at their 5′-ends and cloned in-frame into the BamHI (blunted)-EcoRI sites of pGEX-5X-1 (GE Healthcare Bio-Science Corp, Piscataway, NJ, USA). The PCR primers used were as follows: ∆1-48-F, 5′-AGGGATCCAATGGCTGACAATGAGGTAGATG-3′ and ∆79-112-R, 5′-TTGAATTCCTAATCTCCATCTTCTTCCTC-3′. F-primer contains a BamHI site, while all R-primer contains a stop codon and an EcoRI site. The recombinant proteins were purified using Glutathione-Sepharose™ (GE Healthcare Bio-Science Corp).
Identification of functional active core domain in ProTα
To determine the active core domain in ProTα, we measured the survival activity in primary cultured rat cortical neurons. The preparation and culture of cortical neurons were previously described (Ueda et al. 2007). The culture of neurons was started at low density (1 × 105 cells/cm2) under the serum-free conditions in the presence or absence of GST and GST-ProTα deletion mutants (100 nM). After 12 h from the start of culture, survival activity was evaluated by WST-8 reduction activity (Cell Counting Kit-8; DOJINDO, Kumamoto, Japan). Finally, we successfully obtained the functional active core domain comprised of 30 amino acids in ProTα (a.a. 49–78) and referred as P30 according to number of amino acids.
Intravitreal injection was performed using a 33-gauge needle connected to a microsyringe and the needle was inserted approximately 1 mm behind the corneal limbus, guided under a stereoscopic microscope to avoid lens and retinal injury. Peptide P30 was dissolved in 0.05% dimethyl sulfoxide (DMSO), which was diluted with 0.1 M potassium-free phosphate buffered saline (K+-free PBS). P30 was injected intravitreously (i.vt.) in the eye with doses of 1, 3 and 10 pmol/μL at 24 h after retinal ischemia (n = 5, n = 6 and n = 7, respectively). Vehicle was treated with equal volume of 0.05% DMSO in a similar manner. On the other hand, P30 was injected intracerebroventricularly (0.03 and 3 nmol/5 μL, i.c.v.; n = 6 and n = 7, respectively) in the brain at 1 h after cerebral ischemia (1 h tMCAO). P30 (0.3 and 1 mg/kg) was administered intravenously (i.v.) at 1 h after cerebral ischemia. In addition, P30 was delivered (1 mg/kg, i.v.) at 3 and 6 h after the cerebral ischemic stress.
Retinal ischemia was performed following the method as described previously (Fujita et al. 2009). Briefly, mice were anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg) and pupils were fully dilated with 1% atropine sulfate drops (Nitten, Nagoya, Japan). The anterior chamber of the eye was cannulated with a 33-gauge needle attached to an infusion container of sterile intraocular irrigating solution (BSS PLUS dilution buffer; Alcon, Fort Worth, TX, USA). Retinal ischemia was induced by elevating the IOP to generate a hydrostatic pressure of 130 mm Hg for 45 min by lifting the container. Following 45 min after retinal ischemic stress, the needle was withdrawn and 0.3% ofloxacin (Santen Pharmaceutical Co. Ltd., Osaka, Japan) was applied topically into the eye to avoid infection.
Electroretinogram (ERG) study was performed following the protocol as previously described (Fujita et al. 2009). Briefly, mice were dark-adapted for 3–4 h, then anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg) and pupils were dilated with 1% atropine. A contact electrode (KE–S; Kyoto contact lenses, Kyoto, Japan) was placed topically on the corneal apex and reference electrode was placed near the ipsilateral eye. The ground was a subdermal platinum needle electrode near the abdominal area. ERGs were produced by 20 J flash intensities. The flash stimulus source (SLS-3100; Nihon Kohden, Tokyo, Japan) illuminated the eye by diffuse reflection off the interior surface of the ganzfeld. Maximum flash luminance was measured with detector (MEB–9104; Nihon Kohden). After the intensity series, an incandescent background light sufficient to desensitize the rod system was turned on, and ERGs produced by the standard stimulus were recorded every 2 min for 20 min. The background was then turned off, and ERGs were produced by the standard stimulus every 2 min for the first 30 min of dark adaptation. The a- and b-wave amplitudes were measured online (Neuropack m, QP-903B; Nihon Kohden). ERG was performed at day 7 after retinal ischemia.
Middle cerebral artery occlusion model
The transient middle cerebral artery occlusion (tMCAO) model was induced following the method as described previously (Halder et al. 2012). Briefly, mice were anesthetized by 2% isoflurane (Mylan, Tokyo, Japan), and body temperature was monitored and maintained at 37°C during surgery. After a midline neck incision, the middle cerebral artery was occluded transiently using 8-0 in size monofilament nylon surgical suture (Natsume Co. Ltd., Tokyo, Japan) coated with silicon (Xantopren, Bayer dental, Osaka, Japan) that was inserted through the left common carotid artery and advanced into the left internal carotid artery. Following 1 h tMCAO, the animals were briefly re-anesthetized with isoflurane and the monofilament was withdrawn for reperfusion studies. Cerebral blood flow was monitored by laser Doppler flowmeter (ALF21; Advance Co., Tokyo, Japan) using a probe (diameter 0.5 mm) of a laser Doppler flowmeter (ALF2100; Advance Co.) inserted into the left striatum (anterior: −0.5 mm, lateral: 1.8 mm from bregma; depth: 4.2 mm from the skull surface) through a guide cannula.
Photochemically induced thrombosis (PIT) was produced following the protocol as described previously (Nagai et al. 2007). Briefly, anesthesia was induced with 3% isoflurane, and the rectal temperature was maintained at 37°C. The temporal muscle was dissected, the skull was exposed, and a 1.5-mm opening was made over the middle cerebral artery (MCA). Photo-illumination of green light (wavelength: 540 nm) was achieved with a xenon lamp (model L-4887, Hamamatsu Photonics, Hamamatsu, Japan) with heat-absorbing and green filters, via an optic fiber with a focus of 1 mm, placed on the opening in the skull. Rose Bengal (Wako, Osaka, Japan) was injected (3 mg/kg, i.v.) in mice, and photo-illumination (5000 lx) was applied for 10 min, after which the temporal muscle and skin were replaced. The MCA occlusion time (from the start of light exposure until the flow in the MCA is stopped) was monitored by observation in real time under the microscope.
Retinal and brain tissue preparation
For retinal tissue preparation, mice were deeply anaesthetized with sodium pentobarbital (50 mg/kg, i.p.). Eye was quickly isolated, washed with saline and 4% paraformaldehyde (PFA). Eye was then nicked through pupil, post-fixed in 4% PFA for 24 h and finally transferred to 25% sucrose solution (in 0.1 M K+-free PBS) overnight for cryoprotection. Following frozen in cryoembedding compound, retinal sections were prepared at 10 μm thickness. For brain tissue preparation, mice were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 0.1 M PBS, followed by 4% PFA. Brain was then quickly removed, post-fixed in 4% PFA and transferred immediately to 25% sucrose solution overnight. Brain was frozen in cryoembedding compound and coronal sections were cut at 30 μm thickness for immunohistochemical analysis.
Hematoxylin and eosin staining
For hematoxylin and eosin (H&E) staining, frozen retinal sections were washed with 0.1 M K+-free PBS, immerged in Mayer's hematoxylin solution (Wako) for 5 min at 25°C and then washed with tap water for 20 min. Following brief treatment with 95% ethanol, sections were immerged in eosin-alcohol solution (Wako) for 4 min at 25°C. Sections were dehydrated through a series of ethanol solutions, xylene, and over-slipped with Permount (Fisher Scientific, Waltham, MA, USA). Sections were then analyzed using a BZ-8000 microscope with BZ Image Measurement Software (KEYENCE, Osaka, Japan).
To perform fluorescence immunohistochemistry, retinal sections were washed with 0.1 M K+-free PBS and incubated with 50% methanol followed by 100% methanol for 10 min. Following treatment with blocking buffers [bovine serum albumin (BSA) as well as 10% goat serum with 0.1% Triton X-100 in phosphate buffered saline (PBST)], retinal sections were incubated overnight at 4°C with following primary antibodies: anti-NeuN (1 : 100; mouse monoclonal IgG1, clone A60; Chemicon, Temecula, CA, USA); anti-syntaxin-1 (1 : 500; mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA); and anti-Chx10 (1 : 300; sheep polyclonal; Exalpha Biologicals Inc., MA, USA). Sections were then incubated with Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-sheep IgG secondary antibodies (1 : 300; Molecular Probes, Eugene, OR, USA). The nuclei were visualized with Hoechst 33342 (1 : 10 000; Molecular Probes). Samples were then washed thoroughly with PBS and cover-slipped with Perma Fluor (Thermo Shandon, Pittsburgh, PA, USA). Images were collected using a BZ-8000 microscope with BZ Image Measurement Software.
For blood vessels staining, biotinylated Lycopersicon esculentum (tomato) lectin (Vector Laboratories, Burlingame, CA, USA) is diluted with PBS. Biotinylated tomato lectin was injected (1 mg/mL, 100 μL, i.v.) at 24 h after cerebral ischemia (1 h tMCAO). Mice were perfused 5 min after tomato lectin injection. Following tissue preparation as described in the method section, coronal brain section was blocked with 2% BSA in 0.1% PBST for 2 h, and then incubated with Alexa Fluor 488 streptavidin conjugates for 2 h at 25°C. Sections were washed with PBS and cover-slipped with Perma Fluor. Images were collected using an LSM 710 confocal microscope with ZEN Software (Carl Zeiss, Oberkochen, Germany).
For 2,3,5–triphenyltetrazolium chloride (TTC) staining, brain was quickly removed at 24 h after cerebral ischemia (1 h tMCAO) followed by P30 administration (n = 7), sectioned coronally with a 1-mm thickness and washed with K+-free PBS. Brain slices were incubated in 2% TTC (Sigma-Aldrich) in 0.9% NaCl in dark place for 15–20 min at 25°C and transferred in 4% PFA overnight. Images of brain slices were then collected by scanner, and infarct volume was calculated by Image J software (NIH, Bethesda, MD, USA).
Following P30 administration with doses of 0.03 and 3 nmol/5 μL (i.c.v., n = 6 and n = 7, respectively), 0.3 and 1 mg/kg (i.v., n = 5 and n = 7, respectively) at 1 h as well as 1 mg/kg (i.v.) at 3 and 6 h (n = 5 and n = 7, respectively) after cerebral ischemia (1 h tMCAO), behavioral studies were assessed through 14 days. Clinical score was evaluated from day 1 after ischemia in the following way: 0, no observable deficits; 1, failure to extend the forepaw fully; 2, circling; 3, falling to one side; 4, no spontaneous movement; 5, death. In this study, 0.5 point was added to each score when the motor dysfunction was severe for scores between 1 and 4. Survival rate was evaluated from day 1 after tMCAO and calculated by the percentage of vehicle or P30 post-treated mice that were alive through 14 days after ischemia.
All results are shown as means ± SEM. Two independent groups were compared using the Student's t-test. Multiple groups were compared using Dunnett's multiple comparison test after a one-factor anova or a repeated measure anova. Survival rate was compared using Logrank test after Kaplan–Meyer method. p <0.05 was considered significant.
Characterization of functionally active core peptide in ProTα
The functionally active core domain in rat recombinant ProTα was determined by measuring the survival activity of cultured cortical neurons in the presence of different deletion mutants of GST-fusion ProTα at 12 h after the ischemic (serum-free) stress (Fig. 1a–c). The findings revealed that the N-terminal deletion mutants ProTα (∆1–29 and ∆1–48) as well as C-terminal deletion mutants ProTα (∆79–112 and ∆102–112) elicit its protective effect as like as full-length ProTα against ischemic stress-induced cultured neuronal damages (Fig. 1b, c). However, the deletion mutants of ProTα devoid of central peptide sequence comprised of 30 amino acids (P30: a.a. 49–78) abolished its neuroprotective activity against the ischemic stress (Fig. 1b, c). Interestingly, the core peptide sequence P30 (a.a. 49–78) itself exerted the full survival effect in cultured neurons against ischemic damages, an indication of neuroprotective characteristics of ProTα-derived peptide P30 (Fig. 1b, c).
Blockade of retinal ischemia-induced damages by ProTα-derived peptides
We reported previously that ProTα inhibits the retinal ischemia-induced functional and cellular damages (Fujita et al. 2009). To evaluate whether ProTα-derived peptide has protective activity against ischemic damages in vivo, P30 was injected (i.vt.) with doses of 1, 3 and 10 pmol/μL in the ipsilateral eye at 24 h after retinal ischemia. The hematoxylin and eosin (H&E) staining data showed that the number of cells in different retinal layers as well as the retinal thickness is significantly decreased in the vehicle-treated mice at day 7 after the ischemic stress, whereas 10 pmol P30 maximally and significantly inhibited this cellular loss in retina and decrease in retinal thickness at day 7 (Fig. 2a, b).
In electroretinogram (ERG), the amplitude called a-wave represents the functional activity of photoreceptor cells, whereas b-wave indicates the functions of mixture of cells including bipolar, Muller, amacrine, and ganglion cells (Asi and Perlman 1992; Fujita et al. 2009). Following after retinal ischemia and reperfusion, the ERGs analysis showed that a- and b-wave amplitudes are significantly decreased in the vehicle-treated mice at day 7 after retinal ischemia, compared with the control (Fig. 2c, d). Following P30 treatment, dose-dependent increase in a- and b-wave amplitudes were observed at day 7 after the retinal ischemic stress, and 10 pmol P30 exerted its maximum protective effect against the ischemic damages (Fig. 2c, d). On the other hand, no significant protective effect of thymosin alpha 1 (a.a. 2–29) corresponding to N-terminal sequence of ProTα and the C-terminal peptide (a.a. 102–112) against retinal ischemic damages were observed at day 7 after ischemia (data are not shown).
P30-induced cell type-specific survival against retinal ischemic damages
To examine the cell type-specific protective activity of ProTα-derived peptide in ischemic retina, P30 was injected (10 pmol/μL, i.vt.) in the ipsilateral eye at 24 h after retinal ischemia. The immunohistochemical analysis showed that NeuN-positive neurons (Buckingham et al. 2008) in the ganglion cell layer (GCL) are significantly diminished at day 7 after retinal ischemic stress, compared to the control (Fig. 3a). Following P30 treatment at 24 h after retinal ischemia, the complete recovery of NeuN-positive neuronal cells was observed in the GCL of ischemic retina at day 7 after the ischemic stress (Fig. 3a). On the other hand, treatment of P30 partially, but significantly blocked the loss of Chx10-positive bipolar cells (Rhee et al. 2007) in the inner nuclear layer (INL) (Fig. 3b), syntaxin-1-positive amacrine cells (Sherry et al. 2006), of which the cell bodies and processes are located in the INL and inner plexiform layer (IPL), respectively (Fig. 3c), and photo-receptor cells in the outer nuclear layer (ONL) (Fig. 3d), compared with the respective controls and vehicles.
Inhibition of cerebral ischemia-induced brain damages by P30
To evaluate the protective activity of P30 against ischemic brain damages, mice were post-treated with P30 in time- and dose-dependent manner following different routes of administration, and subsequent 2,3,5-triphenyl tetrazolium chloride (TTC) staining at 24 h and behavioral assessments through 14 days were performed after cerebral ischemia (1 h tMCAO). The TTC staining data showed that the infarct volume is significantly decreased at 24 h in the ischemic brain by intracerebroventicular (i.c.v.) injection of 3 nmol P30 at 1 h after tMCAO (Fig. 4a, b), but not by 0.03 nmol (data are not shown). We also observed that the clinical score is significantly decreased at day 1 after 1 h tMCAO in mice injected with 3 nmol P30 (i.c.v.) at 1 h after the ischemic stress (Fig. 4c). In addition, significant decrease in clinical score and increase in survival rate were observed through 14 days after i.c.v. delivery (1 h after ischemia) of 3 nmol P30, an indication of long-lasting protective effect of P30 against ischemic brain damages (Fig. 4d, e).
On the other hand, P30 was injected intravenously (i.v.) with doses of 0.3 and 1 mg/kg at 1 h after cerebral ischemia (1 h tMCAO). Our TTC staining data revealed that the infarct volume is significantly decreased at 24 h in the ischemic brain treated with 1 mg/kg of P30 treatment at 1 h after the ischemic stress (Fig. 4f, g). Following post-treatment (i.v.) with 1 mg/kg of P30 at the same time point, the clinical score was significantly declined through 7 days and survival rate was maximally increased through 14 days after tMCAO, compared with the vehicle and ischemic mice treated with 0.3 mg/kg of P30 (Fig. 4h–j). The behavioral study also confirmed that systemic (i.v.) P30 delivery with the dose of 1 mg/kg at 1 h after ischemia induces it maximum protective effect at day 7 against the ischemic brain damages, compared to P30 treatment at 3 or 6 h after cerebral ischemia (Fig. 4k).
P30 inhibits the cerebral ischemia-induced blood vessel damages
In the ischemic stroke and cerebrovascular disease, vascular defect is occurred along with neuronal damages (Paul et al. 2001; Fujita and Ueda 2007). To investigate whether P30 protects the ischemia-induced blood vessels damages, P30 was injected (1 mg/kg; i.v.) at 1 h after cerebral ischemia (1 h tMCAO). Following blood vessel immunostaining using biotinylated tomato lectin and Alexa Fluor 488 streptavidin at 24 h after ischemia, the findings revealed that the number blood vessels are markedly decreased in somatosensory cortex in the brain of vehicle-treated mice, compared with the control (Fig. 5a, b). In addition, the decrease in lengths of the blood vessels was observed at 24 h after tMCAO (Fig. 5a, c). This ischemia-induced loss of tomato lectin-stained blood vessels in terms of number and lengths was completely recovered in the somatosensory cortex at 24 h after the ischemic stress in mice post-treated with P30, but the lengths were relatively larger than the vessels in the control brain, an indication of the protective role of P30 against ischemia-induced blood vessel damages (Fig. 5a–c). Similar results of the recovery of cerebral ischemia-induced blood vessels damages by P30 were observed in the striatum and hippocampus at 24 h after 1 h tMCAO (data are not shown).
P30 ameliorates the ischemic brain caused by photochemically induced thrombosis
It is well known that ischemic model because of middle cerebral artery (MCA) occlusion with photochemically induced thrombosis (PIT) is analogous to clinical condition (Tanaka et al. 2007). In this ischemic mouse model, there was a significant behavioral damage evaluated by clinical score (Fig. 6a). This damage was significantly attenuated by systemic post-treatment with P30 (1 mg/kg, i.v.) at 1 h after PIT (Fig. 6a). Following behavioral study after PIT stress, neurological assessments using TTC staining were performed at 24 h. The TTC staining data revealed that there was a marked increase in cerebral infarction observed at 24 h after PIT in vehicle-treated mice (Fig. 6b), but this cerebral brain damage in terms of infarct volume and hemisphere expansion was significantly inhibited by systemic treatment of P30 (Fig. 6c, d).
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 P30in 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.
We thank R. Fujita, J. Sugimoto, and S. Maeda for technical assistance and advice. We also thank M. Moskowitz for the valuable discussion. We acknowledge Parts of this study were supported by Grants-in-Aid for Scientific Research (to H.U.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Health and Labor Sciences Research Grants (to H.U.) on Research from the Ministry of Health, Labor and Welfare. We have no conflict interest to report.