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Tissue plasminogen activator (tPA) administration beyond 4.5 h of stroke symptoms is beneficial for patients but has an increased risk of cerebral hemorrhage. Thus, increasing the therapeutic window of tPA is important for stroke recovery. We previously showed that prothymosin alpha (ProTα) or its mimetic hexapeptide (P6Q) has anti‐ischemic activity. Here, we examined the beneficial effects of ProTα or P6Q against delayed tPA‐induced brain damage following middle cerebral artery occlusion (MCAO) or photochemically induced thrombosis in mice. Brain hemorrhage was observed by tPA administration during reperfusion at 4.5 and 6 h after MCAO. Co‐administration of ProTα with tPA at 4.5 h inhibited hemorrhage and motor dysfunction 2–4 days, but not 7 days after MCAO. ProTα administration at 2 and 4.5 h after MCAO significantly inhibited tPA (4.5 h)‐induced motor dysfunction and death more than 7 days. Administration of tPA caused the loss of tight junction proteins, zona occulden‐1 and occludin, and up‐regulation of matrix metalloproteinase‐2/9, in a ProTα‐reversible manner. P6Q administration abolished tPA (4.5 h)‐induced hemorrhage and reversed tPA (6 h)‐induced vascular damage and matrix metalloproteinase‐2 and 9 up‐regulation. Twice administrations of P6Q at 2 h alone and 6 h with tPA significantly improved motor dysfunction more than 7 days. In photochemically induced thrombosis ischemia, similar vascular leakage and neuronal damage (infarction and motor dysfunction) by late tPA (4.5 or 6 h) were also inhibited by P6Q. Thus, these studies suggest that co‐administration with ProTα or P6Q would be beneficial to inhibit delayed tPA‐induced hemorrhagic mechanisms in acute ischemic stroke. [ABSTRACT FROM AUTHOR]

Background: The cell type, cell status and specific localization of Prothymosin a (PTMA) within cells seemingly determine its function. PTMA undergoes 2 types of protease proteolytic modifications that are useful in elucidating its interactions with other molecules; a factor that typifies its roles. Preferably a nuclear protein, PTMA has been shown to function in the cytoplasm and extracellularly with much evidence leaning on pathognomonic status. As such, determination of its cellular distribution under normal physiological context while utilizing varied techniques is key to illuminating prospective validation of its distinct functions in different tissues. Differential distribution insights at normal physiology would also portent better basis for further clarification of its interactions and proteolytic modifications under pathological conditions like numerous cancer, ischemic stroke and immunomodulation. We therefore raised an antibody against the C terminal of PTMA to use in tandem with available antibody against the N terminal in a murine model to explicate the differences in its distribution in brain cell types and major peripheral organs through western blotting and immunohistochemical approaches. Results: The newly generated antibody was applied against the N-terminal antibody to distinguish truncated versions of PTMA or deduce possible masking of the protein by other interacting molecules. Western blot analysis indicated presence of a truncated form of the protein only in the thymus, while immunohistochemical analysis showed that in brain hippocampus the full-length PTMA was stained prominently in the nucleus whereas in the stomach full-length PTMA staining was not observed in the nucleus but in the cytoplasm. Conclusion: Truncated PTMA could not be detected by western blotting when both antibodies were applied in all tissues examined except the thymus. However, immunohistochemistry revealed differential staining by these antibodies suggesting possible masking of epitopes by interacting molecules. The differential localization patterns observed in the context of nucleic versus cytoplasmic presence as well as punctate versus diffuse pattern in tissues and cell types, warrant further investigations as to the forms of PTMA interacting partners. [ABSTRACT FROM AUTHOR]

Prothymosin-alpha protects the brain and retina from ischemic damage. Although prothymosin-alpha contributes to toll-like receptor (TLR4)-mediated immnunopotentiation against viral infection, the beneficial effects of prothymosin-alpha-TLR4 signaling in protecting against ischemia remain to be elucidated. In this study, intravitreal administration of prothymosinalpha 48 h before induction of retinal ischemia prevented retinal cellular damage as evaluated by histology, and retinal functional deficits as evaluated by electroretinography. Prothymosin- alpha preconditioning completely prevented the ischemia- induced loss of ganglion cells with partial survival of bipolar and photoreceptor cells, but not amacrine cells, in immunohistochemistry experiments. Prothymosin-alpha treatment in the absence of ischemia caused mild activation, proliferation, and migration of retinal microglia, whereas the ischemia-induced microglial activation was inhibited by prothymosin-alpha preconditioning. All these preventive effects of prothymosin-alpha preconditioning were abolished in TLR4 knock-out mice and by pre-treatments with anti-TLR4 antibodies or minocycline, a microglial inhibitor. Prothymosinalpha preconditioning inhibited the retinal ischemia-induced up-regulation of TLR4-related injury genes, and increased expression of TLR4-related protective genes. Furthermore, the prothymosin-alpha preconditioning-induced prevention of retinal ischemic damage was abolished in TIR-domain-containing adapter-inducing interferon-p knock-out mice, but not in myeloid differentiation primary response gene 88 knock-out mice. Taken together, the results of this study suggest that prothymosin-alpha preconditioning selectively drives TLR4-TIR-domain-containing adapter-inducing interferon-p signaling and microglia in the prevention of retinal ischemic damage. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Prothymosin alpha ( Pro Tα), a nuclear protein devoid of signal sequence, has been shown to possess a number of cellular functions including cell survival. Most recently, we demonstrated that Pro Tα is localized in the nuclei of neurons, while it is found in both nuclei and cytoplasm in the astrocytes and microglia of adult brain. However, the cell type-specific non-classical release of Pro Tα under cerebral ischemia is yet unknown. In this study, we report that Pro Tα is non-classically released along with S100A13 from neurons in the hippocampus, striatum and somatosensory cortex at 3 h after cerebral ischemia, but amlexanox (an anti-allergic compound) reversibly blocks this neuronal Pro Tα release. We found that none of Pro Tα is released from astrocytes and microglia under ischemic stress. Indeed, Pro Tα intensity is increased gradually in astrocytes and microglia through 24 h after the cerebral ischemia. Interestingly, Z-Val-Ala-Asp fluoromethyl ketone, a caspase 3 inhibitor, pre-treatment induces Pro Tα release from astrocytes in the ischemic brain, but this release is reversibly blocked by amlexanox. However, Z-Val-Ala-Asp fluoromethyl ketone as well as amlexanox has no effect on Pro Tα distribution in microglia upon cerebral ischemia. Taken together, these results suggest that only neurons have machineries to release Pro Tα upon cerebral ischemic stress in vivo. [ABSTRACT FROM AUTHOR]

Prothymosin α (ProTα) possesses multiple functions for cell robustness. This protein functions intracellularly to stimulate cell proliferation and differentiation through epigenetic or genomic mechanisms. ProTα also regulates the cell defensive mechanisms through an interaction with the Nrf2-Keap1 system. Under the apoptotic conditions, it inhibits apoptosome formation by binding to Apaf-1. Regarding extracellular functions, ProTα is extracellularly released from the nucleus upon necrosis-inducing ischemia stress in a manner of nonclassical release, and thereby inhibits necrosis. However, under the condition of apoptosis, the C-terminus of ProTα is cleaved off and loses binding activity to cargo protein S100A13 for nonclassical release. However, cleaved ProTα is retained in the cytosol and inhibits apoptosome formation. ProTα was recently reported to cause immunological actions through the Toll-like receptor 4. However, the authors also suggest the possible existence of additional receptors for robust cell activities against ischemia stress. [ABSTRACT FROM AUTHOR]

Highlights: [•] Neuroprotective action of 9-amino acid peptide/P9 derived from prothymosin alpha. [•] P9-induced amelioration of ischemic damages in retina and brain. [•] Blockade of ischemia-induced cerebral blood vessels disruption by P9. [Copyright &y& Elsevier]