thymosin and Brain-Ischemia

Following stroke or traumatic damage, neuronal death via both necrosis and apoptosis causes loss of functions including memory, sensory perception and motor skills. Since necrosis has the nature to expand, while apoptosis stops the cell death cascade in the brain, necrosis is considered to be a promising target for rapid treatment for stroke. Pure neuronal necrosis occurs when cortical neurons are cultured under serum-free and low-density conditions. Prothymosin alpha (ProTalpha) isolated from conditioned medium after serum-free culture was found to prevent necrosis by recovering the energy crisis due to endocytosed glucose transporters. At a later time point under the same starvation conditions, ProTalpha causes apoptosis, which in turn seems to inhibit the rapidly occurring necrosis by cleaving poly (ADP-ribose) polymerase, a major machinery involved in ATP consumption. Indeed, ProTalpha administered via systemic routes markedly inhibits the histological and functional damage induced by cerebral and retinal ischemia. Although ProTalpha also causes a cell death mode switch from necrosis to apoptosis in vivo, the induced apoptosis was found to be completely inhibited by endogenously occurring brain-derived neurotrophic factor or erythropoietin. Since forced downregulation of ProTalpha deteriorates the ischemic damage, it is evident that ProTalpha plays in vivo neuroprotective roles after ischemic events. Analyses in terms of the therapeutic time window and potency suggest that ProTalpha could be the prototypic compound to develop the medicine useful for treatment of stroke in clinics.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Death; Drug Delivery Systems; Glucose Transport Proteins, Facilitative; Humans; Ischemia; Necrosis; Neurons; Protein Precursors; Retinal Diseases; Thymosin

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.

Topics: Animals; Biomimetic Materials; Brain Injuries; Brain Ischemia; Male; Mice; Mice, Inbred C57BL; Protein Precursors; Thymosin; Tissue Plasminogen Activator

We previously showed that prothymosin alpha (ProTα) improves cerebral ischemia-induced motor dysfunction. Our recent study also demonstrated that heterozygous ProTα deletion exhibited an enhanced anxiety-like behavior in mice. However, it remains elusive which brain regions or cells are related to these phenotypes. Here we generated conditional Gγ7-specific ProTα knockout mice using G protein γ7 subunit gene (Gng7)-cre promoter to see the brain robustness roles of ProTα in the striatum and hippocampus. The younger conditional ProTα (Gng7) knockout mice at the age of 10 weeks showed no significant phenotypes in motor dysfunction in the Rotarod test and locomotor activity in the open-field test, whereas significant motor dysfunction was obtained by 15 min transient middle cerebral artery occlusion (tMCAO)-induced cerebral ischemia. The aged conditional ProTα (Gng7) knockout mice at the age of 20 weeks showed hypolocomotor activity with less center time in the open-field test and impaired motor coordination in the Rotarod test without ischemia. Thus, this study suggests that ProTα has important roles in the maintenance of motor coordination and anxiety-like behavior.

Topics: Aging; Animals; Anxiety; Brain Ischemia; Female; Gene Deletion; GTP-Binding Protein gamma Subunits; Locomotion; Male; Mice; Mice, Knockout; Protein Precursors; Psychomotor Performance; Thymosin

Prothymosin alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.

Topics: Animals; Brain Ischemia; Depression; Learning; Male; Memory Disorders; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Pain; Peptide Fragments; Protein Precursors; Stroke; Thymosin; Tissue Plasminogen Activator

The aim of the present study was to investigate the protective effects of thymosin β4 (Tβ4) on neuronal apoptosis in rat middle cerebral artery occlusion ischemia/reperfusion (MCAO I/R) injury, and determine the mechanisms involved in this process. Forty‑eight adult male Sprague‑Dawley rats were randomly divided into three groups (n=16 per group): A sham control group, an ischemia/reperfusion group (I/R group), and a Tβ4 group. The focal cerebral I/R model was established by blocking the right MCA for 2 h, followed by reperfusion for 24 h. The Zea‑Longa method was used to assess neurological deficits. Cerebral infarct volume was assessed using 2,3,5‑triphenyltetrazolium chloride staining, and pathological changes were observed via hematoxylin and eosin staining. The terminal dexynucleotidyl transferase (TdT)‑mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis. The expression of glucose‑regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase‑12 (CASP12) protein was assessed using immunohistochemistry and western blotting 24 h after reperfusion. Infarct volume and neuronal damage in the I/R and Tβ4 groups were significantly greater than those observed in the sham group. The Zea‑Longa score, neuronal apoptosis, and expression of GRP78, CHOP, and CASP12 in the I/R and Tβ4 groups were significantly higher than those reported in the sham group. However, the Longa score and neuronal apoptosis were lower in the Tβ4 group compared to the I/R group. The expression of GRP78 was significantly increased, whereas that of CHOP and CASP12 was significantly decreased in the Tβ4 group compared to the I/R group. The present data revealed that Tβ4 can inhibit neuronal apoptosis by upregulating GRP78 and downregulating CHOP and CASP12, thereby reducing cerebral I/R injury.

Topics: Animals; Apoptosis; Brain Ischemia; Disease Models, Animal; Immunohistochemistry; Male; Neurons; Rats; Reperfusion Injury; Thymosin

Prothymosin alpha (ProTα), a nuclear protein, plays multiple functions including cell survival. Most recently, we demonstrated that the active 30-amino acid peptide sequence/P30 (amino acids 49-78) in ProTα 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 ProTα that retains neuroprotective activity. The present study using the experiments of alanine scanning suggested that any amino acid in 9-amino acid peptide sequence/P9 (amino acids 52-60) of P30 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 P9 24h 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 P9 1h after the cerebral ischemia (1h tMCAO) significantly blocks the ischemia-induced brain damages. In addition, systemic P9 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.

Topics: Animals; Brain Ischemia; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Protein Precursors; Thymosin

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.

Topics: Amino Acid Sequence; Animals; Brain Ischemia; Cell Survival; Cells, Cultured; Cerebral Cortex; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neuroprotective Agents; Protein Precursors; Rats; Retinal Diseases; Thymosin

Prothymosin alpha (ProTα), 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 ProTα 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 ProTα under cerebral ischemia is yet unknown. In this study, we report that ProTα 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 ProTα release. We found that none of ProTα is released from astrocytes and microglia under ischemic stress. Indeed, ProTα 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 ProTα 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 ProTα distribution in microglia upon cerebral ischemia. Taken together, these results suggest that only neurons have machineries to release ProTα upon cerebral ischemic stress in vivo.

Topics: Animals; Astrocytes; Brain; Brain Ischemia; Mice; Microglia; Neurons; Neuroprotective Agents; Protein Precursors; Thymosin

Thymosin beta 4 is a protein expressed in most rodent and human tissues, including the brain, and is thought to participate in neurite outgrowth during development by sequestration of G-actin necessary for growth cone extension. Under normal conditions in the adult rat brain, the gene has been suggested to be expressed in microglia and CA1-CA2 hippocampal pyramidal cells. Here we show by using in situ hybridization that t beta 4 is dramatically up-regulated in the infarcted brain area after focal ischaemia. In addition, moderate t beta 4 induction is detected in the perifocal zone, substantia nigra and CA1-CA3 hippocampal pyramidal neurones. Macrophages are probably responsible for t beta 4 induction in the infarcted region, and activated non-neuronal cells probably contribute to the induction seen in the thalamus, substantia nigra and perifocal zone. However, the pyramidal neurones in the hippocampus show t beta 4, upregulation which may be related to restoration of neurite circuits after focal ischemic damage.

Topics: Animals; Autoradiography; Brain Ischemia; Hippocampus; In Situ Hybridization; Male; Rats; Rats, Wistar; RNA, Messenger; Thymosin