Involvement of SNARE Protein Interaction for Non-classical Release of DAMPs/Alarmins Proteins, Prothymosin Alpha and S100A13.

Prothymosin alpha (ProTα) is involved in multiple cellular processes. Upon serum-free stress, ProTα lacking a signal peptide sequence is non-classically released from C6 glioma cells as a complex with Ca2+-binding cargo protein S100A13. Thus, ProTα and S100A13 are conceived to be members of damage-associated molecular patterns (DAMPs)/alarmins. However, it remains to be determined whether stress-induced release of ProTα and S100A13 involves SNARE proteins in the mechanisms underlying membrane tethering of the multiprotein complex. In the present study, we used C6 glioma cells as a model of ProTα release. In pull-down assay, p40 synaptotagmin-1 (Syt-1), a vesicular SNARE, formed a hetero-oligomeric complex with homodimeric S100A13 in a Ca2+-dependent manner. The interaction between p40 Syt-1 and S100A13 was also Ca2+-dependent in surface plasmon resonance (SPR). Immunoprecipitation using conditioned medium (CM) revealed that p40 Syt-1 was co-released with ProTα and S100A13 upon serum-free stress. In in situ proximity ligation assay (PLA), Syt-1 interacted with S100A13 upon serum-free stress in C6 glioma cells. The intracellular delivery of anti-Syt-1 IgG blocked serum free-induced release of ProTα and S100A13. Serum free-induced ProTα-EGFP release was significantly blocked by botulinum neurotoxin/C1 (BoNT/C1), which cleaves target SNARE syntaxin-1 (Stx-1). In immunocytochemistry, the cellular loss of ProTα-EGFP, S100A13, and Syt-1 was also blocked by BoNT/C1. Furthermore, the intracellular delivery of anti-Stx-1 IgG or Stx-1 siRNA treatment blocked Syt-1, S100A13 and ProTα release from C6 glioma cells. All these findings suggest that SNARE proteins play roles in stress-induced non-classical release of DAMPs/alarmins proteins, ProTα and S100A13 from C6 glioma cells.

Prothymosin alpha and its mimetic hexapeptide improve delayed tissue plasminogen activator-induced brain damage following cerebral ischemia.

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.

Gγ7-specific prothymosin alpha deletion causes stress- and age-dependent motor dysfunction and anxiety.

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.

Experimental evidence for the involvement of F0/F1 ATPase and subsequent P2Y12 receptor activation in prothymosin alpha-induced protection of retinal ischemic damage.

The inhibition of retinal ischemia-induced damage by post-ischemic prothymosin alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or ß-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.

Amlexanox Inhibits Cerebral Ischemia-Induced Delayed Astrocytic High-Mobility Group Box 1 Release and Subsequent Brain Damage.

High-mobility group box 1 (HMGB1) is increased in the cerebrospinal fluid (CSF) and serum during the early and late phases of brain ischemia and is known to contribute to brain damage. However, detailed characterization underlying cell type-specific HMGB1 release and pathophysiological roles of extracellularly released HMGB1 in ischemic brain remain unclear. Here, we examined cell type-specific HMGB1 release and the therapeutic potential of amlexanox, an inhibitor of nonclassical release, and of an anti-HMGB1 antibody against ischemic brain damage. HMGB1 depletion from neuronal nuclei was observed within 3 hours after transient middle cerebral artery occlusion (tMCAO), whereas the intracerebroventricular (i.c.v.) pretreatment with amlexanox blocked HMGB1 release from neurons, resulting in HMGB1 redistribution in the nuclei and cytoplasm. HMGB1 was selectively released from astrocytes 27 hours after tMCAO and this HMGB1 release was blocked by late treatment with amlexanox (i.c.v.) 24 hours after tMCAO. Proximity extension assay revealed that the HMGB1 level was elevated in the CSF at 3 and 27 hours after tMCAO. This late treatment with amlexanox significantly protected the brain from ischemic damage, but its pretreatment 30 minutes before tMCAO failed to show any protection. The late treatment (i.c.v.) with anti-HMGB1 antibody 24 hours after tMCAO also ameliorated ischemic brain damage 48 hours after tMCAO. Thus, the inhibition of brain damage by late treatment with amlexanox or anti-HMGB1 antibody indicates that late HMGB1 release plays a role in the maintenance of stroke-induced brain damage, and the inhibition of this release would be a novel therapeutic target for protection of ischemic brain damage.

Prothymosin alpha-deficiency enhances anxiety-like behaviors and impairs learning/memory functions and neurogenesis.

Prothymosin alpha (ProTα) is expressed in various mammalian organs including the neuronal nuclei in the brain, and is involved in multiple functions, such as chromatin remodeling, transcriptional regulation, cell proliferation, and survival. ProTα has beneficial actions against ischemia-induced necrosis and apoptosis in the brain and retina. However, characterizing the physiological roles of endogenous ProTα in the brain without stress remains elusive. Here, we generated ProTα-deficiency mice to explore whether endogenous ProTα is involved in normal brain functions. We successfully generated heterozygous ProTα knockout (ProTα+/- ) mice, while all homozygous ProTα knockout (ProTα-/- ) offspring died at early embryonic stage, suggesting that ProTα has crucial roles in embryonic development. In the evaluation of different behavioral tests, ProTα+/- mice exhibited hypolocomotor activity in the open-field test and enhanced anxiety-like behaviors in the light/dark transition test and the novelty induced hypophagia test. ProTα+/- mice also showed impaired learning and memory in the step-through passive avoidance test and the KUROBOX test. Depression-like behaviors in ProTα+/- mice in the forced swim and tail suspension tests were comparable with that of wild-type mice. Furthermore, adult hippocampal neurogenesis was significantly decreased in ProTα+/- mice. ProTα+/- mice showed an impaired long-term potentiation induction in the evaluation of electrophysiological recordings from acute hippocampal slices. Microarray analysis revealed that the candidate genes related to anxiety, learning/memory-functions, and neurogenesis were down-regulated in ProTα+/- mice. Thus, this study suggests that ProTα has crucial physiological roles in the robustness of brain.

Neuroprotective DAMPs member prothymosin alpha has additional beneficial actions against cerebral ischemia-induced vascular damages.

Prothymosin alpha (ProTα) suppresses stress-induced necrosis of cultured cortical neurons. As neuroprotection alone could not explain the long-lasting protective actions against cerebral ischemia by ProTα, we further examined whether ProTα, in addition to neuroprotective effects, has other anti-ischemic activities. When recombinant mouse ProTα (rmProTα) at 0.3 mg/kg was intravenously (i.v.) given 2 h after the start of tMCAO, all mice survived for more than 14 days. In evaluation of CD31- and tomato lectin-labeling as well as IgG and Evans blue leakage, rmProTα treatment (0.1 mg/kg) largely blocked ischemia-induced vascular damages. Therefore, rmProTα has novel beneficial effects against ischemia-induced brain damage through vascular mechanisms.

Prothymosin-alpha preconditioning activates TLR4-TRIF signaling to induce protection of ischemic retina.

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 prothymosin-alpha 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. Prothymosin-alpha 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-ß 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-ß signaling and microglia in the prevention of retinal ischemic damage. We propose the following mechanism for prothymosin-alpha (ProTα) preconditioning-induced retinal prevention against ischemia: ProTα preconditioning-induced prevention of retinal ischemic damage is mediated by selective activation of the TIR-domain-containing adapter-inducing interferon-ß (TRIF)- interferon regulatory factor 3 (IRF3) pathway downstream of toll-like receptor 4 (TLR4) in microglia, resulting in up-regulation of TRIF-IRF3-dependent protective genes and down-regulation of myeloid differentiation primary response gene 88 (MyD88)-Nuclear factor (NF)κB-dependent injury genes. Detailed investigations would be helpful to test the efficacy of ProTα as a therapeutic agent for the prevention of ischemic disorders.

Novel neuroprotective action of prothymosin α-derived peptide against retinal and brain ischemic damages.

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.

Neuron-specific non-classical release of prothymosin alpha: a novel neuroprotective damage-associated molecular patterns.

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.

Regional distribution and cell type-specific subcellular localization of Prothymosin alpha in brain.

Prothymosin alpha (ProTα) is an acidic nuclear protein implicated in several cellular functions including cell survival. ProTα is found in the central nervous system, but the regional and cell type-specific expression patterns are not known. In this study, our immunohistochemical analysis demonstrated that ProTα is expressed ubiquitously throughout adult brain with difference in the intensity of region-specific protein reactivity. Interestingly, the highest ProTα signals were observed in the brain regions relevant to neurogenesis, such as sub-ventricular zone, granular cell layer of dentate gyrus, as well as granule cell layer of olfactory bulb. Strong immunoreactivity was also found in habenula, ependymal cells lining the dorsal third and fourth ventricle, and in neurons in the Purkinje cell layer of cerebellum. We showed that ProTα was strictly localized in the nuclei of neurons, while it was found in the cytosolic space of astroglial and microglial processes and cell body in the adult brain. To clarify the phenomenon underlying cytosolic localization of ProTα in non-neuronal cells, ZVAD-fmk, a caspase-3 inhibitor, was delivered intracerebroventricularly in the brain. At the follow-up 24 h after ZVAD-fmk injection, we found that nuclear intensity of ProTα was significantly increased in astrocytes, whereas the ProTα expression was not affected in microglia. The present study would contribute toward better understanding of physiological and pathophysiological roles of ProTα in the brain.

Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions.

Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum-free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1. The stress-induced externalization of ANXA2 was inhibited by pretreatment with siRNA for P4-ATPase, ATP8A2, under serum-free conditions, which ablates membrane lipid asymmetry. The stress-induced ProTα release via Stx-1A, ANXA2 and ATP8A2 was also evidenced by the knock-down strategy in the experiments using oxygen glucose deprivation-treated cultured neurons. These findings suggest that starvation stress-induced release of ProTα, S100A13, and p40 Syt-1 from C6 glioma cells is mediated by the ANXA2-flop-out via energy crisis-dependent recovery of membrane lipid asymmetry.

Beneficial actions of prothymosin alpha-mimetic hexapeptide on central post-stroke pain, reduced social activity, learning-deficit and depression following cerebral ischemia in mice.

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.

Expression of fragile X mental retardation protein in neurons and glia of the developing and adult mouse brain.

Fragile X syndrome is the most common inherited form of mental retardation and autism. It is caused by a reduction or elimination of the expression of fragile X mental retardation protein (FMRP). Because fragile X syndrome is a neurodevelopmental disorder, it is important to fully document the cell type expression in the developing CNS to provide a better understanding of the molecular function of FMRP, and the pathogenesis of the syndrome. We investigated FMRP expression in the brain using double-labeling immunocytochemistry and cell type markers for neurons (NeuN), astrocytes (S100ß), microglia (Iba-1), and oligodendrocyte precursor cells (NG2). The hippocampus, striatum, cingulate cortex, retrosplenial cortex, corpus callosum and cerebellum were assessed in wild-type C57/BL6 mice at postnatal days 0, 10, 20, and adult. Our results demonstrate that FMRP is ubiquitously expressed in neurons at all times and brain regions studied, except for corpus callosum where FMRP was predominantly present in astrocytes at all ages. FMRP expression in Iba-1 and NG2-positive cells was detected at postnatal day 0 and 10 and gradually decreased to very low or undetectable levels in postnatal day 20 and adult mice. Our results reveal that in addition to continuous and extensive expression in neurons in the immature and mature brain, FMRP is also present in astrocytes, oligodendrocyte precursor cells, and microglia during the early and mid-postnatal developmental stages of brain maturation. Prominent expression of FMRP in glia during these crucial stages of brain development suggests an important contribution to normal brain function, and in its absence, to the fragile X phenotype.

Therapeutic benefits of 9-amino acid peptide derived from prothymosin alpha against ischemic damages.

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.

Prothymosin α plays multifunctional cell robustness roles in genomic, epigenetic, and nongenomic mechanisms.

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.