Sebok Kumar Halder

Retinal cell type-specific prevention of ischemia-induced damages by LPS-TLR4 signaling through microglia.

Reprogramming of toll‐like receptor 4 (TLR4) by brief ischemia or lipopolysacharide (LPS) contributes to superintending tolerance against destructive ischemia in brain. However, beneficial roles of TLR4 signaling in ischemic retina are not well known. This study demonstrated that preconditioning with LPS 48 h prior to the retinal ischemia prevents the cellular damage in morphology with hematoxylin and eosin (H&E) staining and functions of retina with electroretinogram (ERG), while post‐ischemia treatment deteriorated it. The preventive effects of LPS preconditioning showed the cell type‐specificity of retinal cells. There was complete rescue of ganglion cells, partial rescue of bipolar and photoreceptor cells or no rescue of amacrine cells, respectively. LPS treatment caused the proliferation and migration of retinal microglia and its preconditioning prevented the ischemia‐induced microglial activation. Preventive actions from cell damages following LPS preconditioning prior to retinal ischemia were abolished in TLR4 knock‐out mice, and by pre‐treatments with anti‐TLR4 antibody or minocycline, a microglia inhibitor, which themselves had no effects on the retinal ischemia‐induced damages or microglia activation. Thus, this study revealed that TLR4 mediates the LPS preconditioning‐induced preventive effects through microglial activation in the retinal ischemia model.

Novel neuroprotective action of prothymosin alpha-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.

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.

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‐β 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‐β signaling and microglia in the prevention of retinal ischemic damage.

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.

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 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 impact of prothymosin alpha-derived hexapeptide against retinal ischemia-reperfusion.

Prothymosin alpha (ProTα) has robustness roles against brain and retinal ischemia or serum-starvation stress. In the ProTα sequence, the active core 30-amino acid peptide/P30 (a.a.49-78) is necessary for the original neuroprotective actions against ischemia. Moreover, the 9-amino acid peptide sequence/P9 (a.a.52-60) in P30 still shows neuroprotective activity against brain and retinal ischemia, though P9 is less potent than P30. As the previous structure-activity relationship study for ProTα may not be enough, the possibility still exists that any sequence smaller than P9 retains potent neuroprotective activity. When different P9- and P30-related peptides were intravitreally injected 24h after retinal ischemia in mice, the 6-amino acid peptide/P6 (NEVDEE, a.a.51-56) showed potent protective effects against ischemia-induced retinal functional deficits, which are equipotent to the level of P30 peptide in electroretinography (ERG) and histological damage in Hematoxylin and Eosin (HE) staining. Further studies using ERG and HE staining suggested that intravitreal or intravenous (i.v.) injection with modified P6 peptide/P6Q (NEVDQE) potently inhibited retinal ischemia-induced functional and histological damage. In an immunohistochemical analysis, the ischemia-induced loss of retinal ganglion, bipolar, amacrine and photoreceptor cells were inhibited by a systemic administration with P6Q peptide 24h after the ischemic stress. In addition, systemic post-treatment with P6Q peptide significantly inhibited retinal ischemia-induced microglia and astrocyte activation in terms of increased ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) intensity, respectively, as well as their morphological changes, increased number and migration. Thus, this study demonstrates the therapeutic significance of modified P6 peptide P6Q (NEVDQE) derived from 6-amino acid peptide (P6) in ProTα against ischemic damage.

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.