Hayato Matsunaga

Lysophosphatidic acid: chemical signature of neuropathic pain.

Acute inflammatory pain signal originates from transient hypersensitivity in afferent fibers when depolarized via injured tissues or proinflammatory cells-derived pronociceptive ligand binding. This pain is sensitive to opioids and NSAIDs. In neuropathic pain, however, damage to the nerve along the pain pathway results in spontaneous generation of action potential and lowered nociceptive threshold, as seen in allodynia and hyperalgesia. This abnormal pain transmission had been linked to LPA production in the spinal cord, through activation of NMDA and NK1 activation by glutamate and SP in iPLA(2)/cPLA(2)/ATX-dependent pathway. In a bifurcated response involving G(q/11) and G(12/13) coupling, Schwann cell LPA(1) mediates degradation and transcriptional suppression of myelin proteins, respectively. The loss of contact inhibition on axonal growth creates cytoskeletal framework for axonal sprouting. LPA causes an amplification of LPA production through activation of LPA(3) signaling in microglia immediately after nerve injury. LPA(1) deficient mice (LPA(1)(-/-)) show no neuropathic-pain behavior or demyelination in response to intrathecal LPA injection or nerve injury. Given these bodies of research evidence, LPA therefore presents as the chemical signature for the initiation of neuropathic pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Identification of prothymosin-α1, the necrosis–apoptosis switch molecule in cortical neuronal cultures

We initially identified a nuclear protein, prothymosin-α1 (ProTα), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTα inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl2-family proteins. The apoptosis caused by ProTα was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTα-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCβII expression prevented this cell death mode switch. Collectively, these results suggest that ProTα is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors.

Stress-induced non-vesicular release of prothymosin- α initiated by an interaction with S100A13, and its blockade by caspase-3 cleavage

The nuclear protein prothymosin-α (ProTα), which lacks a signal peptide sequence, is released from neurons and astrocytes on ischemic stress and exerts a unique form of neuroprotection through an anti-necrotic mechanism. Ischemic stress-induced ProTα release is initiated by a nuclear release, followed by extracellular release in a non-vesicular manner, in C6 glioma cells. These processes are caused by ATP loss and elevated Ca2+, respectively. S100A13, a Ca2+-binding protein, was identified to be a major protein co-released with ProTα in an immunoprecipitation assay. The Ca2+-dependent interaction between ProTα and S100A13 was found to require the C-terminal peptide sequences of both proteins. In C6 glioma cells expressing a Δ88–98 mutant of S100A13, serum deprivation caused the release of S100A13 mutant, but not of ProTα. When cells were administered apoptogenic compounds, ProTα was cleaved by caspase-3 to generate a C-terminal peptide-deficient fragment, which lacks the nuclear localization signal (NLS). However, there was no extracellular release of ProTα. All these results suggest that necrosis-inducing stress induces an extacellular release of ProTα in a non-vesicular manner, whereas apoptosis-inducing stress does not, owing to the loss of its interaction with S100A13, a cargo molecule for extracellular release.

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.

Evidence for serum-deprivation-induced co-release of FGF-1 and S100A13 from astrocytes.

Since fibroblast growth factor (FGF)-1 lacks conventional amino-terminal signal peptide essential for endoplasmic reticulum (ER)-Golgi pathway, the mode of release of this polypeptide remains to be fully understood. We attempted to characterize the non-classical (non-vesicular) mode of FGF-1 release in the analyses using immunocytochemistry and immunoblot of conditioned medium (CM) from astrocytes. FGF-1 was completely released from astrocytes upon serum-deprivation stress in a Brefeldin A-insensitive manner. In the immunoprecipitation study using anti-FGF-1 IgG, S100A13 was identified to be the major protein co-eluted with FGF-1. The interaction between GST-FGF-1 and Strep-tag II-S100A13 was found to be Ca(2+)-sensitive, and to require the C-terminal 11 amino acid peptide sequence of S100A13. The overexpression of Delta88-98 mutant of S100A13 selectively inhibited the serum-deprivation stress-induced release of FGF-1, but not the release of S100A13 mutant from C6 glioma cells. However, amlexanox, anti-allergic drug whose target is S100A13, completely inhibited the stress-induced release of FGF-1 as well as S100A13. The stress-induced release of both proteins was also abolished by BAPTA-AM, an intracellular Ca(2+) chelating agent. The serum-deprivation caused Ca(2+) spikes in omega-conotoxin GVIA and thapsigargin-sensitive manner. All these results suggest that S100A13 is a cargo molecule for the serum-deprivation stress-induced non-classical release of FGF-1, and that its driving force of protein-protein interaction and release is possibly mediated by Ca(2+)-induced Ca(2+) release (CICR) coupled to N-type Ca(2+) channel activity.

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.

Endocrine disrupting chemicals bind to a novel receptor, microtubule‐associated protein 2, and positively and negatively regulate dendritic outgrowth in hippocampal neurons

J. Neurochem. (2010) 114, 1333–1343.

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.