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.

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. Keywords: ischemia, lipopolysacharide, microglia, neuroprotection, toll-like receptor 4. J. Neurochem. (2013) 126, 243-260. Ischemia in the central nervous system including retina is one of the most well-known pathophysiological condition, which leads to extensive neuronal damages and functional disorders by triggering diverse types of self-reinforcing destructive mechanisms, such as necrosis and apoptosis (Lipton 1999;Bernstein et al. 2003;Danton and Dietrich 2003;Osborne et al. 2004;Ueda and Fujita 2004;Arumugam et al. 2006;Kaur et al. 2008;Lakhan et al. 2009;Neroev et al. 2010;Iadecola and Anrather 2011). These neuronal damages in retina are caused by ischemia-induced activation of detrimental cascades including up-regulation and secretion of injuryrelated cytokines derived from retinal glial cells (Neufeld et al. Address correspondence and reprint requests to Dr Hiroshi Ueda, Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyomachi, Nagasaki 852-8521, Japan. E-mail: ueda@nagasaki-u.ac.jpAbbreviations used: Chx10, ceh-10 homeodomain-containing homolog; ERG, electroretinogram; GCL, the ganglion cell layer; GFAP, glial fibrilary acidic protein; H&E, hematoxylin and eosin; i.vt., intravitreously; Iba-1, ionized calcium binding adaptor molecule 1; INL, inner nuclear layer; IPL, inner plexiform layer; LPS, lipopolysacharide; NeuN, neuronal nuclei; ONL, outer nuclear layer; OPL, outer plexiform layer; pp38 MAPK, phospho-p38 mitogen-activated protein kinase; TLR4, toll-like receptor 4; WT, wild-type.

The nuclear protein prothymosin-a (ProTa), 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 ProTa 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 Ca 2 þ , respectively. S100A13, a Ca 2 þ -binding protein, was identified to be a major protein co-released with ProTa in an immunoprecipitation assay. The Ca 2 þ -dependent interaction between ProTa and S100A13 was found to require the C-terminal peptide sequences of both proteins. In C6 glioma cells expressing a D88-98 mutant of S100A13, serum deprivation caused the release of S100A13 mutant, but not of ProTa. When cells were administered apoptogenic compounds, ProTa 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 ProTa. All these results suggest that necrosisinducing stress induces an extacellular release of ProTa 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.

No abstract