Ischemia preconditioning protects astrocytes from ischemic injury through 14‐3‐3γ

Stroke is a leading cause of death and disability, and new strategies are required to reduce neuronal injury and improve prognosis. Ischemia preconditioning (IPC) is an intrinsic phenomenon that protects cells from subsequent ischemic injury and might provide promising mechanisms for clinical treatment. In this study, primary astrocytes exhibited significantly less cell death than control when exposed to different durations of IPC (15, 30, 60, or 120 min). A 15‐min duration was the most effective IPC to protect astrocytes from 8‐hr‐ischemia injury. The protective mechanisms of IPC involve the upregulation of protective proteins, including 14‐3‐3γ, and attenuation of malondialdehyde (MDA) content and ATP depletion. 14‐3‐3γ is an antiapoptotic intracellular protein that was significantly upregulated for up to 84 hr after IPC. In addition, IPC promoted activation of the c‐Jun N‐terminal kinase (JNK), extracellular signal‐related kinase (ERK)?1/2, p38, and protein kinase B (Akt) signaling pathways. When JNK was specifically inhibited with SP600125, the upregulation of 14‐3‐3γ induced by IPC was almost completely abolished; however, there was no effect on ATP or MDA levels. This suggests that, even though both energy preservation and 14‐3‐3γ up‐regulation were turned on by IPC, they were controlled by different pathways. The ERK1/2, p38, and Akt signaling pathways were not involved in the 14‐3‐3γ upregulation and energy preservation. These results indicate that IPC could protect astrocytes from ischemia injury by inducing 14‐3‐3γ and by alleviating energy depletion through different pathways, suggesting multiple protection of IPC and providing new insights into potential stroke therapies. © 2015 Wiley Periodicals, Inc.     

α‐Synuclein activates innate immunity but suppresses interferon‐γ expression in murine astrocytes

Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α‐Synuclein (α‐syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α‐syn, as well as its mutants (A53T, A30P, and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wild‐type α‐syns to stimulate primary astrocytes in dose‐ and time‐dependent manners (0.5, 2, 8, and 20 μg/ml for 24 hr or 3, 12, 24, and 48 hr at 2 μg/ml), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wild‐type α‐syn significantly upregulated mRNA expression of toll‐like receptor (TLR)2, TLR3, nuclear factor‐κB and interleukin (IL)‐1β, displaying a pattern of positive dose–effect correlation or negative time–effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/ml), TLR3 (at most doses), and IL‐1β (at 3 hr) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL‐1β mRNA expressions. By contrast, interferon (IFN)‐γ was significantly downregulated at mRNA, protein, and protein release levels, especially at high concentrations of α‐syns or early time‐points. These findings indicate that α‐syn was a TLRs‐mediated immunogenic agent (A53T mutant stronger than wild‐type α‐syn). The stimulation patterns suggest that persistent release and accumulation of α‐syn is required for the maintenance of innate immunity activation, and IFN‐γ expression inhibition by α‐syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases.     

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimer's disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes. © 2008 Wiley‐Liss, Inc.     

Hypoxia‐independent mechanisms of HIF‐1α expression in astrocytes after ischemic preconditioning

We recently demonstrated that ischemic tolerance was dependent on astrocytes, for which HIF‐1α had an essential role. The mild ischemia (preconditioning; PC) increased HIF‐1α in a biphasic pattern, that is, a quick and transient increase in neurons, followed by a slow and sustained increase in astrocytes. However, mechanisms underlying such temporal difference in HIF‐1α increase remain totally unknown. Here, we show that unlike a hypoxia‐dependent mechanism in neurons, astrocytes increase HIF‐1α via a novel hypoxia‐independent but P2X7‐dependent mechanism. Using a middle cerebral artery occlusion (MCAO) model of mice, we found that the PC (a 15‐min MCAO period)‐evoked increase in HIF‐1α in neurons was quick and transient (from 1 to 3 days after PC), but that in astrocytes was slow‐onset and long‐lasting (from 3 days to at least 2 weeks after PC). The neuronal HIF‐1α increase was dependent on inhibition of PHD2, an oxygen‐dependent HIF‐1α degrading enzyme, whereas astrocytic one was independent of PHD2. Astrocytes even do not possess this enzyme. Instead, they produced a sustained increase in P2X7 receptors, activation of which resulted in HIF‐1α increase. The hypoxia‐independent but P2X7‐receptor‐dependent mechanism could allow astrocytes to cause long‐lasting HIF‐1α expression, thereby leading to induction of ischemic tolerance efficiently. GLIA 2017;65:523–530     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia‐inducible factor‐2α

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood–brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side‐effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia‐induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia‐induced increase in EPO mRNA expression in a concentration‐dependent manner. TNFα inhibition of hypoxia‐induced EPO expression was mediated primarily by hypoxia‐inducible factor (HIF)‐2α rather than HIF‐1α. The effects of TNFα in reducing hypoxia‐induced upregulation of EPO mRNA expression probably involve destabilization of HIF‐2α, which is regulated by the nuclear factor (NF)‐κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.     

Role of ERK map kinase and CRM1 in IL‐1β‐stimulated release of HMGB1 from cortical astrocytes

Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high‐mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL‐1β for 24 h elicited a dose‐dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL‐1β induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho‐ERK were increased by IL‐1β, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL‐1β increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL‐1β‐stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL‐1β stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke. © 2010 Wiley‐Liss, Inc.     

Identification and topography of neuronal cell populations expressing; TNFα and IL-1α in response to hippocampal lesion

In previous studies, we have shown that a traumatic lesion to the hippocampus of adult mice induces the transitory expression of TNFα and IL-1α by neurons of different brain areas and also by glial cells at the site of injury. The aim of the present study was to establish whether the expression of TNFα and IL-1α is restricted to defined subpopulations, or else is common to most of the central neuronal populations. Using polyclonal anti-GAD 67, anti-TH and monoclonal anti-ChAT, and anti-5-HT antibodies in a double-labeling immunohistochemical procedure in combination with murine anti-TNFα and anti-IL-1α polyclonal antibodies, we show that most GABAergic, catecholaminergic, and serotoninergic neurons, and a subgroup of the cholinergic neurons, express these cytokines. Although not immunohistochemically characterized, neurons in some glutamatergic structures such as the hippocampus and the prefrontal cortex also express these cytokines. Thus, we conclude that the capacity of central neurons to express cytokines like TNFα and IL-1α in reaction to a brain Injury is not restricted to peculiar neuronal subtypes, but could include most of the neuronal populations of the brain. © 1996 Wiley-Liss, Inc.     

Glycogen synthase kinase‐3β phosphorylates synphilin‐1 in vitro

α‐Synuclein is known to be a major component of Lewy bodies and glial cytoplasmic inclusions in the brains of patients with α‐synucleinopathies. Synphilin‐1, an α‐synuclein‐associated protein, is also present in these inclusions. However, little is known about the post‐translational modifications of synphilin‐1. In the present study, it is reported that synphilin‐1 is phosphorylated by glycogen synthase kinase‐3βin vitro. It is well known that protein phosphorylation is involved in various physiological phenomena, including signal transduction and protein degradation. Therefore, phosphorylation of synphilin‐1 may play an important role in the function of this protein in the brain.     

Implication of Akt‐dependent Prp19α/14‐3‐3β/Cdc5L complex formation in neuronal differentiation

PRP19α and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19α/14‐3‐3β/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)‐induced neuronal differentiation of PC12 cells. Analysis of PRP19α mutants revealed that the phosphorylation of PRP19α at Thr 193 by Akt was critical for its binding with 14‐3‐3β to translocate into the nuclei and for PRP19α/14‐3‐3β/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19α or sense 14‐3‐3β RNAs promoted NGF‐induced neuronal differentiation, whereas down‐regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19αT193A lost its binding ability with 14‐3‐3β and acted as a dominant‐negative mutant in neuronal differentiation. These results imply that Akt‐dependent phosphorylation of PRP19α at Thr193 triggers PRP19α/14‐3‐3β/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre‐mRNAs. © 2010 Wiley‐Liss, Inc.     

Expression of macrophage inflammatory protein‐1α in Schwann cell tumors

It is well known that tumors originating from Schwann cells often demonstrate degeneration, such as myxoid areas in Schwannoma (Antoni B) and granular cell tumors. However, the mechanism has never been understood in detail. Recruitment of circulating macrophages plays a role in degeneration, however, the factors leading to that process are not well known. Macrophage inflammatory protein‐1α (MIP‐1α), which belongs to the C‐C family of chemokines, is thought to be involved in the recruitment of inflammatory cells and might play a role in the degenerative change of Schwann cell tumors. To elucidate the role of MIP‐1α in such conditions, we employed immunohistochemistry and in situ hybridization analyses. Strongly positive staining of MIP‐1α was revealed in the myxoid areas of tested tumors and in situ hybridization detected the existence of MIP‐1α mRNA in some of the tumor cells. These results suggest that MIP‐1α produced from tumorigenic cells plays a role in the auto‐degenerative process of Schwann cell tumors. Furthermore, various levels of MIP‐1α expression in these tumors were implicated to be correlated with histological variations.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Metformin increases peroxisome proliferator–activated receptor γ Co‐activator‐1α and utrophin a expression in dystrophic skeletal muscle

Introduction: Metformin (MET) stimulates skeletal muscle AMP‐activated protein kinase (AMPK), a key phenotype remodeling protein with emerging therapeutic relevance for Duchenne muscular dystrophy (DMD). Our aim was to identify the mechanism of impact of MET on dystrophic muscle. Methods: We investigated the effects of MET in cultured C₂C₁₂ muscle cells and mdx mouse skeletal muscle. Expression of potent phenotypic modifiers was assessed, including peroxisome proliferator–activated receptor (PPAR)γ coactivator‐1α (PGC‐1α), PPARδ, and receptor‐interacting protein 140 (RIP140), as well as that of the dystrophin‐homolog, utrophin A. Results: In C₂C₁₂ cells, MET augmented expression of PGC‐1α, PPARδ, and utrophin A, whereas RIP140 content was reciprocally downregulated. MET treatment of mdx mice increased PGC‐1α and utrophin A and normalized RIP140 levels. Conclusions: In this study we identify the impact of MET on skeletal muscle and underscore the timeliness and importance of investigating MET and other AMPK activators as relevant therapeutics for DMD. Muscle Nerve 52 : 139–142, 2015