S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

Neuroinflammation following traumatic brain injury (TBI) is increasingly recognized to contribute to chronic tissue loss and neurologic dysfunction. Circulating levels of S100B increase after TBI and have been used as a biomarker. S100B is produced by activated astrocytes and can promote microglial activation; signaling by S100B through interaction with the multiligand advanced glycation end product-specific receptor (AGER) has been implicated in brain injury and microglial activation during chronic neurodegeneration. We examined the effects of S100B inhibition in a controlled cortical impact model, using S100B knockout mice or administration of neutralizing S100B antibody. Both interventions significantly reduced TBI-induced lesion volume, improved retention memory function, and attenuated microglial activation. The neutralizing antibody also significantly reduced sensorimotor deficits and improved neuronal survival in the cortex. However, S100B did not alter microglial activation in BV2 cells or primary microglial cultures stimulated by lipopolysaccharide or interferon gamma. Further, proximity ligation assays did not support direct interaction in the brain between S100B and AGER following TBI. Future studies are needed to elucidate specific pathways underlying S100B-mediated neuroinflammatory actions after TBI. Our results strongly implicate S100B in TBI-induced neuroinflammation, cell loss, and neurologic dysfunction, thereby indicating that it is a potential therapeutic target for TBI.     

Microglial activation induced by the alarmin S100B is regulated by poly(ADP‐ribose) polymerase‐1

Brain injury resulting from stroke or trauma can be exacerbated by the release of proinflammatory cytokines, proteases, and reactive oxygen species by activated microglia. The microglial activation resulting from brain injury is mediated in part by alarmins, which are signaling molecules released from damaged cells. The nuclear enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1) has been shown to regulate microglial activation after brain injury, and here we show that signaling effects of the alarmin S100B are regulated by PARP‐1. S100B is a protein localized predominantly to astrocytes. Exogenous S100B added to primary microglial cultures induced a rapid change in microglial morphology, upregulation of IL‐1β, TNFα, and iNOS gene expression, and release of matrix metalloproteinase 9 and nitric oxide. Most, though not all of these effects were attenuated in PARP‐1^‐/‐ microglia and in wild‐type microglia treated with the PARP inhibitor, veliparib. Microglial activation and gene expression changes induced by S100B injected directly into brain were likewise attenuated by PARP‐1 inhibition. The anti‐inflammatory effects of PARP‐1 inhibitors in acutely injured brain may thus be mediated in part through effects on S100B signaling pathways. GLIA 2016;64:1869–1878     

Autocrine S100B effects on astrocytes are mediated via RAGE

To find out if the astrocytic protein S100B involves its autocrine effects via RAGE we investigated the capacity of astrocytes to upregulate IL-6 and TNF-alpha expression by stimulation with S100B. The subcellular localization of RAGE expression at the cell surface membrane of cultured astrocytes was demonstrated by immunofluorescence microscopy, flow cytometry and Western blotting. S100B was able to stimulate IL-6 and TNF-alpha secretion in cultured astrocytes in a concentration- and time-dependent manner as shown by ELISA. S100B induced IL-6 and TNF-alpha secretion was blocked by the use of RAGE siRNA specific for knocking down RAGE expression.     

The role of receptor for advanced glycation end products (RAGE) in neuronal differentiation

The receptor for advanced glycation end products (RAGE) is a multiligand receptor protein thought to play an important role in neuronal differentiation. RAGE can bind a number of ligands and activate a variety of signalling pathways that lead to diverse downstream effects. Amphoterin and S100B are endogenous ligands, the interaction of which with RAGE is known to be involved in defined physiological processes. The present study investigated the spatiotemporal pattern of the expression for RAGE and its ligands, amphoterin and S100B, during neuronal differentiation of NT2/D1 cells. In this study, all three proteins were shown to increase with progression of neuronal differentiation as determined by Western blotting, raising the possibility that both amphoterin and S100B may interact with RAGE and have important functions during the process of cell differentiation. Moreover, blocking the activation of RAGE with neutralizing antibody in the presence of retinoic acid disrupted the progression of normal neuronal differentiation. Immunocytochemistry (ICC) studies showed that amphoterin partially colocalized with RAGE within differentiating NT2 cells, whereas S100B showed a high degree of colocalization. This result suggests that S100B is more likely to be the principal ligand for RAGE during the differentiation process and that RAGE and amphoterin might have both independent and combined roles. Moreover, RAGE was expressed only in cells that were committed to a neuronal phenotype, suggesting direct involvement of RAGE in mediating cellular changes within differentiating neuronal cells. Further detailed studies are now required to characterize fully the role of RAGE during the neuronal differentiation period.     

S100B attenuates microglia activation in gliomas: possible role of STAT3 pathway

Despite significant infiltration into tumors, the effector function of macrophages (MPs) and microglia (MG) appears to be suppressed in gliomas. Although STAT3 pathway is thought to play a role in this process, the exact mechanism by which gliomas induce STAT3 activation in MPs and MG is not known. Because activation of receptor for advanced glycation end products (RAGE) can induce STAT3, and because gliomas express high levels of S100B, a RAGE ligand, we hypothesized that MP/MG STAT3 activity may be modulated through S100B-RAGE interaction. Exposure of N9 MG and bone marrow-derived monocytes (BMM) to GL261 glioma condition medium (GCM) and low (nM) levels of S100B increased RAGE expression, induced STAT3 and suppressed MG function in vitro. Furthermore, neutralization of S100B in GCM, partially reversed IL-1β suppression in BMM, suggesting that the inhibitory effect of GCM to be in part due to S100B. Finally, blockage of S100B-RAGE interaction inhibited STAT3 activation in N9 MG and in glioma MG/MP in vivo. These findings suggest that the RAGE pathway may play an important role in STAT3 induction in glioma-associated MG/MPs, and that this process may be mediated through S100B.     

Vitamin E suppression of microglial activation is neuroprotective

Neurotoxic microglial-neuronal interactions have been implicated in the pathogenesis of various neurodegenerative diseases such as Alzheimer's disease, and vitamin E has been shown to have direct neuroprotective effects. To determine whether vitamin E also has indirect neuroprotective effects through suppression of microglial activation, we used a microglial-neuronal coculture. Lipopolysaccharide (LPS) treatment of a microglial cell line (N9) induced a time-dependent activation of both p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappaB (NFkappaB), with consequent increases in interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production. Differentiated neuronal cells (PC12 cells treated with nerve growth factor) exhibited marked loss of processes and decreased survival when cocultured with LPS-activated microglia. Preincubation of microglia with vitamin E diminished this neurotoxic effect, independently of direct effects of the antioxidant on the neuronal cells. Microglial NO production and the induction of IL-1alpha and TNFalpha expression also were attenuated by vitamin E. Such antiinflammatory effects of vitamin E were correlated with suppression of p38 MAPK and NFkappaB activation and were mimicked by an inhibition of either p38 MAPK (by SB203580) or NFkappaB (by decoy oligonucleotides). These results suggest that, in addition to the beneficial effects of providing direct antioxidant protection to neurons reported by others, vitamin E may provide neuroprotection in vivo through suppression of signaling events necessary for microglial activation.     

S100B expression in and effects on microglia

We evaluated the intracellular and extracellular biological role of S100B protein with respect to microglia. S100B, which belongs to the multigenic family of Ca2+-binding proteins, is abundant in astrocytes where it is found diffusely in the cytoplasm and is associated with membranes and cytoskeleton constituents. S100B protein is also secreted by astrocytes and acts on these cells to stimulate nitric oxide secretion in an autocrine manner. However, little is known about the relationship between S100B and microglia. To address this issue, we used primary microglia from newborn rat cortex and the BV-2 microglial cell line, a well-established cell model for the study of microglial properties. S100B expression was assessed by immunofluorescence in primary microglia and by RT-PCR, Western blotting, and immunofluorescence in BV-2 cells. S100B was found in microglia in the form of a filamentous network as well as diffusely in the cytoplasm and associated with intracellular membranes. S100B relocated around phagosomes during BV-2 phagocytosis of opsonized Cryptococcus neoformans. Furthermore, interferon-gamma (IFN-gamma) treatment caused cell shape changes and redistribution of S100B, and downregulation of S100B mRNA expression in BV-2 cells. Treatment of BV-2 cells with nanomolar to micromolar amounts of S100B resulted in increased IFN-gamma-induced expression of inducible nitric oxide synthase mRNA as well as nitric oxide secretion. Taken together, these data suggest a possible role for S100B in the accomplishment/regulation of microglial cell functions.     

S100B protects LAN-5 neuroblastoma cells against Abeta amyloid-induced neurotoxicity via RAGE engagement at low doses but increases Abeta amyloid neurotoxicity at high doses

At the concentrations normally found in the brain extracellular space the glial-derived protein, S100B, protects neurons against neurotoxic agents by interacting with the receptor for advanced glycation end products (RAGE). It is known that at relatively high concentrations S100B is neurotoxic causing neuronal death via excessive stimulation of RAGE. S100B is detected within senile plaques in Alzheimer's disease, where its role is unknown. The present study was undertaken to evaluate a putative neuroprotective role of S100B against Abeta amyloid-induced neurotoxicity. We treated LAN-5 neuroblastoma cultures with toxic amounts of Abeta25-35 amyloid peptide. Our results show that at nanomolar concentrations S100B protects cells against Abeta-mediated cytotoxicity, as assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein isothiocyanate nick end-labeling (TUNEL) experiments, by countering the Abeta-mediated decrease in the expression of the anti-apoptotic factor Bcl-2. This effect depends on S100B binding to RAGE because S100B is unable to contrast Abeta-mediated neurotoxicity in neurons overexpressing a signaling-deficient RAGE mutant lacking the cytosolic and transducing domain. Our data suggest that at nanomolar doses S100B counteracts Abeta peptide neurotoxicity in a RAGE-mediated manner. However, at micromolar doses S100B is toxic to LAN-5 cells and its toxicity adds to that of the Abeta peptide, suggesting that additional molecular mechanisms may be involved in the neurotoxic process.     

Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein

Abstract  In the central nervous system glial-derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end-products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon-gamma (IFN-γ) in the presence of anti-RAGE or anti-S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti-RAGE blocking antibody. Incubation with LPS + IFN-γ induced a significant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN-γ-induced S100B up-regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti-RAGE and anti-S100B blocking antibodies. Enteroglial-derived S100B up-regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.     

Glial S100B Positive Vacuoles In Purkinje Cells: Earliest Morphological Abnormality In SCA1 Transgenic Mice

Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-1. The overexpression of mutant ataxin-1 in SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in Purkinje neurons (PKN) of the cerebellum. PKN are closely associated with neighboring Bergmann glia. To elucidate the role of Bergmann glia in SCA1 pathogenesis, cerebellar tissue from 7 days to 6 wks old SCA1 transgenic and wildtype mice were used. We observed that Bergmann glial S100B protein is localized to the cytoplasmic vacuoles in SCA1 PKN. These S100B positive cytoplasmic vacuoles began appearing much before the onset of behavioral abnormalities, and were negative for other glial and PKN marker proteins. Electron micrographs revealed that vacuoles have a double membrane. In the vacuoles, S100B colocalized with receptors of advanced glycation end-products (RAGE), and S100B co-immunoprecipated with cerebellar RAGE. In SCA1 PKN cultures, exogenous S100B protein interacted with the PKN membranes and was internalized. These data suggest that glial S100B though extrinsic to PKN is sequestered into cytoplasmic vacuoles in SCA1 mice at early postnatal ages. Further, S100B may be binding to RAGE on Purkinje cell membranes before these membranes are internalized.     

Modulation of glial activation by astrocyte-derived protein S100B: differential responses of astrocyte and microglial cultures

The astrocyte-derived protein S100B stimulates production of inducible nitric oxide synthase and nitric oxide (NO) in astrocytes [Hu et al., 1996, J. Biol. Chem. 271:2543], but its effect on microglia is not known. In addition, S100B's ability to modulate the activity of other glial activating agents has not been defined. In this study, we compared the ability of S100B to stimulate NO in cultures of rat primary astrocytes and the BV-2 murine microglial cell line, and investigated the effect of the combined action of S100B and other stimuli known to activate glial cells. S100B itself stimulated the production of NO in astrocytes, and did not modify or potentiated only weakly the NO production induced by interleukin-1 beta, tumor necrosis factor alpha, dibutyryl cyclic AMP, zymosan A or lipid A. In contrast, S100B alone did not induce NO in BV-2 cells but strongly potentiated NO production in the presence of lipid A but not zymosan A. The deletion of eight C-terminal amino acid residues in S100B leads to a loss of the effect of S100B on microglia but not on astrocytes. These results demonstrate that responses of glial cells to extracellular S100B can vary depending on the cell type, and suggest that different structural features of S100B are important for the protein's effects on microglia and astrocytes.     

In vitro S100B secretion is reduced by apomorphine: effects of antipsychotics and antioxidants

Astrocytes express dopamine receptors and respond to dopamine stimulation. However, the role of astrocytes in psychiatric disorders and the effects of antipsychotics on astroglial cells have only been investigated recently. S100B is a glial-derived protein, commonly used as a marker of astroglial activation in psychiatric disorders, particularly schizophrenia. We investigated S100B secretion in three different rat brain preparations (fresh hippocampal slices, C6 glioma cells and primary astrocyte cultures) exposed to apomorphine and antipsychotics (haloperidol and risperidone), aiming to evaluate, ex vivo and in vitro, whether dopamine activation and dopaminergic antagonists modulate astroglial activation, as measured by changes in the extracellular levels of S100B. The serum S100B elevation observed in schizophrenic patients is not reflected by the in vitro decrease of S100B secretion that we observed in hippocampal slices, cortical astrocytes and C6 glioma cells treated with apomorphine, which mimics dopaminergic hyperactivation. This decrease in S100B secretion can be explained by a stimulation of D2 receptors negatively coupled to adenyl cyclase. Antipsychotic medications and antioxidant supplementation were able to prevent the decline in S100B secretion. Findings reinforce the benefits of antioxidant therapy in psychiatric disorders. Based on our results, in hippocampal slices exposed to apomorphine, it may be suggested that antipsychotics could help to normalize S100B secretion by astrocytes.     

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.     

S100B protein stimulates calcineurin activity

S100B is a calcium binding protein from astrocytes that regulates protein phosphorylation by binding to substrates and protein kinases. S100B might also regulate protein phosphatases and this was investigated for protein phosphatase 2B (calcineurin). The results indicate that S100B (5-10 microM) increased the activity of both purified and cytoskeletal calcineurin in a Ca-dependent manner. This effect was blocked by a specific inhibitor of calcineurin activity, but not by TRTK-12 (an inhibitor of S100B binding to other protein targets). The present results and the known co-localization of S100B and calcineurin in the astrocyte cytoskeleton suggest that S100B may play a role in the phosphorylation state of cytoskeletal proteins.     

Effect of the branched-chain alpha-keto acids accumulating in maple syrup urine disease on S100B release from glial cells

Accumulation of the branched-chain alpha-keto acids (BCKA), alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) and their respective branched-chain alpha-amino acids (BCAA) occurs in tissues and biological fluids of patients affected by the neurometabolic disorder maple syrup urine disease (MSUD). The objective of this study was to verify the effect of the BCKA on S100B release from C6 glioma cells. The cells were exposed to 1, 5 or 10 mM BCKA for different periods and the S100B release was measured afterwards. The results indicated that KIC and KIV, but not KMV, significantly enhanced S100B liberation after 6 h of exposure. Furthermore, the stimulatory effect of the BCKA on S100B release was prevented by coincubation with the energetic substrate creatine and with the N-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, indicating that energy deficit and nitric oxide (NO) were probably involved in this effect. Furthermore, the increase of S100B release was prevented by preincubation with the protein kinase inhibitors KN-93 and H-89, indicating that KIC and KIV altered Ca2+/calmodulin (PKCaMII)- and cAMP (PKA)-dependent protein kinases activities, respectively. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble vitamin E) were not able to prevent KIC- and KIV-induced increase of S100B liberation, suggesting that the alteration of S100B release caused by the BCKA is not mediated by oxidation of sulfydryl or other essential groups of the enzyme as well as by lipid peroxyl radicals. Considering the importance of S100B for brain regulation, it is conceivable that enhanced liberation of this protein by increased levels of BCKA may contribute to the neurodegeneration characteristic of MSUD patients.     

Inhibition of phosphodiesterase-4 suppresses HMGB1/RAGE signaling pathway and NLRP3 inflammasome activation in mice exposed to chronic unpredictable mild stress

Inhibition of phosphodiesterase-4 (PDE4) produces robust anti-inflammatory and antidepressant-like effects in multiple animal models. However, the detailed mechanisms have not been well studied. Receptor for advanced glycation endproducts (RAGE) and inflammasome activation are implicated in the etiology of depression. Here, we aimed to investigate the involvement of RAGE and nucleotide-binding domain (NOD)–like receptor protein 3 (NLRP3) inflammasome in the antidepressant-like effects of PDE4 inhibition in mice. We found that inhibition of PDE4 by roflupram (ROF, 0.5, and 1.0 mg/kg, i.g.) exerted antidepressant-like effects in mice subjected to chronic unpredictable mild stress (CUMS). Simultaneously, ROF inhibited CUMS-induced microglial activation and restored the morphology of microglial cells in the hippocampus, as evidenced by reduced total process length, area, volume, number of branching points, number of terminal points and total sholl intersections of microglia. ROF also decreased the expression of ionized calcium-binding adapter molecule-1 and the level of interleukin-1β. Western blot analysis showed that PDE4 inhibition suppressed the high-mobility group box 1 protein (HMGB1)/RAGE signaling pathway, as the levels of HMGB1, RAGE, toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and nuclear factor κ-B were decreased in both hippocampus and cortex in mice after treatment with ROF. Moreover, ROF also attenuated the protein levels of NLRP3, the apoptosis-associated speck-like protein containing (ASC), and cysteine-requiring aspartate protease-1 (Caspase-1), which are key proteins in the NLRP3-mediated inflammasome signaling pathway. In summary, these results demonstrate that the down-regulation of HMGB1/RAGE signaling pathway and inflammasome suppression possibly contribute to the antidepressant-like effects of PDE4 inhibitors. And, ROF has potential as a candidate drug in the treatment of depression.     

RAGE: a journey from the complications of diabetes to disorders of the nervous system - striking a fine balance between injury and repair

The Receptor for Advanced Glycation End Products (RAGE) is a multiligand member of the immunoglobulin superfamily. RAGE interacts with AGEs, the products of nonenzymatic glycation/oxidation of proteins and lipids that accumulate in diverse settings, such as diabetes, inflammation, renal failure, pro-oxidant states and natural aging. In addition, RAGE is also a receptor for amyloid-beta peptide and beta-sheet fibril species. Recent studies underscore the premise that RAGE interacts with pro-inflammatory molecules, including S100/calgranulins and amphoterin, the latter also known as high mobility group box 1 (HMGB1). In chronic neurodegenerative disorders as well as in nerve tissue upon acute injury, evidence points to upregulation of both RAGE and these ligand families. In this review, we will discuss the implications of transient/self-limited upregulation of RAGE and its ligands, vs sustained/chronic upregulation of this axis in neurodegeneration vs repair in both the central and peripheral nervous systems. Experimental evidence supports the premise that RAGE bears both homeostatic and injurious properties in the nervous system, thereby highlighting "yin/yang" features of this receptor and its ligand families.     

S100B and schizophrenia

The research for peripheral biological markers of schizophrenia, although abundant, has been unfruitful. In the last 2 decades, the S100B protein has made its own room in this area of research. S100B is a calcium‐binding protein that has been proposed as a marker of astrocyte activation and brain dysfunction. Research results on S100B concentrations and schizophrenia clinical diagnosis are very consistent; patients with schizophrenia have higher S100B concentrations than healthy controls. The results regarding schizophrenia subtypes and clinical characteristics are not as conclusive. Age of patients, body mass index, illness duration and age at onset have been found to show no correlation, a positive correlation or a negative correlation with S100B levels. With respect to psychopathology, S100B data are inconclusive. Positive, negative and absence of correlation between S100B concentrations and positive and negative psychopathology have been reported. Methodological biases, such as day/night and seasonal variations, the use of anticoagulants to treat biological samples, the type of analytical technique to measure S100B and the different psychopathological scales to measure schizophrenia symptoms, are some of the factors that should be taken into account when researching into this area in order to reduce the variability of the reported results. The clinical implications of S100B changes in schizophrenia remain to be elucidated.