Extracellular S100B Research Articles

Oneyear longitudinal study on biomarkers of blood–brain barrier permeability in COVID-19 patients

The pathophysiology behind neurological and cognitive sequelae of COVID-19 may be related to dysfunction of the blood-brain barrier (BBB) and previous research indicate transient neuronal injury and glial activation. The aim of this study was to investigate if COVID-19 is related to increased BBB permeability by analyzing leakage of biomarkers such as astrocyte-derived extracellular vesicles (EVs) and S100B. We also investigated whether levels of these biomarkers correlated with self-reported symptoms that persisted > 2 months. The samples in this 1-year follow-up study came from an ongoing longitudinal study of unvaccinated patients hospitalized for COVID-19 at Danderyd University Hospital, Stockholm, Sweden, between April and June 2020. Blood samples were collected at baseline and 4, 8, and 12 months after hospitalization. Information on self-reported clinical symptoms was collected at follow-up visits. A total of 102 patients were enrolled, and 47 completed all follow-up measurements. Peak levels of both biomarkers were observed at 4 months in the subset of 55 patients who were measured at this timepoint. At 12 months, the biomarkers had returned to baseline levels. The biomarkers were not correlated with any of the long-term self-reported symptoms. COVID-19 is associated with transient increased BBB permeability, shown by elevated levels of astrocyte biomarkers in plasma. However, these levels return to baseline 12 months post-infection and do not correlate with long-term symptoms. Further research is needed to unravel the underlying mechanisms causing long-term symptoms in COVID-19 patients.

Comparison of physiological state and conditions imitating the comorbidity of type 2 diabetes with Alzheimer's disease. The impact on: proliferation, H2O2, Aβ42, S100A8, S100B levels, neuronal protrusion and neurogenesis

Abstract Aim: Recent studies demonstrate that individuals with T2DM more likely to develop AD. While, disturbed glucose or insulin homeostasis, are at the forefront of AD research, not much is known about extra- and intracellular interplay between different forms of Aβ and microenviromental fluctuations of glucose or insulin concentrations in neuronal cells. Methods: We create conditions imitating the coexistence of T2DM with AD and compare the results with state where the neuropathological changes are yet not developed. We have investigated the effect of the physiological (Aβ40) and toxic amyloid form (Aβ25–35) and its co-incubation with glucose or insulin on neuronal proliferation, H2O2, Aβ42, S100B, S100A8 protein concentrations, mature neuronal protrusions and neurogenesis. Results: Aβ40 and Aβ25-35 with hyperglycaemia provoked stronger cytotoxic effect comparing to Aβ alone, while hyperinsulinemia dampen this effect. Opposite results were obtained in H2O2 measurement. Insulin stimulated Aβ42 generation when co-incubated with both Aβ forms. Aβ40 and Aβ25-35 caused similar pattern of extracellular S100B protein influx an concomitant cytosolic efflux. Neuronal protrusions and neurogenesis were initiated by co-incubation of Aβ40 with hyperglycaemia while reduced in Aβ25-35 and hyperglycemia or insulinemia. Significance: Our finding suggest that for understanding the biochemical origins of neuropathological amyloid β progression and potential involvement of metabolic disturbances in this process, it’s crucial to gasp preliminary interactions on cellular level. Our data can lead to hypothesis that S100B protein could be a potential modulator as well as indicator of prodromal neuropathological alterations.

Extracellular S100B inhibits A-type voltage-gated potassium currents and increases L-type voltage-gated calcium channel activity in dopaminergic neurons.

Parkinson's disease (PD) is associated with an increase in secreted S100B within the midbrain and cerebrospinal fluid. In addition, S100B overexpression in mice accelerates the loss of substantia nigra pars compacta dopaminergic (DA) neurons, suggesting a role for this protein in PD pathogenesis. We found that in the mouse SNc, S100B labeled astrocytic processes completely envelop the somata of tyrosine hydroxylase (TH) expressing DA neurons only in male mice. These data suggest that an increase in S100B secretion by astrocytes within the midbrain could play a role in DA dysfunction during early PD. We therefore asked if acute exposure to extracellular S100B alters the activity of identified TH expressing DA neurons in primary mouse midbrain cultures. Acute exposure to 50 pM S100B specifically inhibited A-type voltage-gated potassium currents in TH+ , but not TH- neurons. This was accompanied by ~2-fold increases in the frequency of both intrinsic firing, as well as L-type voltage-gated calcium channel (VGCC)-mediated calcium fluxes only in TH+ neurons. Further, exposure to 100 μM 4-aminopyridine (4-AP), an A-type voltage-gated potassium channel inhibitor, mimicked the S100B mediated increase in intrinsic firing and L-type VGCC-mediated calcium fluxes in TH+ neurons. Taken together, our finding that extracellular S100B alters the activity of native DA neurons via an inhibition of A-type voltage-gated potassium channels has important implications for understanding the pathophysiology of early PD.

Intracellular Protein S-Nitrosylation-A Cells Response to Extracellular S100B and RAGE Receptor.

Human S100B is a small, multifunctional protein. Its activity, inside and outside cells, contributes to the biology of the brain, muscle, skin, and adipocyte tissues. Overexpression of S100B occurs in Down Syndrome, Alzheimer’s disease, Creutzfeldt–Jakob disease, schizophrenia, multiple sclerosis, brain tumors, epilepsy, melanoma, myocardial infarction, muscle disorders, and sarcopenia. Modulating the activities of S100B, related to human diseases, without disturbing its physiological functions, is vital for drug and therapy design. This work focuses on the extracellular activity of S100B and one of its receptors, the Receptor for Advanced Glycation End products (RAGE). The functional outcome of extracellular S100B, partially, depends on the activation of intracellular signaling pathways. Here, we used Biotin Switch Technique enrichment and mass-spectrometry-based proteomics to show that the appearance of the S100B protein in the extracellular milieu of the mammalian Chinese Hamster Ovary (CHO) cells, and expression of the membrane-bound RAGE receptor, lead to changes in the intracellular S-nitrosylation of, at least, more than a hundred proteins. Treatment of the wild-type CHO cells with nanomolar or micromolar concentrations of extracellular S100B modulates the sets of S-nitrosylation targets inside cells. The cellular S-nitrosome is tuned differently, depending on the presence or absence of stable RAGE receptor expression. The presented results are a proof-of-concept study, suggesting that S-nitrosylation, like other post-translational modifications, should be considered in future research, and in developing tailored therapies for S100B and RAGE receptor-related diseases.

Time-Dependent Internalization of S100B by Mesenchymal Stem Cells via the Pathways of Clathrin- and Lipid Raft-Mediated Endocytosis.

Mesenchymal stem cells (MSCs) are promising tools for cancer therapy, but there is a risk of malignant transformation in their clinical application. Our previous work revealed that the paracrine protein S100B in the glioma microenvironment induces malignant transformation of MSCs and upregulates intracellular S100B, which could affect cell homeostasis by interfering with p53. The purpose of this study was to investigate whether extracellular S100B can be internalized by MSCs and the specific endocytic pathway involved in S100B internalization. By using real-time confocal microscopy and structured illumination microscopy (SIM), we visualized the uptake of fluorescently labeled S100B protein (S100B-Alexa488) and monitored the intracellular trafficking of internalized vesicles. The results showed that S100B-Alexa488 was efficiently internalized into MSCs in a time-dependent manner and transported through endolysosomal pathways. After that, we used chemical inhibitors and RNA interference approaches to investigate possible mechanisms involved in S100B-Alexa488 uptake. The internalization of S100B-Alexa488 was inhibited by pitstop-2 or dyngo-4a treatment or RNA-mediated silencing of clathrin or dynamin, and the lipid raft-mediated endocytosis inhibitors nystatin and MβCD. In conclusion, our findings show that clathrin and lipid rafts contribute to the internalization of S100B-Alexa488, which provides promising interventions for the safe application of MSCs in glioma therapy.

Arundic acid (ONO-2526) inhibits stimulated-S100B secretion in inflammatory conditions

Astrocytes respond to injury by modifying the expression profile of several proteins, including the S100 calcium-binding protein B (S100B), assumed to be a marker as well as a mediator of brain injury. AA is an inhibitor of S100B synthesis and plays a protective role in different models of brain injury, as decreases in S100B expression cause decreases in extracellular S100B. However, S100B mRNA expression, S100B protein content and S100B secretion do not always occur in association; as such, we herein investigated the effect of AA on S100B secretion, using different approaches with three stimulating conditions for S100B secretion, namely, low potassium medium, TNF-α (in hippocampal slices) and LPS exposure (in astrocyte cultures). Our data indicate that AA directly affects S100B secretion, indicating that the extracellular levels of this astroglial protein may be mediating the action of this compound. More importantly, AA had no effect on basal S100B secretion, but inhibited stimulated S100B secretion (stimulated either by the proinflammatory molecules, LPS or TNF-α, or by low potassium medium). Data from hippocampal slices that were directly exposed to AA, or from animals that received the acid by intracerebroventricular infusion, contribute to understanding its neuroprotective effect.

Association Between S100b Levels and COVID-19 Pneumonia: A Case Control Study.

Extracellular S100b effects are mediated by the receptor for advanced glycation end products (RAGE), which is the S100b membrane receptor. RAGE belongs to the immunoglobulin superfamily of cell surface molecules and serves as a multiligand receptor and is expressed in high abundance by alveolar type I (AT-I) cells in adult pulmonary tissue. This study aimed to provide an insight into the association between the severity of COVID-19 disease and serum S100b levels during admission to the emergency department (ED). A total of 64 patients (34 mild cases; 30 severe cases) were diagnosed with COVID-19 pneumonia and 30 healthy volunteers were admitted to study. Serum S100b levels were measured by using enzymle linked immunoassay method from blood serum samples. Serum S100b levels showed a significantly higher mean value in mild and severe disease cohorts than in healthy controls (p=0.036 and p=0.028 respectively). Receiver operating characteristic (ROC) analysis indicated greater area under the curve (AUC) for serum S100b levels of the COVID-19 patients (AUC=0.663, 95% CI=0.541-0.785; p=0.014). In addition, serum S100b concentration was measured as 151.7 ng/ml at 79.3% sensitivity and 51.7% specificity (p=0.014). Serum S100b protein levels can serve as a valuable clinical marker in establishing diagnosis of patients. Though not useful in identifying different stages of COVID-19 infection, serum S100b concentration along with other known markers can be utilized to reliably predict clinical severity along with other clinical parameters.

The contribution of S100B to the glioprotective effects of valproic and arundic acids.

Objective(s):Valproic and arundic acids are astrocytes-modulating agents with potential effects in the treatment of Alzheimer’s disease (AD). S100B is an astrocytic cytokine with a possible role in the pathogenesis of AD. In this study, we aimed to assess the glioprotective effects of valproic and arundic acids against amyloid-β-peptide (Aβ)-induced glial death and contribution of S100B to the glioprotective effects of these agents in an astrocytic culture. Materials and Methods:We used Aβ25–35 at a concentration of 200 μM in 1321N1 astrocyte cells. We treated the cells with valproic acid (0.5 and 1 mM) and/or arundic acid 50 µM for 24 hr. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) test was used to measure cell viability. The intracellular and extracellular S100B levels were measured using an ELISA kit. The data were analyzed using one-way analysis of variance followed by the Tukey’s test. Results:Aβ (200 µM) decreased the cell viability compared to the control group (P<0.001). Valproic acid (0.5 and 1 mM) and arundic acid (50 µM) ameliorated the gliotoxic effects of Aβ (P<0.05). The Aβ-treated group had higher S100B levels (both intracellular and extracellular) compared to the negative control groups (P<0.001). Arundic and valproic acids (0.5 and 1 mM) decreased both the intracellular and extracellular S100B levels compared to the Aβ-treated group (P<0.001).Conclusion:By considering homeostatic and neuroprotective functions of astrocyte, the astroprotective effects and the attenuation of S100B level may be responsible, at least in part, for the beneficial effects of valproic and arundic acids in AD.

GABAA Modulation of S100B Secretion in Acute Hippocampal Slices and Astrocyte Cultures.

Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABAA receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABAA communication between astrocytes and neurons. The effects of two putative agonists of GABAA, β-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABAA receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response.

S100B protein activates a RAGE-dependent autocrine loop in astrocytes: implications for its role in the propagation of reactive gliosis.

Extracellular S100B dramatically increases after brain injury. While low S100B levels are neuroprotective, micromolar S100B levels have shown in vitro to activate microglia and facilitate neuronal death. In astrocytes, S100B exposure activates nuclear factor kappa B (NF-κB) and induces pro-inflammatory mediators. On microglia and neurons S100B effects are essentially mediated by receptor for advanced glycation end products (RAGE)/NF-κB, but it is not clear if these intracellular cascades are activated by different S100B levels in astrocytes and whether increased extracellular S100B is sufficient to induce reactive gliosis. A better understanding of these pathways is essential for developing successful strategies to preserve the beneficial S100B effects after brain injury. Here, we show that microglia-depleted cultured astrocytes exposed to S100B mimicked several features of reactive gliosis by activating RAGE/Rac-1-Cdc42, RAGE/Erk-Akt or RAGE/NF-κB-dependent pathways. S100B effects include RAGE/Rac1-Cdc42-dependent astroglial hypertrophy and facilitation of migration as well as increased mitosis. S100B exposure improved the astrocytic survival to oxidative stress, an effect that requires Erk/Akt. S100B also activates NF-κB in a dose-dependent manner; increases RAGE proximal promoter transcriptional activity and augmented endogenous RAGE expression. S100B-exposed astrocytes showed a pro-inflammatory phenotype with expression of Toll-like receptor 2 (TLR 2), inducible nitric oxide synthase (iNOS) and interleukin 1-beta (IL-1β), and facilitated neuronal death induced by oxygen-glucose deprivation. In vivo, intracerebral infusion of S100B was enough to induce an astroglial reactive phenotype. Together, these findings demonstrate that extracellular S100B in the micromolar level activates different RAGE-dependent pathways that turn astrocytes into a pro-inflammatory and neurodegenerative phenotype. We propose that S100B turns astrocytes into a reactive phenotype in a RAGE-dependent manner but engaging different intracellular pathways. While both nanomolar and micromolar S100B turn astrocytes into a reactive phenotype, micromolar S100B induces a conversion into a pro-inflammatory-neurodegenerative profile that facilitates neuronal death of OGD-exposed neurons. We think that S100B/RAGE interaction is essential to expand reactive gliosis in the injured brain being a tempting target for limiting reactive gliosis to prevent the glial conversion into the neurodegenerative profile.

S100B as a potential biomarker for the detection of cytotoxicity of melanocytes

Skin irritation is one of the most common adverse reactions in hydroquinone (HQ) and retinoic acid (RA). Although melanocytes have rarely been considered to be involved in skin irritation, RA and particularly HQ could induce melanocyte toxicity, resulting in depigmentation. We chose S100B as a candidate gene for melanocytotoxicity from a genome-wide transcriptional profiling analysis after applying irritant doses of HQ, RA and sodium lauryl sulphate (SLS) to cultures of keratinocytes and/or melanocytes. In this study, the role of S100B on melanocyte viability and cytotoxicity was examined. S100B was detected in melanocytes, but not in keratinocytes or fibroblasts. Melanocytes after treatment with increasing concentrations of HQ, RA, SLS and urushiol showed significant increases in intracellular and extracellular S100B expression with reduced viable cell number and increased release of lactate dehydrogenase. No RAGE expression and no significant function of CD166/ALCAM in melanocyte survival and cytotoxicity favoured the role of intracellular S100B in chemically irritated melanocytes. S100B knock-down increased apoptosis through inhibition of PI3K/AKT, NF-κB and ERK activation, suggesting the increased intracellular S100B expression by chemical irritation as a compensatory reaction to reduce cytotoxicity. The numerical decrease in S100B/c-kit-double-positive melanocytes was also examined in human skin epidermis irritated by HQ or RA with stronger staining intensities of S100B. Collectively, the decrease in viable cell number by reduced intracellular S100B levels in vitro and by chemical irritation in vivo suggests that S100B could be a potential biomarker for melanocytes cytotoxicity.

Structural insights into the interaction of human S100B and basic fibroblast growth factor (FGF2): Effects on FGFR1 receptor signaling

S100B is a calcium sensing protein belonging to the S100 protein family with intracellular and extracellular roles. It is one of the EF hand homodimeric proteins, which is known to interact with various protein targets to regulate varied biological functions. Extracellular S100B has been recently reported to interact with FGF2 in a RAGE-independent manner. However, the recognition mechanism of S100B–FGF2 interaction at the molecular level remains unclear. In this study, the critical residues on S100B–FGF2 interface were mapped by combined information derived from NMR spectroscopy and site directed mutagenesis experiments. Utilizing NMR titration data, we generated the structural models of S100B–FGF2 complex from the computational docking program, HADDOCK which were further proved stable during 15ns unrestrained molecular dynamics (MD) simulations. Isothermal titration calorimetry studies indicated S100B interaction with FGF2 is an entropically favored process implying dominant role of hydrophobic contacts at the protein–protein interface. Residue level information of S100B interaction with FGF2 was useful to understand the varied target recognition ability of S100B and further explained its role in effecting extracellular signaling diversity. Mechanistic insights into the S100B–FGF2 complex interface and cell-based assay studies involving mutants led us to conclude the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation.

S100B Induces the Release of Pro-Inflammatory Cytokines in Alveolar Type I-like Cells

S100B, a 21kDa cytosolic calcium-binding protein of the EF-hand type, present in high abundance in the brain, stimulates inflammatory responses in different cellular types inside and outside the central nervous system. Most of extracellular S100B effects are mediated by Receptor for Advanced Glycation End-products (RAGE). RAGE is highly expressed in lung by Alveolar Type-I (AT-I) cells and its activation contributes to ALI/ARDS pathogenesis. In this in-vitro study, we tested the hypothesis that S100B stimulates an ATI-derived cell line (R3/1) to secrete inflammatory mediators involved in lung inflammation. Our main result is that S100B stimulates R3/1 cells to secrete TNF-alpha and IL-6 (well-known pro-inflammatory cytokines in lung inflammation and neurogenic pulmonary edema), but not sICAM-1, CINC-1 or CINC-3. Soluble RAGE (sRAGE) reduced S100B-dependent secretion of TNF-alpha but did not decrease S100B-dependent secretion of IL-6. Moreover, in absence of S100B, sRAGE enhanced IL-6 release. This study demonstrates that in vitro S100B dose-dependently stimulated R3/1 cells, to enhance the secretion of TNF-alpha and IL-6; S100B pro-inflammatory activity might be mediated at least in part by RAGE. Besides acting as decoy receptor, sRAGE could have pro-inflammatory properties.

Outcome following severe traumatic brain injury TBI correlates with serum S100B but not brain extracellular fluid S100B: An intracerebral microdialysis study

S100B protein is released by astrocytes into the brain extracellular fluid following acute brain injury and elevated levels in CSF and serum have been shown to correlate with patient outcome following traumatic brain injury. A prospective study of brain extracellular fluid (ECF) and serum S100B levels in 12 patients with severe head injury (GCS ≤ 8) was undertaken using intracerebral microdialysis to investigate whether a correlation with ECF S100B and outcome could be confirmed. Patient outcomes were assessed at 6 months using the Glasgow Outcome Scale (GOS) and divided into two outcome groups: group A, 8 survivors with either a good recovery or moderate disability (GOS scores of 4 or 5); and group B, 4 patients who died (GOS 1). Peak serum levels of S100B were significantly greater in group B (mean 6.03 ng/ml) compared with group A (mean 0.73 ng/ml) (P = 0.009). Group A had a mean peak S100B in the extracellular compartment of 186 ng/ml compared to 150 ng/ml in group B. There was no significant difference between the mean peak brain ECF S100B concentrations for the 2 outcome groups (P = 0.932). We confirm that intracerebral microdialysis can be used to sample S100B concentrations from brain extracellular fluid and our results suggest that the ECF S100B levels were variable and that there was no significant difference between the good outcome and poor outcome groups. In contrast, the serum levels of S100B of patients with a poor outcome were significantly higher than those with a good outcome.

S100B protein in tissue development, repair and regeneration

The Ca(2+)-binding protein of the EF-hand type, S100B, exerts both intracellular and extracellular regulatory activities. As an intracellular regulator, S100B is involved in the regulation of energy metabolism, transcription, protein phosphorylation, cell proliferation, survival, differentiation and motility, and Ca(2+) homeostasis, by interacting with a wide array of proteins (i.e., enzymes, enzyme substrates, cytoskeletal subunits, scaffold/adaptor proteins, transcription factors, ubiquitin E3 ligases, ion channels) in a restricted number of cell types. As an extracellular signal, S100B engages the pattern recognition receptor, receptor for advanced glycation end-products (RAGE), on immune cells as well as on neuronal, astrocytic and microglial cells, vascular smooth muscle cells, skeletal myoblasts and cardiomyocytes. However, RAGE may not be the sole receptor activated by S100B, the protein being able to enhance bFGF-FGFR1 signaling by interacting with FGFR1-bound bFGF in particular cell types. Moreover, extracellular effects of S100B vary depending on its local concentration. Increasing evidence suggests that at the concentration found in extracellular fluids in normal physiological conditions and locally upon acute tissue injury, which is up to a few nM levels, S100B exerts trophic effects in the central and peripheral nervous system and in skeletal muscle tissue thus participating in tissue homeostasis. The present commentary summarizes results implicating intracellular and extracellular S100B in tissue development, repair and regeneration.

Functions of S100 Proteins

The S100 protein family consists of 24 members functionally distributed into three main subgroups: those that only exert intracellular regulatory effects, those with intracellular and extracellular functions and those which mainly exert extracellular regulatory effects. S100 proteins are only expressed in vertebrates and show cell-specific expression patterns. In some instances, a particular S100 protein can be induced in pathological circumstances in a cell type that does not express it in normal physiological conditions. Within cells, S100 proteins are involved in aspects of regulation of proliferation, differentiation, apoptosis, Ca2+ homeostasis, energy metabolism, inflammation and migration/invasion through interactions with a variety of target proteins including enzymes, cytoskeletal subunits, receptors, transcription factors and nucleic acids. Some S100 proteins are secreted or released and regulate cell functions in an autocrine and paracrine manner via activation of surface receptors (e.g. the receptor for advanced glycation end-products and toll-like receptor 4), G-protein-coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and N-glycans. Extracellular S100A4 and S100B also interact with epidermal growth factor and basic fibroblast growth factor, respectively, thereby enhancing the activity of the corresponding receptors. Thus, extracellular S100 proteins exert regulatory activities on monocytes/macrophages/microglia, neutrophils, lymphocytes, mast cells, articular chondrocytes, endothelial and vascular smooth muscle cells, neurons, astrocytes, Schwann cells, epithelial cells, myoblasts and cardiomyocytes, thereby participating in innate and adaptive immune responses, cell migration and chemotaxis, tissue development and repair, and leukocyte and tumor cell invasion.

Functions of S100 Proteins

The S100 protein family consists of 24 members functionally distributed into three main subgroups: those that only exert intracellular regulatory effects, those with intracellular and extracellular functions and those which mainly exert extracellular regulatory effects. S100 proteins are only expressed in vertebrates and show cell-specific expression patterns. In some instances, a particular S100 protein can be induced in pathological circumstances in a cell type that does not express it in normal physiological conditions. Within cells, S100 proteins are involved in aspects of regulation of proliferation, differentiation, apoptosis, Ca2+ homeostasis, energy metabolism, inflammation and migration/invasion through interactions with a variety of target proteins including enzymes, cytoskeletal subunits, receptors, transcription factors and nucleic acids. Some S100 proteins are secreted or released and regulate cell functions in an autocrine and paracrine manner via activation of surface receptors (e.g. the receptor for advanced glycation end-products and toll-like receptor 4), G-protein-coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and N-glycans. Extracellular S100A4 and S100B also interact with epidermal growth factor and basic fibroblast growth factor, respectively, thereby enhancing the activity of the corresponding receptors. Thus, extracellular S100 proteins exert regulatory activities on monocytes/macrophages/microglia, neutrophils, lymphocytes, mast cells, articular chondrocytes, endothelial and vascular smooth muscle cells, neurons, astrocytes, Schwann cells, epithelial cells, myoblasts and cardiomyocytes, thereby participating in innate and adaptive immune responses, cell migration and chemotaxis, tissue development and repair, and leukocyte and tumor cell invasion. Keywords: S100 protein, calcium binding, calcium homeostasis, DAMPs, inflammation, cancer, tissue repair/regeneration, RAGE, TLRs, signaling pathways

Effects of the putative antipsychotic alstonine on glutamate uptake in acute hippocampal slices

A dysfunctional glutamatergic system is thought to be central to the negative symptoms and cognitive deficits recognized as determinant to the poor quality of life of people with schizophrenia. Modulating glutamate uptake has, thus, been suggested as a novel target for antipsychotics. Alstonine is an indole alkaloid sharing with atypical antipsychotics the profile in animal models relevant to schizophrenia, though divergent in its mechanism of action. The aim of this study was to evaluate the effects of alstonine on glutamate uptake. Additionally, the effects on glutathione content and extracellular S100B levels were assessed. Acute hippocampal slices were incubated with haloperidol (10μM), clozapine (10 and 100μM) or alstonine (1–100μM), alone or in combination with apomorphine (100μM), and 5-HT2 receptor antagonists (0.01μM altanserin and 0.1μM SB 242084). A reduction in glutamate uptake was observed with alstonine and clozapine, but not haloperidol. Apomorphine abolished the effect of clozapine, whereas 5-HT2A and 5-HT2C antagonists abolished the effects of alstonine. Increased levels of glutathione were observed only with alstonine, also the only compound that failed to decrease the release of S100B. This study shows that alstonine decreases glutamate uptake, which may be beneficial to the glutamatergic deficit observed in schizophrenia. Noteworthily, the decrease in glutamate uptake is compatible with the reversal of MK-801-induced social interaction and working memory deficits. An additional potential benefit of alstonine as an antipsychotic is its ability to increase glutathione, a key cellular antioxidant reported to be decreased in the brain of patients with schizophrenia. Adding to the characterization of the novel mechanism of action of alstonine, the lack of effect of apomorphine in alstonine-induced changes in glutamate uptake reinforces that D2 receptors are not primarily implicated. Though clearly mediated by 5-HT2A and 5-HT2C serotonin receptors, the precise mechanisms that result in the effects of alstonine on glutamate uptake warrant elucidation.

Poster #214 STUDY ON THE CONTROL OF SYMPTOMS AND IMPACT ON FUNCTIONING IN HOSPITAL INTERVENTION DURING A FIRST PSYCHOTIC EPISODE

A dysfunctional glutamatergic system is thought to be central to the negative symptoms and cognitive deficits recognized as determinant to the poor quality of life of people with schizophrenia. Modulating glutamate uptake has, thus, been suggested as a novel target for antipsychotics. Alstonine is an indole alkaloid sharing with atypical antipsychotics the profile in animal models relevant to schizophrenia, though divergent in its mechanism of action. The aim of this study was to evaluate the effects of alstonine on glutamate uptake. Additionally, the effects on glutathione content and extracellular S100B levels were assessed. Acute hippocampal slices were incubated with haloperidol (10 μM), clozapine (10 and 100 μM) or alstonine (1–100 μM), alone or in combination with apomorphine (100 μM), and 5-HT2 receptor antagonists (0.01 μM altanserin and 0.1 μM SB 242084). A reduction in glutamate uptake was observed with alstonine and clozapine, but not haloperidol. Apomorphine abolished the effect of clozapine, whereas 5-HT2A and 5-HT2C antagonists abolished the effects of alstonine. Increased levels of glutathione were observed only with alstonine, also the only compound that failed to decrease the release of S100B. This study shows that alstonine decreases glutamate uptake, which may be beneficial to the glutamatergic deficit observed in schizophrenia. Noteworthily, the decrease in glutamate uptake is compatible with the reversal of MK-801-induced social interaction and working memory deficits. An additional potential benefit of alstonine as an antipsychotic is its ability to increase glutathione, a key cellular antioxidant reported to be decreased in the brain of patients with schizophrenia. Adding to the characterization of the novel mechanism of action of alstonine, the lack of effect of apomorphine in alstonine-induced changes in glutamate uptake reinforces that D2 receptors are not primarily implicated. Though clearly mediated by 5-HT2A and 5-HT2C serotonin receptors, the precise mechanisms that result in the effects of alstonine on glutamate uptake warrant elucidation.

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.