Decreased Astrocytic CCL2 Accounts for BAF-312 Effect on PBMCs Transendothelial Migration Through a Blood Brain Barrier in Vitro Model.

Disruption of the blood brain barrier (BBB) is a common event in several neurological diseases and in particular, in multiple sclerosis (MS), it contributes to the infiltration of the central nervous system by peripheral inflammatory cells. Sphingosine-1-phosphate (S1P) is a bioactive molecule with pleiotropic effects. Agonists of S1P receptors such as fingolimod and siponimod (BAF-312) are in clinical practice for MS and have been shown to preserve BBB function in inflammatory conditions. Using an in vitro BBB model of endothelial-astrocytes co-culture exposed to an inflammatory insult (tumor necrosis factor-α and interferon-γ; T&I), we show that BAF-312 reduced the migration of peripheral blood mononuclear cells (PBMCs) through the endothelial layer, only in the presence of astrocytes. This effect was accompanied by decreased expression of the adhesion molecule ICAM-1. BAF-312 also reduced the activation of astrocytes, by controlling NF-kB and NLRP3 induction and preventing the increase of proinflammatory cytokine and chemokines. Reduction of CCL2 by BAF-312 may be responsible for the observed effects and, accordingly, addition of exogenous CCL2 was able to counteract BAF-312 effects and rescued T&I responses on PBMC migration, ICAM-1 expression and astrocyte activation. The present results further point out BAF-312 effects on BBB properties, suggesting also the key role of astrocytes in mediating drug effects on endothelial function.

ß-amyloid and Oxidative Stress: Perspectives in Drug Development.

Alzheimer's Disease (AD) is a slow-developing neurodegenerative disorder in which the main pathogenic role has been assigned to ß-amyloid protein (Aß) that accumulates in extracellular plaques. The mechanism of action of Aß has been deeply analyzed and several membrane structures have been identified as potential mediators of its effect. The ability of Aß to modify neuronal activity, receptor expression, signaling pathways, mitochondrial function, and involvement of glial cells have been analyzed. In addition, extensive literature deals with the involvement of oxidative stress in Aß effects. Herein we focus more specifically on the reciprocal regulation of Aß, that causes oxidative stress, that favors Aß aggregation and toxicity and negatively affects the peptide clearance. Analysis of this strict interaction may offer novel opportunities for therapeutic intervention. Both common and new molecules endowed with antioxidant properties deserve attention in this regard.

Carnosine Prevents Aß-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-ß1.

Carnosine (ß-alanyl-L-histidine), a dipeptide, is an endogenous antioxidant widely distributed in excitable tissues like muscles and the brain. Carnosine is involved in cellular defense mechanisms against oxidative stress, including the inhibition of amyloid-beta (Aß) aggregation and the scavenging of reactive species. Microglia play a central role in the pathogenesis of Alzheimer's disease, promoting neuroinflammation through the secretion of inflammatory mediators and free radicals. However, the effects of carnosine on microglial cells and neuroinflammation are not well understood. In the present work, carnosine was tested for its ability to protect BV-2 microglial cells against oligomeric Aß1-42-induced oxidative stress and inflammation. Carnosine prevented cell death in BV-2 cells challenged with Aß oligomers through multiple mechanisms. Specifically, carnosine lowered the oxidative stress by decreasing NO and O2-• intracellular levels as well as the expression of iNOS and Nox enzymes. Carnosine also decreased the secretion of pro-inflammatory cytokines such as IL-1ß, simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-ß1. Carnosine also prevented Aß-induced neurodegeneration in mixed neuronal cultures challenged with Aß oligomers, and these neuroprotective effects were completely abolished by SB431542, a selective inhibitor of the type-1 TGF-ß receptor. Our data suggest a multimodal mechanism of action of carnosine underlying its protective effects on microglial cells against Aß toxicity with a key role of TGF-ß1 in mediating these protective effects.

Shedding of Microvesicles from Microglia Contributes to the Effects Induced by Metabotropic Glutamate Receptor 5 Activation on Neuronal Death.

Metabotropic glutamate (mGlu) receptor 5 is involved in neuroinflammation and has been shown to mediate reduced inflammation and neurotoxicity and to modify microglia polarization. On the other hand, blockade of mGlu5 receptor results in inhibition of microglia activation. To dissect this controversy, we investigated whether microvesicles (MVs) released from microglia BV2 cells could contribute to the communication between microglia and neurons and whether this interaction was modulated by mGlu5 receptor. Activation of purinergic ionotropic P2X7 receptor with the stable ATP analog benzoyl-ATP (100 µM) caused rapid MVs shedding from BV2 cells. Ionic currents through P2X7 receptor increased in BV2 cells pretreated for 24 h with the mGlu5 receptor agonist CHPG (200 µM) as by patch-clamp recording. This increase was blunted when microglia cells were activated by exposure to lipopolysaccharide (LPS; 0.1 µg/ml for 6 h). Accordingly, a greater amount of MVs formed after CHPG treatment, an effect prevented by the mGlu5 receptor antagonist MTEP (100 µM), as measured by expression of flotillin, a membrane protein enriched in MVs. Transferred MVs were internalized by SH-SY5Y neurons where they did not modify neuronal death induced by a low concentration of rotenone (0.1 µM for 24 h), but significantly increased rotenone neurotoxicity when shed from CHPG-treated BV2 cells. miR146a was increased in CHPG-treated MVs, an effect concealed in MVs from LPS-activated BV2 cells that showed per se an increase in miRNA146a levels. The present data support a role for microglia-shed MVs in mGlu5-mediated modulation of neuronal death and identify miRNAs as potential critical mediators of this interaction.

Effects of neuromyelitis optica-IgG at the blood-brain barrier in vitro.

OBJECTIVE: To address the hypothesis that physiologic interactions between astrocytes and endothelial cells (EC) at the blood-brain barrier (BBB) are afflicted by pathogenic inflammatory signaling when astrocytes are exposed to aquaporin-4 (AQP4) antibodies present in the immunoglobulin G (IgG) fraction of serum from patients with neuromyelitis optica (NMO), referred to as NMO-IgG. METHODS: We established static and flow-based in vitro BBB models incorporating co-cultures of conditionally immortalized human brain microvascular endothelial cells and human astrocyte cell lines with or without AQP4 expression. RESULTS: In astrocyte-EC co-cultures, exposure of astrocytes to NMO-IgG decreased barrier function, induced CCL2 and CXCL8 expression by EC, and promoted leukocyte migration under flow, contingent on astrocyte expression of AQP4. NMO-IgG selectively induced interleukin (IL)-6 production by AQP4-positive astrocytes. When EC were exposed to IL-6, we observed decreased barrier function, increased CCL2 and CXCL8 expression, and enhanced leukocyte transmigration under flow. These effects were reversed after application of IL-6 neutralizing antibody. CONCLUSIONS: Our results indicate that NMO-IgG induces IL-6 production by AQP4-positive astrocytes and that IL-6 signaling to EC decreases barrier function, increases chemokine production, and enhances leukocyte transmigration under flow.

Fluoxetine Prevents Aß1-42-Induced Toxicity via a Paracrine Signaling Mediated by Transforming-Growth-Factor-ß1.

Selective reuptake inhibitors (SSRIs), such as fluoxetine and sertraline, increase circulating Transforming-Growth-Factor-ß1 (TGF-ß1) levels in depressed patients, and are currently studied for their neuroprotective properties in Alzheimer's disease. TGF-ß1 is an anti-inflammatory cytokine that exerts neuroprotective effects against ß-amyloid (Aß)-induced neurodegeneration. In the present work, the SSRI, fluoxetine, was tested for the ability to protect cortical neurons against 1 µM oligomeric Aß1-42-induced toxicity. At therapeutic concentrations (100 nM-1 µM), fluoxetine significantly prevented Aß-induced toxicity in mixed glia-neuronal cultures, but not in pure neuronal cultures. Though to a lesser extent, also sertraline was neuroprotective in mixed cultures, whereas serotonin (10 nM-10 µM) did not mimick fluoxetine effects. Glia-conditioned medium collected from astrocytes challenged with fluoxetine protected pure cortical neurons against Aß toxicity. The effect was lost in the presence of a neutralizing antibody against TGF-ß1 in the conditioned medium, or when the specific inhibitor of type-1 TGF-ß1 receptor, SB431542, was added to pure neuronal cultures. Accordingly, a 24 h treatment of cortical astrocytes with fluoxetine promoted the release of active TGF-ß1 in the culture media through the conversion of latent TGF-ß1 to mature TGF-ß1. Unlike fluoxetine, both serotonin and sertraline did not stimulate the astrocyte release of active TGF-ß1. We conclude that fluoxetine is neuroprotective against Aß toxicity via a paracrine signaling mediated by TGF-ß1, which does not result from a simplistic SERT blockade.

Sphingosine 1-phosphate signaling at the blood-brain barrier.

The characterization of molecular pathways that modulate blood-brain barrier (BBB) function and integrity has been fueled by a growing body of literature implicating BBB dysfunction in a wide range of neurologic diseases. Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that has been effectively targeted by the immunomodulatory S1P1 functional antagonist fingolimod in the treatment of multiple sclerosis (MS). Investigation into the pathways modulated by S1P has revealed its important role in regulating BBB integrity via signaling through receptor isoforms on astrocytes and endothelial cells (ECs). Current evidence supports a significant role for S1P signaling as a key determinant of BBB permeability and hence as a potential pathogenic player or therapeutic target in diseases characterized by BBB dysfunction.

High mobility group box 1 contributes to wound healing induced by inhibition of dipeptidylpeptidase 4 in cultured keratinocytes.

Dipeptidyl peptidase 4 (DPP4) is expressed in various tissues, including the skin, and DPP4 inhibitors, that are currently used for the treatment of diabetes, may be effective also for complications of diabetes that affect the skin. To assess the role of DPP4 in keratinocytes, after creating a scratch wound in a monolayer of NTCC 2544 cells, we evaluated DPP4 expression and monitored wound repair over time, after treatment with the DPP4 inhibitor 1(((1-(hydroxymethyl)cyclopentyl)amino)acetyl)2,5-cis-pyrrolidinedicarbonitrile (DPP4-In). Expression of DPP4 increased early and was maintained up to 48 h following the scratch as shown by western blot and immunostaining. Treatment with 10 µM DPP4-In reduced DPP4 expression and significantly accelerated wound repair. This effect did not involve enhanced cell proliferation as shown by MTT proliferation assay, the lack of changes of cell cycle profiles and the slight inhibition of ERK phosphorylation. Enhancement of wound repair by DPP4 inhibition was prevented by the non-specific MMPs inhibitor GM6100 (5 µM). Treatment with DPP4-In increased the expression of high mobility group box 1 (HMGB1), a substrate of this enzyme, and exposure of NCTC 2544 cells to DPP4-In and exogenous HMGB1 (10 nM) produced a non-additive effect. Finally the healing promoting effect of DPP4-In was prevented by pretreatment with a neutralizing anti-HMGB1 antibody. The present results suggest that DPP4 inhibition contributes to enhanced wound healing by inducing keratinocytes to migrate into a scratched area. This effect seems to be independent of cell proliferation and involves enhanced production of HMGB1.

Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

The ability of the Blood Brain Barrier (BBB) to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS) activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS): fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF) that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.