Norepinephrine Protects against Methamphetamine Toxicity through ß2-Adrenergic Receptors Promoting LC3 Compartmentalization.

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane ß2-adrenergic receptors (ARs). Evidence indicates that ß2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

The Mechanisms Mediated by α7 Acetylcholine Nicotinic Receptors May Contribute to Peripheral Nerve Regeneration.

Due to the microenvironment created by Schwann cell (SC) activity, peripheral nerve fibers are able to regenerate. Inflammation is the first response to nerve damage and the removal of cellular and myelin debris is essential in preventing the persistence of the local inflammation that may negatively affect nerve regeneration. Acetylcholine (ACh) is one of the neurotransmitters involved in the modulation of inflammation through the activity of its receptors, belonging to both the muscarinic and nicotinic classes. In this report, we evaluated the expression of α7 nicotinic acetylcholine receptors (nAChRs) in rat sciatic nerve, particularly in SCs, after peripheral nerve injury. α7 nAChRs are absent in sciatic nerve immediately after dissection, but their expression is significantly enhanced in SCs after 24 h in cultured sciatic nerve segments or in the presence of the proinflammatory neuropeptide Bradykinin (BK). Moreover, we found that activation of α7 nAChRs with the selective partial agonist ICH3 causes a decreased expression of c-Jun and an upregulation of uPA, MMP2 and MMP9 activity. In addition, ICH3 treatment inhibits IL-6 transcript level expression as well as the cytokine release. These results suggest that ACh, probably released from regenerating axons or by SC themselves, may actively promote through α7 nAChRs activation an anti-inflammatory microenvironment that contributes to better improving the peripheral nerve regeneration.

Autophagy in trimethyltin-induced neurodegeneration.

Autophagy is a degradative process playing an important role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria and endoplasmic reticulum, as well as eliminating intracellular pathogens. The autophagic process is important for balancing sources of energy at critical developmental stages and in response to nutrient stress. Recently, autophagy has been involved in the pathophysiology of neurodegenerative diseases although its beneficial (pro-survival) or detrimental (pro-death) role remains controversial. In the present review, we discuss the role of autophagy following intoxication with trimethyltin (TMT), an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. TMT is considered a useful tool to study the molecular mechanisms occurring in human neurodegenerative diseases such as Alzheimer's disease and temporal lobe epilepsy. This is also relevant in the field of environmental safety, since organotin compounds are used as heat stabilizers in polyvinyl chloride polymers, industrial and agricultural biocides, and as industrial chemical catalysts.

mTOR-Related Cell-Clearing Systems in Epileptic Seizures, an Update.

Recent evidence suggests that autophagy impairment is implicated in the epileptogenic mechanisms downstream of mTOR hyperactivation. This holds true for a variety of genetic and acquired epileptic syndromes besides malformations of cortical development which are classically known as mTORopathies. Autophagy suppression is sufficient to induce epilepsy in experimental models, while rescuing autophagy prevents epileptogenesis, improves behavioral alterations, and provides neuroprotection in seizure-induced neuronal damage. The implication of autophagy in epileptogenesis and maturation phenomena related to seizure activity is supported by evidence indicating that autophagy is involved in the molecular mechanisms which are implicated in epilepsy. In general, mTOR-dependent autophagy regulates the proliferation and migration of inter-/neuronal cortical progenitors, synapse development, vesicular release, synaptic plasticity, and importantly, synaptic clustering of GABAA receptors and subsequent excitatory/inhibitory balance in the brain. Similar to autophagy, the ubiquitin-proteasome system is regulated downstream of mTOR, and it is implicated in epileptogenesis. Thus, mTOR-dependent cell-clearing systems are now taking center stage in the field of epilepsy. In the present review, we discuss such evidence in a variety of seizure-related disorders and models. This is expected to provide a deeper insight into the molecular mechanisms underlying seizure activity.

Quantitative Ultrastructural Morphometry and Gene Expression of mTOR-Related Mitochondriogenesis within Glioblastoma Cells.

In glioblastoma (GBM) cells, an impairment of mitochondrial activity along with autophagy suppression occurs. Autophagy suppression in GBM promotes stemness, invasion, and poor prognosis. The autophagy deficit seems to be due, at least in part, to an abnormal up-regulation of the mammalian target of rapamycin (mTOR), which may be counteracted by pharmacological mTORC1 inhibition. Since autophagy activation is tightly bound to increased mitochondriogenesis, a defect in the synthesis of novel mitochondria is expected to occur in GBM cells. In an effort to measure a baseline deficit in mitochondria and promote mitochondriogenesis, the present study used two different GBM cell lines, both featuring mTOR hyperactivity. mTORC1 inhibition increases the expression of genes and proteins related to autophagy, mitophagy, and mitochondriogenesis. Autophagy activation was counted by RT-PCR of autophagy genes, LC3- immune-fluorescent puncta and immune-gold, as well as specific mitophagy-dependent BNIP3 stoichiometric increase in situ, within mitochondria. The activation of autophagy-related molecules and organelles after rapamycin exposure occurs concomitantly with progression of autophagosomes towards lysosomes. Remarkably, mitochondrial biogenesis and plasticity (increased mitochondrial number, integrity, and density as well as decreased mitochondrial area) was long- lasting for weeks following rapamycin withdrawal.

Role of the protease-activated receptor 1 in regulating the function of glial cells within central and peripheral nervous system.

Protease-activated receptor 1 (PAR1) is a cell surface receptor, which belongs to a family of G protein-coupled receptors and signals in response to multiple extracellular proteases. PAR1 is widely distributed in mammalian cells and tissues, including human glial cells. Within this context, PAR1 may participate to various activities promoted by glial cells. In fact, glia does not represent merely a glue in the nervous system but affects significantly various neuronal functions and activities being also significantly involved in the pathophysiology of various nervous system disorders. In this review, we summarize the current understanding of PAR1 expression and functions within glial cells both in the central and peripheral nervous system.

PAR1 activation affects the neurotrophic properties of Schwann cells.

Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR.

Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.

Lithium limits trimethyltin-induced cytotoxicity and proinflammatory response in microglia without affecting the concurrent autophagy impairment.

Trimethyltin (TMT) is a highly toxic molecule present as an environmental contaminant causing neurodegeneration particularly of the limbic system both in humans and in rodents. We recently described the occurrence of impairment in the late stages of autophagy in TMT-intoxicated astrocytes. Here we show that similarly to astrocytes also in microglia, TMT induces the precocious block of autophagy indicated by the accumulation of the autophagosome marker, microtubule associated protein light chain 3. Consistent with autophagy impairment we observe in TMT-treated microglia the accumulation of p62/SQSTM1, a protein specifically degraded through this pathway. Lithium has been proved effective in limiting neurodegenerations and, in particular, in ameliorating symptoms of TMT intoxication in rodents. In our in vitro model, lithium displays a pro-survival and anti-inflammatory action reducing both cell death and the proinflammatory response of TMT-treated microglia. In particular, lithium exerts these activities without reducing TMT-induced accumulation of light chain 3 protein. In fact, the autophagic block imposed by TMT is unaffected by lithium administration. These results are of interest as defects in the execution of autophagy are frequently observed in neurodegenerative diseases and lithium is considered a promising therapeutic agent for these pathologies. Thus, it is relevant that this cation can still maintain its pro-survival and anti-inflammatory role in conditions of autophagy block. Copyright © 2016 John Wiley & Sons, Ltd.

Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFß Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.

Rab11A Depletion in Microglia-Derived Extracellular Vesicle Proteome upon Beta-Amyloid Treatment.

Microglia, the macrophage-like glial cells, behave as sentinels against exogenous pathogens invading the neural tissue. Their commitment is not only confined to the defensive function, but they also perform balancing trophic activities such as neuronal postnatal development, remodeling and pruning of synapses. Likewise, microglia-derived extracellular vesicles (EVs) can play strategic roles in maintaining a healthy brain by modulating neuronal activity and by controlling neurite outgrowth as well as innate immune response. Nevertheless, strong evidence also points to their role in the development of neurodegenerative pathologies such as Alzheimer's disease (AD). Here, we explored EV protein content released by BV2 microglial cells in a resting state and after stimulation with beta-amyloid peptides (Aß), mimicking conditions occurring in AD. In the resting BV2 cells, we extended the list of proteins present in mouse microglia EV cargo with respect to those reported in the Vesiclepedia exosome database while, in amyloid-triggered microglia, we highlighted a pronounced drop in EV protein content. Focusing on Rab11A, a key factor in the recycling routes of amyloid species, we observed a dramatic decrease of this protein in Aß-treated microglia EV cargo with respect to the EVs from the untreated sample. This decrease might affect the delivery of Rab11A to neurons thus increasing the harmful amyloid burden in neuronal cells that eventually may lead to their death. We tentatively proposed that alterations observed in EVs derived from Aß-treated microglia may represent molecular features that, among others, shape the disease-associated microglial phenotype, a recently proposed subset of microglial population, present in neurodegenerative pathologies.

14-3-3ε marks the amyloid-stimulated microglia long-term activation.

Microglia-mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimer's disease. Microglial cells activation follows the deposition of amyloid ß fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimer's disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid ß peptides in triggering microglial activation. The response was monitored at the proteome level through a two-dimensional gel electrophoresis-based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14-3-3ε protein, found significantly upregulated in Aß-activated cells. 14-3-3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.

Protease Activated Receptor 1 and Its Ligands as Main Regulators of the Regeneration of Peripheral Nerves.

In contrast with the brain and spinal cord, peripheral nerves possess a striking ability to regenerate after damage. This characteristic of the peripheral nervous system is mainly due to a specific population of glial cells, the Schwann cells. Schwann cells promptly activate after nerve injury, dedifferentiate assuming a repair phenotype, and assist axon regrowth. In general, tissue injury determines the release of a variety of proteases which, in parallel with the degradation of their specific targets, also activate plasma membrane receptors known as protease-activated receptors (PARs). PAR1, the prototypical member of the PAR family, is also known as thrombin receptor and is present at the Schwann cell plasma membrane. This receptor is emerging as a possible regulator of the pro-regenerative capacity of Schwann cells. Here, we summarize the most recent literature data describing the possible contribution of PAR1 and PAR1-activating proteases in regulating the regeneration of peripheral nerves.

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide.

Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS.

Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.

Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

Potential Antidepressant Effects of Scutellaria baicalensis, Hericium erinaceus and Rhodiola rosea.

Recent studies focused on the pharmacology and feasibility of herbal compounds as a potential strategy to target a variety of human diseases ranging from metabolic to brain disorders. Accordingly, bioactive ingredients which are found within a variety of herbal compounds are reported to produce both neuroprotective and psychotropic activities which may help to combat mental disorders such as depression, anxiety, sleep disturbances and cognitive alterations. In the present manuscript, we focus on three herbs which appear effective in mitigating anxiety or depression with favourable risk-benefit profiles, namely Scutellaria baicalensis (S. baicalensis), Hericium erinaceus (H. erinaceus) and Rhodiola rosea (R. rosea). These three traditional folk medicinal herbs target the main biochemical events that are implicated in mental disorders, mimicking, to some extent, the mechanisms of action of conventional antidepressants and mood stabilizers with a wide margin of tolerability. In detail, they rescue alterations in neurotransmitter and neuro-endocrine systems, stimulate neurogenesis and the synthesis of neurotrophic factors, and they counteract oxidative stress, mitochondrial dysfunction and inflammation. Albeit the encouraging results that emerge from both experimental and clinical evidence, further studies are needed to confirm and better understand the mental-health promoting, and specifically, the antidepressant effects of these herbs.

Thrombin regulates the ability of Schwann cells to support neuritogenesis and to maintain the integrity of the nodes of Ranvier.

Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone-like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve ex vivo explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion.

Protease-activated receptor-1 regulates cytokine production and induces the suppressor of cytokine signaling-3 in microglia.

Protease-activated receptors (PARs) are cleaved and activated by thrombin and other extracellular proteases which are released during tissue trauma and inflammation. PAR-1 is the prototypic member of the PAR family and has been shown to be upregulated in several brain pathologies being expressed by neurons and glial cells. The present experiments show that the administration of the PAR-1 activating peptides (TRAP6 and TFLLR) inhibits the production of the pro-inflammatory cytokines TNF-alpha and IL-6 in microglial cells treated with lipopolysaccharide (LPS) while promoting the release of the anti-inflammatory cytokine IL-10. Conversely, the addition of the specific PAR-2 agonist SLIGRL had no effect on the amount of cytokines released following LPS treatment. Consistent with these data PAR-1, but not PAR-2, stimulation upregulates the expression of the suppressor of cytokine signaling-3 (SOCS-3). The present data support the hypothesis that in microglia PAR-1 may be involved in the regulation of inflammatory reactions modulating the balance between pro- and anti-inflammatory cytokines possibly through SOCS induction.

Blockade of chloride intracellular ion channel 1 stimulates Abeta phagocytosis.

In amyloid-beta (Abeta)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-alpha and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce Abeta phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on Abeta-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, Abeta25-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of Abeta phagocytosis, we treated BV-2 cells with biotinylated Abeta1-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased Abeta phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested Abeta1-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed Abeta1-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (iNOS), stimulated by Abeta, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates Abeta phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD.

2-Aminotetraline derivative protects from ischemia/reperfusion brain injury with a broad therapeutic window.

The effect of ST1942, a 2-aminotetraline derivative with anti-inflammatory properties, was evaluated in ischemia/reperfusion injury in CD1 and C57BL/6 mice. ST1942 or saline were injected intraperitoneally 30 min and 6, 24, 36 h after ischemia. Forty-eight hours after ischemia, ST1942 (25 mg/kg) reduced the infarct volume by 50% in CD1 and 61% in C57BL/6 mice. All subsequent data were obtained from the latter strain. The ischemic lesion was significantly reduced by 30% when the first injection was administered 6 h after ischemia, revealing a broad effective window. Degenerating neurons in striatum, cortex and hippocampus of ischemic mice were markedly decreased by ST1942. Also examined was the effect of ST1942 on general and focal neurological deficits for 4 days after ischemia. Mice receiving the drug twice daily showed constantly reduced deficits. We then investigated the cortical mRNA expression of some inflammatory and apoptotic genes by real-time PCR. Forty-eight hours after ischemia ST1942 treatment significantly counteracted ischemia-induced activation of IL-1beta, TNFalpha, and Bax, and enhanced the expression of the antiapoptotic gene, Bcl-2, showing in vivo anti-inflammatory and antiapoptotic actions. The microglial activation/macrophage recruitment in the ischemic lesion was strongly prevented in mice receiving ST1942. In neuron-microglia cocultures, ST1942 significantly counteracted LPS-induced cytotoxicity. Binding data and experiments on microglial cell cultures indicate that the anti-inflammatory effect of ST1942 may be due to its action on 5-HT2B receptors, thus highlighting the possibility that this 5-HT receptor subtype may represent a novel target for neuroprotective drugs in ischemic injury.