MicroRNA-451 regulates LKB1/AMPK signaling and allows adaptation to metabolic stress in glioma cells.

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Failure to Target RANKL Signaling Through p38-MAPK Results in Defective Osteoclastogenesis in the Microphthalmia Cloudy-Eyed Mutant.

The Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C-terminal end. Here we show that the Mitf(ce) allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitf(ce/ce) mice. The loss of the leucine zipper domain in Mitf(ce/ce) mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitf(ce). This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site-directed mutations in the docking site interfered with the interaction between MITF and its co-factors FUS and BRG1. MITF-ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation.

Extracellular Vesicles and MicroRNAs: Their Role in Tumorigenicity and Therapy for Brain Tumors.

MicroRNAs are small non-coding RNAs which mediate post-transcriptional gene regulation. Recently, microRNAs have also been found to be localized to the extracellular space, often encapsulated in secreted extracellular vesicles (EVs). This tandem of EVs and tissue-specific expressed/secreted microRNAs that can be taken up by neighboring or distant recipient cells, leading to changes in gene expression-suggests a cell-specialized role in physiological and pathological conditions. The complexity of solid tumors and their distinct pathophysiology relies on interactive communications between the various cell types in the neoplasm (tumor, endothelial, or macrophages, for instance). Understanding how such EV/microRNA-mediated communication occurs may actually lead to avenues for therapeutic exploitation and/or intervention, particularly for the most formidable cancers, such as those in the brain. In this review, the role of microRNAs/EVs in brain tumors will be discussed with emphasis on how these molecules could be utilized for tumor therapy.

The multifunctional protein fused in sarcoma (FUS) is a coactivator of microphthalmia-associated transcription factor (MITF).

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a signaling effector engaged by macrophage colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). MITF exerts its regulatory functions through its association with cofactors. Discovering the identity of its various partners will provide insights into the mechanisms governing gene expression during osteoclastogenesis. Here, we demonstrate that the proto-oncogene fused in sarcoma (FUS), the chromatin remodeling ATPase BRG1, and MITF form a trimeric complex that is regulated by phosphorylation of MITF at Ser-307 by p38 MAPK during osteoclast differentiation. FUS was recruited to MITF target gene promoters Acp5 and Ctsk during osteoclast differentiation, and FUS knockdown abolished efficient transcription of Acp5 and Ctsk. Furthermore, sumoylation of MITF at Lys-316, known to negatively regulate MITF transcriptional activity, inhibited MITF interactions with FUS and BRG1 in a p38 MAPK phosphorylation-dependent manner. These results demonstrate that FUS is a coregulator of MITF activity and provide new insights into how the RANKL/p38 MAPK signaling nexus controls gene expression in osteoclasts.

MEK-1 activates C-Raf through a Ras-independent mechanism.

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using (32)P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating the C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the down-regulation of RKIP and MST2.

MiR-128-3p - a gray eminence of the human central nervous system.

MicroRNA-128-3p (miR-128-3p) is a versatile molecule with multiple functions in the physiopathology of the human central nervous system. Perturbations of miR-128-3p, which is enriched in the brain, contribute to a plethora of neurodegenerative disorders, brain injuries, and malignancies, as this miRNA is a crucial regulator of gene expression in the brain, playing an essential role in the maintenance and function of cells stemming from neuronal lineage. However, the differential expression of miR-128-3p in pathologies underscores the importance of the balance between its high and low levels. Significantly, numerous reports pointed to miR-128-3p as one of the most depleted in glioblastoma, implying it is a critical player in the disease's pathogenesis and thus may serve as a therapeutic agent for this most aggressive form of brain tumor. In this review, we summarize the current knowledge of the diverse roles of miR-128-3p. We focus on its involvement in the neurogenesis and pathophysiology of malignant and neurodegenerative diseases. We also highlight the promising potential of miR-128-3p as an antitumor agent for the future therapy of human cancers, including glioblastoma, and as the linchpin of brain development and function, potentially leading to the development of new therapies for neurological conditions.

Circular RNAs-New Kids on the Block in Cancer Pathophysiology and Management.

The ever-increasing number of cancer cases and persistently high mortality underlines the urgent need to acquire new perspectives for developing innovative therapeutic approaches. As the research on protein-coding genes brought significant yet only incremental progress in the development of anticancer therapy, much attention is now devoted to understanding the role of non-coding RNAs (ncRNAs) in various types of cancer. Recent years have brought about the awareness that ncRNAs recognized previously as "dark matter" are, in fact, key players in shaping cancer development. Moreover, breakthrough discoveries concerning the role of a new group of ncRNAs, circular RNAs, have evidenced their high importance in many diseases, including malignancies. Therefore, in the following review, we focus on the role of circular RNAs in cancer, particularly in cancer stem-like cells, summarize their mechanisms of action, and provide an overview of the state-of-the-art toolkits to study them.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-12.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Pretreatment with mGluR2 or mGluR3 Agonists Reduces Apoptosis Induced by Hypoxia-Ischemia in Neonatal Rat Brains.

Hypoxia-ischemia (HI) in an immature brain results in energy depletion and excessive glutamate release resulting in excitotoxicity and oxidative stress. An increase in reactive oxygen species (ROS) production induces apoptotic processes resulting in neuronal death. Activation of group II mGluR was shown to prevent neuronal damage after HI. The application of agonists of mGluR3 (N-acetylaspartylglutamate; NAAG) or mGluR2 (LY379268) inhibits the release of glutamate and reduces neurodegeneration in a neonatal rat model of HI, although the exact mechanism is not fully recognized. In the present study, the effects of NAAG (5 mg/kg) and LY379268 (5 mg/kg) application (24 h or 1 h before experimental birth asphyxia) on apoptotic processes as the potential mechanism of neuroprotection in 7-day-old rats were investigated. Intraperitoneal application of NAAG or LY379268 at either time point before HI significantly reduced the number of TUNEL-positive cells in the CA1 region of the ischemic brain hemisphere. Both agonists reduced expression of the proapoptotic Bax protein and increased expression of Bcl-2. Decreases in HI-induced caspase-9 and caspase-3 activity were also observed. Application of NAAG or LY379268 24 h or 1 h before HI reduced HIF-1α formation likely by reducing ROS levels. It was shown that LY379268 concentration remains at a level that is required for activation of mGluR2 for up to 24 h; however, NAAG is quickly metabolized by glutamate carboxypeptidase II (GCPII) into glutamate and N-acetyl-aspartate. The observed effect of LY379268 application 24 h or 1 h before HI is connected with direct activation of mGluR2 and inhibition of glutamate release. Based on the data presented in this study and on our previous findings, we conclude that the neuroprotective effect of NAAG applied 1 h before HI is most likely the result of a combination of mGluR3 and NMDA receptor activation, whereas the beneficial effects of NAAG pretreatment 24 h before HI can be explained by the activation of NMDA receptors and induction of the antioxidative/antiapoptotic defense system triggered by mild excitotoxicity in neurons. This response to NAAG pretreatment is consistent with the commonly accepted mechanism of preconditioning.

Oncolytic Virus Therapy Alters the Secretome of Targeted Glioblastoma Cells.

Oncolytic virus (OV) therapy, which is being tested in clinical trials for glioblastoma, targets cancer cells, while triggering immune cells. Yet OV sensitivity varies from patient to patient. As OV therapy is regarded as an anti-tumor vaccine, by making OV-infected cancer cells secrete immunogenic proteins, linking these proteins to transcriptome would provide a measuring tool to predict their sensitivity. A set of six patient-derived glioblastoma cells treated ex-vivo with herpes simplex virus type 1 (HSV1) modeled a clinical setting of OV infection. The cellular transcriptome and secreted proteome (separated into extracellular vesicles (EV) and EV-depleted fractions) were analyzed by gene microarray and mass-spectroscopy, respectively. Data validation and in silico analysis measured and correlated the secretome content with the response to infection and patient survival. Glioblastoma cells reacted to the OV infection in a seemingly dissimilar fashion, but their transcriptomes changed in the same direction. Therefore, the upregulation of transcripts encoding for secreted proteins implies a common thread in the response of cancer cells to infection. Indeed, the OV-driven secretome is linked to the immune response. While these proteins have distinct membership in either EV or EV-depleted fractions, it is their co-secretion that augments the immune response and associates with favorable patient outcomes.

Anti-EGFR VHH-armed death receptor ligand-engineered allogeneic stem cells have therapeutic efficacy in diverse brain metastatic breast cancers.

Basal-like breast cancer (BLBC) shows brain metastatic (BM) capability and overexpresses EGFR and death-receptors 4/5 (DR4/5); however, the anatomical location of BM prohibits efficient drug-delivery to these targetable markers. In this study, we developed BLBC-BM mouse models featuring different patterns of BMs and explored the versatility of estem cell (SC)-mediated bi-functional EGFR and DR4/5-targeted treatment in these models. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, EVDRL [anti-EGFR VHH (EV) fused to DR ligand (DRL)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the EV domain facilitated in augmenting DR4/5-DRL binding and enhancing DRL-induced apoptosis. EVDRL secreting stem cells alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. This study reports mechanism based simultaneous targeting of EGFR and DR4/5 in BLBC and defines a new treatment paradigm for treatment of BM.

Eos, MITF, and PU.1 recruit corepressors to osteoclast-specific genes in committed myeloid progenitors.

Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.

Oxidative Stress-Part of the Solution or Part of the Problem in the Hypoxic Environment of a Brain Tumor.

Rapid growth of brain tumors such as glioblastoma often results in oxygen deprivation and the emergence of hypoxic zones. In consequence, the enrichment of reactive oxygen species occurs, harming nonmalignant cells and leading them toward apoptotic cell death. However, cancer cells survive such exposure and thrive in a hypoxic environment. As the mechanisms responsible for such starkly different outcomes are not sufficiently explained, we aimed to explore what transcriptome rearrangements are used by glioblastoma cells in hypoxic areas. Using metadata analysis of transcriptome in different subregions of the glioblastoma retrieved from the Ivy Glioblastoma Atlas Project, we created the reactive oxygen species-dependent map of the transcriptome. This map was then used for the analysis of differential gene expression in the histologically determined cellular tumors and hypoxic zones. The gene ontology analysis cross-referenced with the clinical data from The Cancer Genome Atlas revealed that the metabolic shift is one of the major prosurvival strategies applied by cancer cells to overcome hypoxia-related cytotoxicity.

Hypoxic Roadmap of Glioblastoma-Learning about Directions and Distances in the Brain Tumor Environment.

Malignant brain tumor-glioblastoma is not only difficult to treat but also hard to study and model. One of the reasons for these is their heterogeneity, i.e., individual tumors consisting of cancer cells that are unlike each other. Such diverse cells can thrive due to the simultaneous co-evolution of anatomic niches and adaption into zones with distorted homeostasis of oxygen. It dampens cytotoxic and immune therapies as the response depends on the cellular composition and its adaptation to hypoxia. We explored what transcriptome reposition strategies are used by cells in the different areas of the tumor. We created the hypoxic map by differential expression analysis between hypoxic and cellular features using RNA sequencing data cross-referenced with the tumor's anatomic features (Ivy Glioblastoma Atlas Project). The molecular functions of genes differentially expressed in the hypoxic regions were analyzed by a systematic review of the gene ontology analysis. To put a hypoxic niche signature into a clinical context, we associated the model with patients' survival datasets (The Cancer Genome Atlas). The most unique class of genes in the hypoxic area of the tumor was associated with the process of autophagy. Both hypoxic and cellular anatomic features were enriched in immune response genes whose, along with autophagy cluster genes, had the power to predict glioblastoma patient survival. Our analysis revealed that transcriptome responsive to hypoxia predicted worse patients' outcomes by driving tumor cell adaptation to metabolic stress and immune escape.

Microphthalmia-associated transcription factor interactions with 14-3-3 modulate differentiation of committed myeloid precursors.

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a target for signaling pathways engaged by colony stimulating factor (CSF)-1 and receptor-activator of nuclear factor-kappaB ligand (RANKL). Work presented here demonstrates that MITF can shuttle from cytoplasm to nucleus dependent upon RANKL/CSF-1 action. 14-3-3 was identified as a binding partner of MITF in osteoclast precursors, and overexpression of 14-3-3 in a transgenic model resulted in increased cytosolic localization of MITF and decreased expression of MITF target genes. MITF/14-3-3 interaction was phosphorylation dependent, and Ser173 residue, within the minimal interaction region of amino acid residues 141-191, was required. The Cdc25C-associated kinase (C-TAK)1 interacted with an overlapping region of MITF. C-TAK1 increased MITF/14-3-3 complex formation and thus promoted cytoplasmic localization of MITF. C-TAK1 interaction was disrupted by RANKL/CSF-1 treatment. The results indicate that 14-3-3 regulates MITF activity by promoting the cytosolic localization of MITF in the absence of signals required for osteoclast differentiation. This work identifies a mechanism that regulates MITF activity in monocytic precursors that are capable of undergoing different terminal differentiation programs, and it provides a mechanism that allows committed precursors to rapidly respond to signals in the bone microenvironment to promote specifically osteoclast differentiation.

MicroRNA-451 Inhibits Migration of Glioblastoma while Making It More Susceptible to Conventional Therapy.

Malignant glioblastoma (GBM, glioma) is the most common and aggressive primary adult brain tumor. The prognosis of GBM patients remains poor, despite surgery, radiation and chemotherapy. The major obstacles for successful remedy are invasiveness and therapy resistance of GBM cells. Invasive glioma cells leave primary tumor core and infiltrate surrounding normal brain leading to inevitable recurrence, even after surgical resection, radiation and chemotherapy. Therapy resistance allowing for selection of more aggressive and resistant sub-populations including GBM stem-like cells (GSCs) upon treatment is another serious impediment to successful treatment. Through their regulation of multiple genes, microRNAs can orchestrate complex programs of gene expression and act as master regulators of cellular processes. MicroRNA-based therapeutics could thus impact broad cellular programs, leading to inhibition of invasion and sensitization to radio/chemotherapy. Our data show that miR-451 attenuates glioma cell migration in vitro and invasion in vivo. In addition, we have found that miR-451 sensitizes glioma cells to conventional chemo- and radio-therapy. Our data also show that miR-451 is regulated in vivo by AMPK pathway and that AMPK/miR-451 loop has the ability to switch between proliferative and migratory pattern of glioma cells behavior. We therefore postulate that AMPK/miR-451 negative reciprocal feedback loop allows GBM cells/GSCs to adapt to tumor "ecosystem" by metabolic and behavioral flexibility, and that disruption of such a loop reduces invasiveness and diminishes therapy resistance.

Statins affect human glioblastoma and other cancers through TGF-ß inhibition.

The cholesterol-lowering statins have known anti-cancer effects, but the mechanisms and how to utilize statins in oncology have been unclear. We noted in the CellMiner database that statin activity against cancer lines correlated with higher expression of TGF-ß target genes such as SERPINE1 and ZYX. This prompted us to assess whether statins affected TGF-ß activity in glioblastoma (GBM), a cancer strongly influenced by TGF-ß and in dire need of new therapeutic approaches. We noted that statins reduced TGF-ß activity, cell viability and invasiveness, Rho/ROCK activity, phosphorylation and activity of the TGF-ß mediator Smad3, and expression of TGF-ß targets ZYX and SERPINE1 in GBM and GBM-initiating cell (GIC) lines. Statins were most potent against GBM, GIC, and other cancer cells with high TGF-ß activity, and exogenous TGF-ß further sensitized mesenchymal GICs to statins. Statin toxicity was rescued by addition of exogenous mevalonolactone or geranylgeranyl pyrophosphate, indicating that the observed effects reflected inhibition of HMG CoA-reductase by the statins. Simvastatin significantly inhibited the growth of subcutaneous GIC grafts and prolonged survival in GIC intracranially grafted mice. These results indicate where the statins might best be applied as adjunct therapies in oncology, against GBM and other cancers with high TGF-ß activity, and have implications for other statin roles outside of oncology.

The functional synergism of microRNA clustering provides therapeutically relevant epigenetic interference in glioblastoma.

MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies.

Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding.

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.

The activation of group II metabotropic glutamate receptors protects neonatal rat brains from oxidative stress injury after hypoxia-ischemia.

Birth asphyxia resulting in brain hypoxia-ischemia (H-I) can cause neonatal death or lead to persistent brain damage. Recent investigations have shown that group II metabotropic glutamate receptor (mGluR2/3) activation can provide neuroprotection against H-I but the mechanism of this effect is not clear. The aim of this study was to investigate whether mGluR2/3 agonists applied a short time after H-I reduce brain damage in an experimental model of birth asphyxia, and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I in 7-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intra peritoneally with mGluR2 (LY 379268) or mGluR3 (NAAG) agonists 1 h or 6 h after H-I (5 mg/kg). The weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content levels, antioxidant enzymes activity and the concentrations of reduced glutathione (GSH) were measured. Both agonists reduced weight loss in the ischemic hemisphere and mitigated neuronal degeneration in the CA1 hippocampal region and cerebral cortex. Both agonists reduced the elevated levels of ROS in the ipsilateral hemisphere observed after H-I and prevented an increase in antioxidant enzymes activity in the injured hemisphere restoring them to control levels. A decrease in GSH level was also restored after agonists application. The results show that the activation of mGluR2 and mGluR3 a short time after H-I triggers neuroprotective mechanisms that act through the inhibition of oxidative stress and ROS production. The prevention of ROS production by the inhibition of glutamate release and decrease in its extracellular concentration is likely the main mechanism involved in the observed neuroprotection.