Redox regulation of STAT1 in microglia M1 activation

STAT1 is a transcription factor implicated in the regulation of various cell processes such as immune response and apoptosis. Some authors report that hyper-activation of STAT1 signaling is involved in the development of neuroinflammation, a process closely related to oxidative stress. Although the role of oxidative-stress in neuroinflammation and in the pathogenesis of neurodegenerative disorders is clearly described, its influence in the regulation of STAT1 pathway is poorly understood. Herein, it is demonstrated that oxidative stress induces rapid activation of STAT1 signaling in murine microglia BV2 cells using H2O2 and hypoxia treatment. The molecular mechanism of its activation is related to S-glutathionylation on Cys324 and Cys492 residues of STAT1. These results reveal that STAT1 is a redox-sensitive protein and that its activation involves both tyrosine phosphorylation and S- glutathionylation under oxidative stress condition. Several studies report that microglia M1 activation is the hallmark of neuroinflammation and contributes to neurodegeneration and loss of neurological function. Here, the mechanisms that drive M1 microglia activation in BV2 cells under hypoxic stimulus have been analysed and correlate it to STAT1 activation. Silencing of STAT1 protein expression, hypoxia-induced M1 microglia phenotype is counteracted suggesting the strong link between STAT1 and microglia activation triggered by hypoxia. Moreover, the ability of hypoxia-activated microglia to induce neuronal apoptosis is shown using in vitro cross-talk model between BV2 and SH-SY5Y cell lines. Finally, it is revealed that a specific anti-STAT1 flavonoid myricetin is able to counteract microglia activation under hypoxia preventing neuronal death in the cross-talk cellular model

Natural sesquiterpene lactones enhance chemosensitivity of tumor cells through redox regulation of STAT3 signaling

STAT3 is a nuclear transcription factor that regulates genes involved in cell cycle, cell survival, and immune response. Although STAT3 activation drives cells to physiological response, its deregulation is often associated with the development and progression of many solid and hematological tumors as well as with drug resistance. STAT3 is a redox-sensitive protein, and its activation state is related to intracellular GSH levels. Under oxidative conditions, STAT3 activity is regulated by S-glutathionylation, a reversible posttranslational modification of cysteine residues. Compounds able to suppress STAT3 activation and, on the other hand, to modulate intracellular redox homeostasis may potentially improve cancer treatment outcome. Nowadays, about 35% of commercial drugs are natural compounds that derive from plant extracts used in phytotherapy and traditional medicine. Sesquiterpene lactones are an interesting chemical group of plant-derived compounds often employed in traditional medicine against inflammation and cancer. This review focuses on sesquiterpene lactones able to downmodulate STAT3 signaling leading to an antitumor effect and correlates the anti-STAT3 activity with their ability to decrease GSH levels in cancer cells. These properties make them lead compounds for the development of a new therapeutic strategy for cancer treatment

Immunoprecipitation methods to identify S-glutathionylation in target proteins

S-glutathionylation is a reversible post-translational modification of proteins that generate a mixed disulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylation has been extensively studied since it represents the cellular response to oxidative stress, in physiological as well as pathological conditions. This modification may be a protective mechanism from irreversible oxidative damage and, on the other hand, may modulate protein folding and function. Due to the importance of S-glutathionylation in cellular redox signaling, various methods have been developed to identify S-gluthationylated proteins. Herein, we describe two easy methods to recognized S-glutathionylation of a target protein after oxidative stress in cellular extracts based on different immunoprecipitation procedures. The immunoprecipitation assay allows the capture of one glutathionylated protein using a specific antibody that binds to the target protein. The presence of S-glutathionylation in the immunoprecipitated protein is identified using anti-glutathione antibody. The second type of approach is based on the detection of the glutathionylated protein with biotin/streptavidin technique. After different steps of protection of non-oxidized thiolic groups and reduction of S-glutathionylated groups, the newly-formed protein free-thiols are labeled with biotin-GSH. The modified protein can be isolate with streptavidin-beads and recognized using an antibody against target protein. \u2022S-glutathionylation is a reversible post-translational modification of proteins that recently has been emerged as important signaling in the redox regulation of protein function.\u2022Both methods to identify glutathionylated proteins are economic, easy and do not require particular equipment.\u2022The setups of both methods guarantee high reproducibility

Increased expression of autophagy-related genes in Alzheimer's Disease-Type 2 Diabetes Mellitus comorbidity models in cells

The association between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) has been extensively demonstrated, but despite this, the pathophysiological mechanisms underlying it are still unknown. In previous work, we discovered a central role for the autophagy pathway in the common alterations observed between AD and T2DM. In this study, we further investigate the role of genes belonging to this pathway, measuring their mRNA expression and protein levels in 3xTg-AD transgenic mice, an animal model of AD. Moreover, primary mouse cortical neurons derived from this model and the human H4Swe cell line were used as cellular models of insulin resistance in AD brains. Hippocampal mRNA expression showed significantly different levels for Atg16L1, Atg16L2, GabarapL1, GabarapL2, and Sqstm1 genes at different ages of 3xTg-AD mice. Significantly elevated expression of Atg16L1, Atg16L2, and GabarapL1 was also observed in H4Swe cell cultures, in the presence of insulin resistance. Gene expression analysis confirmed that Atg16L1 was significantly increased in cultures from transgenic mice when insulin resistance was induced. Taken together, these results emphasise the association of the autophagy pathway in AD-T2DM co-morbidity, providing new evidence about the pathophysiology of both diseases and their mutual interaction

In vivo morphological alterations of TAMs during KCa3.1 inhibition—by using in vivo two-photon time-lapse technology

Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs

Tumor dormancy and interplay with hypoxic tumor microenvironment

The tumor microenvironment is a key factor in disease progression, local resistance, immune-escaping, and metastasis. The rapid proliferation of tumor cells and the aberrant structure of the blood vessels within tumors result in a marked heterogeneity in the perfusion of the tumor tissue with regions of hypoxia. Although most of the tumor cells die in these hypoxic conditions, a part of them can adapt and survive for many days or months in a dormant state. Dormant tumor cells are characterized by cell cycle arrest in G0/G1 phase as well as a low metabolism, and are refractive to common chemotherapy, giving rise to metastasis. Despite these features, the cells retain their ability to proliferate when conditions improve. An understanding of the regulatory machinery of tumor dormancy is essential for identifying early cancer biomarkers and could provide a rationale for the development of novel agents to target dormant tumor cell populations. In this review, we examine the current knowledge of the mechanisms allowing tumor dormancy and discuss the crucial role of the hypoxic microenvironment in this process

STAT1 drives M1 microglia activation and neuroinflammation under hypoxia

Microglia are resident immune cells that act as the first active defence in the central nervous system. These cells constantly monitor the tissue microenvironment and rapidly react in response to hypoxia, infection and injuries. Hypoxia in the brain has been detected in several neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Hypoxic conditions activate microglia cells towards M1 phenotype resulting in oxidative stress and the release of pro-inflammatory cytokines. Recently, we have demonstrated that oxidative stress induces S-glutathionylation of the STAT1 and hyper-activates its signaling in microglia BV2 cells pointing out the importance of this transcription factor in neuroinflammation. In this paper we analyse the cellular mechanisms that drive M1 microglia activation in BV2 cells in response to hypoxia correlating it to STAT1 activation. The analysis of the molecular mechanism of STAT1 signaling reveals that hypoxia generates oxidative stress and induces both phosphorylation and S-glutathionylation of STAT1 that are responsible of its aberrant activation. The silencing of STAT1 protein expression counteracts hypoxia-M1 microglia phenotype suggesting the strong link between hypoxia-STAT1 and STAT1-microglia activation

Plausible biochemical mechanisms of chemotherapy-induced cognitive impairment ("chemobrain"), a condition that significantly impairs the quality of life of many cancer survivors

Increasing numbers of cancer patients survive and live longer than five years after therapy, but very often side effects of cancer treatment arise at same time. One of the side effects, chemotherapy-induced cognitive impairment (CICI), also called "chemobrain" or "chemofog" by patients, brings enormous challenges to cancer survivors following successful chemotherapeutic treatment. Decreased abilities of learning, memory, attention, executive function and processing speed in cancer survivors with CICI, are some of the challenges that greatly impair survivors' quality of life. The molecular mechanisms of CICI involve very complicated processes, which have been the subject of investigation over the past decades. Many mechanistic candidates have been studied including disruption of the blood-brain barrier (BBB), DNA damage, telomere shortening, oxidative stress and associated inflammatory response, gene polymorphism of neural repair, altered neurotransmission, and hormone changes. Oxidative stress is considered as a vital mechanism, since over 50% of FDA-approved anti-cancer drugs can generate reactive oxygen species (ROS) or reactive nitrogen species (RNS), which lead to neuronal death. In this review paper, we discuss these important candidate mechanisms, in particular oxidative stress and the cytokine, TNF-alpha and their potential roles in CICI

The anti-STAT1 polyphenol myricetin inhibits M1 microglia activation and counteracts neuronal death

Microglia activation toward M1 pro-inflammatory phenotype represents one of the earliest events of neurological disorders. Therefore, reducing microglia activation should inhibit neuroinflammation, thereby delaying the progression of neurodegeneration. Recently, we pointed out the role of STAT1 signaling in hypoxia-induced M1 activation and proposed STAT1 as a suitable molecular target for the prevention and treatment of neurodegeneration. Myricetin is a natural flavonoid that exhibits a specific anti-STAT1 activity correlated with its direct interaction with STAT1 protein itself. Herein, we investigated the anti-inflammatory effect of myricetin and its ability to protect neurons from death in an in vitro model of neurotoxicity using the neuroblast-like SH-SY5Y cells that were exposed to conditioned media from hypoxia-activated microglia BV2 cells. We demonstrate that myricetin pretreatment is able to switch off hypoxia-induced M1 microglia polarization through the inhibition of STAT1 signaling. The analysis of the molecular mechanism suggests that the direct interaction of myricetin with STAT1 impairs its S-glutathionylation and phosphorylation. Moreover, treatment of SH-SY5Y cells with conditioned medium from hypoxia-activated microglia pretreated with myricetin produced a significant reduction in neuronal viability. Our data indicate that myricetin may represent a promising candidate for prevention and treatment of neuroinflammation in neurodegenerative disorders