Oxidative Stress In Astrocytes Research Articles

Astrocytic autophagy plasticity modulates Aβ clearance and cognitive function in Alzheimer’s disease

BackgroundAstrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer’s disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD.MethodsWhole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR).ResultsHere, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and GFAP-positive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Aβ aggregates in the brain of APP/PS1 mice. An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients.ConclusionsTaken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice.

Total Saponins of Panax Notoginseng Modulate the Astrocyte Inflammatory Signaling Pathway and Attenuate Inflammatory Injury Induced by Oxygen- Glucose Deprivation/Reperfusion Injury in Rat Brain Microvascular Endothelial Cells.

Reperfusion after cerebral ischemia causes brain injury. Total saponins of Panax notoginseng (PNS) have potential roles in protecting against cerebral ischemia-reperfusion injury. However, whether PNS regulates astrocytes on oxygen-glucose deprivation/reperfusion (OGD/R) injury in rat brain microvascular endothelial cells (BMECs) and its mechanism still need further clarification. Rat C6 glial cells were treated with PNS at different doses. Cell models were established by exposing C6 glial cells and BMECs to OGD/R. Cell viability was assessed, and levels of nitrite concentration, inflammatory factors (iNOS, IL-1β, IL-6, IL-8, TNF-α), and oxidative stress-related factors (MDA, SOD, GSH-Px, T-AOC) were subsequently measured through CCK8, Grice analysis, Western blot, and ELISA, respectively. The co-cultured C6 and endothelial cells were treated with PNS for 24 hours before model establishment. Then transendothelial electrical resistance (TEER), lactate dehydrogenase (LDH) activity, brain-derived neurotrophic factor (BDNF) content, and mRNA and protein levels and positive rates of tight junction proteins [Claudin-5, Occludin, ZO-1] were measured by a cell resistance meter, corresponding kits, ELISA, RT-qPCR, Western blot, and immunohistochemistry, respectively. PNS had no cytotoxicity. PNS reduced iNOS, IL-1β, IL-6, IL-8, and TNF-α levels in astrocytes, promoted T-AOC level and SOD and GSH-Px activities, and inhibited MDA levels, thus inhibiting oxidative stress in astrocytes. In addition, PNS alleviated OGD/R injury, reduced Na-Flu permeability, and enhanced TEER, LDH activity, BDNF content, and levels of tight junction proteins Claudin-5, Occludin, ZO-1 in the culture system of astrocytes and rat BMECs after OGD/R. PNS repressed astrocyte inflammation and attenuated OGD/R injury in rat BMECs.

Inhibition of the Rho/ROCK pathway promotes the expression of developmental and migration-related genes in astrocytes exposed to alcohol

Astrocytes are an important regulator of alcohol dependence. Furthermore, the downregulation of Rho-associated coiled coil-containing protein kinase 2 (ROCK2) attenuates alcohol-induced inflammation and oxidative stress in astrocytes. On the basis of these findings, we examined the effects of alcohol and a Rho/RACK kinases inhibitor on astrocyte function and investigated their effects on mRNA expression to further explore the protective mechanisms of a Rho/RACK kinases inhibitor in astrocytes after alcohol exposure. CTX TNA2 astrocytes were cultured with alcohol and Rho/RACK kinases inhibitor intervention before undergoing transcriptome sequencing, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and wound healing assays. Alcohol exposure modulated cell morphology and inhibited astrocyte migration, whereas Fasudil improved cell morphology and promoted astrocyte migration after alcohol exposure. Transcriptome sequencing results indicated that alcohol exposure modulates the expression of genes involved in astrocyte development. Fasudil reversed the effects of alcohol exposure on the astrocyte developmental process. Four genes related to the developmental process and migration – Ccl2, Postn, Itga8, and Serpine1 – with the highest protein–protein interaction correlations (node degree >7) were selected for verification by qRT-PCR, and the results were consistent with those of the sequencing and wound healing assays. Our results suggest that the Rho/ROCK pathway is essential for alcohol to be able to interfere with astrocyte development and migration gene expression. The Rho/ROCK pathway inhibitor Fasudil reversed the adverse effects of alcohol exposure on astrocytes and may have clinical applications.

TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

Neuroinflammation and oxidative stress have been implicated in the pathogenesis of Alzheimer’s disease (AD). Neuroinflammation and oxidative stress are associated with neuronal death in AD. Astrocytes are linked to neuroinflammation during AD. Astrocytes are important contributors to AD progression. Although the role of thioredoxin-interacting protein (TXNIP) has been identified in inflammation and oxidative stress, the mechanism by which TXNIP regulates inflammation and oxidative stress in astrocytes during AD remains unclear. In the present study, we found that TXNIP gene levels were elevated in cerebral cortex of patients with AD. The protein levels of TXNIP were elevated in GFAP-positive astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. Our results showed that TXNIP increased expression of genes related to pro-inflammatory reactive astrocytes and pro-inflammatory cytokines and chemokines in human astrocytes. Moreover, TXNIP increased production of pro-inflammatory cytokines and chemokines in human astrocytes. TXNIP induced activation of NK-kB signaling and over-production of mitochondrial reactive oxygen species (mtROS) in human astrocytes. TXNIP also induced mitochondrial oxidative stress by reduction of mitochondrial respiration and ATP production in human astrocytes. Furthermore, elevated TXNIP levels are correlated with caspase-3 activation of GFAP-positive astrocytes in patients with AD and mouse AD. TXNIP induced mitochondria-dependent apoptosis via caspase-9 and caspase-3 activation in human astrocytes. These results suggest that TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during AD.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke

Stroke is the second leading cause of death worldwide, and oxidative stress plays a crucial role. Celastrol exhibits strong antioxidant properties in several diseases; however, whether it can affect oxidation in cerebral ischemic-reperfusion injury (CIRI) remains unclear. This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms. Here, we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2 (Nrf2). Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry, we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4 (Nedd4) and then released Nrf2 from Nedd4 in astrocytes. Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI, which was significantly blocked by celastrol. Furthermore, by inhibiting oxidative stress and astrocyte activation, celastrol effectively rescued neurons from axon damage and apoptosis. Our study uncovered Nedd4 as a direct target of celastrol, and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

Copper regulation disturbance linked to oxidative stress and cell death during Zika virus infection in human astrocytes.

The Zika virus (ZIKV) caused neurological abnormalities in more than 3500 Brazilian newborns between 2015 and 2020. Data have pointed to oxidative stress in astrocytes as well as to dysregulations in neural cell proliferation and cell cycle as important events accounting for the cell death and neurological complications observed in Congenital Zika Syndrome. Copper imbalance has been shown to induce similar alterations in other pathologies, and disturbances in copper homeostasis have already been described in viral infections. Here, we investigated copper homeostasis imbalance as a factor that could contribute to the cytotoxic effects of ZIKV infection in astrocytes. Humaninduced pluripotent stem cell-derived astrocytes were infected with ZIKV; changes in the gene expression of copper homeostasis proteins were analyzed. The effect of the administration of CuCl2 or a copper chelator on oxidative stress, cell viability and percentage of infection were also studied. ZIKV infection leads to a downregulation of one of the transporters mediating copper release, ATP7B protein. We also observed the activation of mechanisms that counteract high copper levels, including the synthesis of copper chaperones and the reduction of the copper importer protein CTR1. Finally, we show that chelator-mediated copper sequestration in ZIKV-infected astrocytes reduces the levels of reactive oxygen species and improves cell viability, but does not change the overall percentage of infected cells. In summary, our results show that copper homeostasis imbalance plays a role in the pathology of ZIKV in astrocytes, indicating that it may also be a factor accounting for the developmental abnormalities in the central nervous system following viral infection. Evaluating micronutrient levels and the use of copper chelators in pregnant women susceptible to ZIKV infection may be promising strategies to manage novel cases of congenital ZIKV syndrome.

Role of Glial Cell-Derived Oxidative Stress in Blood-Brain Barrier Damage after Acute Ischemic Stroke.

The integrity of the blood-brain barrier (BBB) is mainly maintained by endothelial cells and basement membrane and could be regulated by pericytes, neurons, and glial cells including astrocytes, microglia, oligodendrocytes (OLs), and oligodendrocyte progenitor cells (OPCs). BBB damage is the main pathological basis of hemorrhage transformation (HT) and vasogenic edema after stroke. In addition, BBB damage-induced HT and vasogenic edema will aggravate the secondary brain tissue damage. Of note, after reperfusion, oxidative stress-initiated cascade plays a critical role in the BBB damage after acute ischemic stroke (AIS). Although endothelial cells are the target of oxidative stress, the role of glial cell-derived oxidative stress in BBB damage after AIS also should receive more attention. In the current review, we first introduce the physiology and pathophysiology of the BBB, then we summarize the possible mechanisms related to BBB damage after AIS. We aim to characterize the role of glial cell-derived oxidative stress in BBB damage after AIS and discuss the role of oxidative stress in astrocytes, microglia cells and oligodendrocytes in after AIS, respectively.

Age-Associated Glia Remodeling and Mitochondrial Dysfunction in Neurodegeneration: Antioxidant Supplementation as a Possible Intervention.

Aging induces substantial remodeling of glia, including density, morphology, cytokine expression, and phagocytic capacity. Alterations of glial cells, such as hypertrophy of lysosomes, endosomes and peroxisomes, and the progressive accumulation of lipofuscin, lipid droplets, and other debris have also been reported. These abnormalities have been associated with significant declines of microglial processes and reduced ability to survey the surrounding tissue, maintain synapses, and recover from injury. Similarly, aged astrocytes show reduced capacity to support metabolite transportation to neurons. In the setting of reduced glial activity, stressors and/or injury signals can trigger a coordinated action of microglia and astrocytes that may amplify neuroinflammation and contribute to the release of neurotoxic factors. Oxidative stress and proteotoxic aggregates may burst astrocyte-mediated secretion of pro-inflammatory cytokines, thus activating microglia, favoring microgliosis, and ultimately making the brain more susceptible to injury and/or neurodegeneration. Here, we discuss the contribution of microglia and astrocyte oxidative stress to neuroinflammation and neurodegeneration, highlight the pathways that may help gain insights into their molecular mechanisms, and describe the benefits of antioxidant supplementation-based strategies.

Abstract WP252: Tamoxifen Treatment Worsens Astrocyte Activation And Mitochondrial Stress From Oxygen Glucose Deprivation

Introduction: Tamoxifen (TAM), a selective estrogen receptor modifier, is a first-line treatment option for hormone receptor positive breast cancer. TAM inhibits the downstream estrogen receptor signaling in tumor cells. Stroke risk is increased in women with low estrogen levels, such as early-onset menopause, and doubles in women after the age of 55, which corresponds with the average age of menopause. Women with breast cancer who were treated with tamoxifen had an increased risk for ischemic stroke (82%). Since stroke severity is associated with increased blood brain barrier (BBB) damage, we investigated the effects of tamoxifen on activation and mitochondrial stress in astrocytes, which is a cellular component of the BBB. Methods: Cultured female human astrocytes were treated with tamoxifen (1uM), 17ß-estradiol (10nM), or vehicle (DMSO) for 24 hours. Cells were separated into two groups: normoxic (21% O2, 25mM glucose) or oxygen and glucose deprivation (OGD) conditions 6 hours before analysis. ROS production was measured using the Cellular ROS Assay Kit (Red) and aquaporin-4 levels were visualized via immunocytochemistry. Mitochondrial function was assessed by using a Seahorse XFe96 Analyzer. Results: Tamoxifen treatment increased Aquaporin-4 protein levels (p <0.0001) in cultured astrocytes after OGD compared to both vehicle (DMSO) and 17ß-estradiol treatment. While there was no significant effect, tamoxifen treated cells showed a trend toward (p =0.0732) increased ROS production after OGD compared to vehicle. Additionally, tamoxifen reduced oxygen consumption rate (p =0.0197) and basal respiration under normoxic conditions in astrocytes when compared with DMSO or 17ß-estradiol treatment. Conclusions: Tamoxifen treatment altered the expression of a water channel protein and decreased the ability to respond to oxidative stress in astrocytes. Aquaporin-4, when localized to the astrocytic end feet, facilitates edema after stroke, and ischemic stroke by definition puts brain tissue under oxidative stress. Our findings suggest tamoxifen may increase edema, which is known to impair stroke recovery. These data suggest a mechanism by which breast cancer treatment may increase the risk for severe ischemic stroke. Supported by RFAG042189 to FS

Benzaldehyde Attenuates the Fifth Stage Larval Excretory-Secretory Product of Angiostrongylus cantonensis-Induced Injury in Mouse Astrocytes via Regulation of Endoplasmic Reticulum Stress and Oxidative Stress.

Excretory–secretory products (ESPs) are the main research targets for investigating the hosts and helminths interaction. Parasitic worms can migrate to parasitic sites and avoid the host immune response by secreting this product. Angiostrongylus cantonensis is an important food-borne zoonotic parasite that causes severe neuropathological damage and symptoms, including eosinophilic meningitis or meningoencephalitis in humans. Benzaldehydes are organic compounds composed of a benzene ring and formyl substituents. This compound has anti-inflammatory and antioxidation properties. Previous studies showed that 3-hydroxybenzaldehyde (3-HBA) and 4-hydroxybenzaldehyde (4-HBA) can reduce apoptosis in A. cantonensis ESP-treated astrocytes. These results on the protective effect underlying benzaldehyde have primarily focused on cell survival. The study was designed to investigate the molecular mechanisms of endoplasmic reticulum stress (ER stress) and oxidative stress in astrocytes in A. cantonensis ESP-treated astrocytes and to evaluate the therapeutic consequent of 3-HBA and 4-HBA. First, we initially established the RNA-seq dataset in each group, including normal, ESPs, ESPs + 3-HBA, and ESPs + 4-HBA. We also found that benzaldehyde (3-HBA and 4-HBA) can stimulate astrocytes to express ER stress-related molecules after ESP treatment. The level of oxidative stress could also be decreased in astrocytes by elevating antioxidant activity and reducing ROS generation. These results suggested that benzaldehyde may be a potential therapeutic compound for human angiostrongyliasis to support brain cell survival by inducing the expression levels of ER stress- and oxidative stress-related pathways.

Polychlorinated biphenyls induce oxidative stress and metabolic responses in astrocytes

Exposure to environmental toxicants is prevalent, hazardous and linked to varied detrimental health outcomes and disease. Polychlorinated biphenyls (PCBs), a class of hazardous organic chlorines once widely used for industrial purposes, are associated with neurodegenerative disease and oxidative stress in both in vitro and in vivo models. Here, we investigated the impact of Aroclor 1254, a commercially available PCB mixture, on primary murine astrocytes to determine the response to this once ubiquitously used toxicant on the most numerous cells of the central nervous system (CNS). Astrocytes are a critical component of homeostasis throughout the CNS, including at the blood-brain barrier, where they serve as the primary defense against xenobiotics entering the CNS, and at the synapse, where they are closely coupled to neurons through several metabolic pathways. We hypothesized that PCBs cause astrocytic oxidative stress and related dysfunction including altered metabolism. We exposed primary murine cortical astrocytes to PCBs and report an increased expression of antioxidant genes (Prdx1, Gsta2, Gfap, Amigo2) in response to oxidative stress. Our data show increased ATP production and spare respiratory capacity in astrocytes exposed to 10 μM (∼ 3 ppm) PCBs. This dose also causes an increase in glucose uptake that is not seen at a higher dose (50 μM) suggesting that, at a lower dose, astrocytes are able to engage compensatory mechanisms to promote survival. Together, these data suggest that exposure to PCBs impact astrocytic metabolism, which is important to consider both in the context of human health and disease and in in vitro and in vivo disease models.

Differential production of interleukin‐6 and tumor necrosis factor‐α in primary rat astrocyte cultures using two distinct methods of microglia elimination

AbstractHepatic encephalopathy (HE) is a neurocognitive disorder due to acute or chronic liver failure as well as portosystemic bypass. Both neuroinflammation and oxidative stress have been strongly implicated in the pathogenesis of HE. An increasing body of evidence suggests that in the development of these mechanisms astrocytes may be involved. With regard of HE, this aspect of astrocytes is, however, neglected. While several studies focus on astrocytes related to the above‐mentioned pathological processes, the applied in vitro models are generated by diverse methods, which make the results difficult to compare with each other. In addition, these astrocyte cultures frequently contain microglia as well, complicating the interpretation of results. Therefore, we applied enriched primary rat astrocyte cultures generated by microglia elimination via shaking or the combination of cytosine β‐d‐arabinofuranoside hydrochloride (AraC) and l‐leucine methyl ester (LME). We examine whether some HE‐relevant pathological factors, such as ammonia, manganese (Mn), lipopolysaccharide (LPS), and H2O2 could elicit oxidative stress and cytokine production in astrocytes. Mn exposure increased the oxidative stress in astrocytes in a time‐dependent manner; furthermore, we could detect tumor necrosis factor (TNF)‐α production in both cultures at resting state. In addition, it was around 10‐fold higher in the cultures after shaking. LPS increased TNF‐α secretion in cultures after shaking, but not in cultures after AraC + LME treatment. In contrast, interleukin (IL)‐6 was detectable only in the AraC + LME‐treated cultures. In conclusion, we have demonstrated that HE‐relevant factors evoke oxidative stress and cytokine production, albeit depending on the elimination method used.

Pretreatment of Ascorbic Acid Inhibits MPTP-Induced Astrocytic Oxidative Stress through Suppressing NF-κB Signaling

Astrocytes are a major constituent of the central nervous system (CNS). Astrocytic oxidative stress contributes to the development of Parkinson's disease (PD). Maintaining production of antioxidant and detoxification of reactive oxygen and nitrogen species (ROS/RNS) in astrocytes is critical to prevent PD. Study has illuminated that ascorbic acid (AA) stimulates dopamine synthesis and expression of tyrosine hydroxylase in human neuroblastoma cells. However, the role and regulatory mechanisms of AA on detoxification of astrocytes are still unclear. The purpose of our study is in-depth study of the regulatory mechanism of AA on detoxification of astrocytes. We found that AA pretreatment decreased the expression of ROS and inducible nitric oxide synthase (iNOS) in MPP+-treated astrocytes. In contrast, the expression levels of antioxidative substances—including superoxide dismutase (SOD), glutathione (GSH), and glutamate-cysteine ligase modifier (GCLM) subunit—were upregulated after AA pretreatment in MPP+-treated astrocytes. However, inhibition of NF-κB prevented such AA induced increases in antioxidative substances following MPP+ treatment in astrocytes, suggesting that AA improved antioxidative function of astrocytes through inhibiting NF-κB-mediated oxidative stress. Furthermore, in vivo studies revealed that AA preadministration also suppressed NF-κB and upregulated the expression levels of antioxidative substances in the midbrain of MPTP-treated mice. Additionally, pretreatment of AA alleviated MPTP-induced PD-like pathology in mice. Taken together, our results demonstrate that preadministration of AA improves the antioxidative function of astrocytes through suppressing NF-κB signaling, following alleviated the pathogenesis of PD which induced by MPTP. Hence, our findings elucidate a novel protective mechanism of AA in astrocytes.

Downregulation of ROCK2 attenuates alcohol-induced inflammation and oxidative stress in astrocytes

Objective Alcohol abuse can cause severe injury to human brain. Astrocytes are the most abundant nonneuronal cells that function to maintain the brain homeostasis. In present study, we aimed to investigate the role of ROCK2 in astrocytes exposed to alcohol. Methods Astrocytes were transfected with lentivirus (LV)-anti-ROCK2 vector to downregulate the expression of ROCK2. The ROCK2 expression in mRNA and protein level was analyzed by real-time PCR and Western blotting, respectively. Cytokines or indicators involved in inflammation and oxidative stress were determined by assay kits. Proteins involved in nuclear factor kappa B (NF-κB) signaling pathway and NOD-like receptor protein 3 (NLRP3) inflammasome were analyzed by Western blotting. Results Alcohol exposure dramatically upregulated ROCK2 expression and lactate dehydrogenase (LDH) activity in astrocytes. On the contrary, transfecting with LV-anti-ROCK2 vector downregulated ROCK2 expression and LDH activity in astrocytes, demonstrating that downregulation of ROCK2 alleviated alcohol-induced astrocytic injury. Furthermore, downregulation of ROCK2 attenuated alcohol-induced inflammation by reducing the levels of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) and enhanced the level of anti-inflammatory IL-10. Downregulation of ROCK2 also attenuated alcohol-induced oxidative stress by reducing the reactive oxygen species (ROS) production, as well as enhancing the activity of anti-oxidative superoxide dismutase (SOD) and glutathione (GSH). More importantly, downregulation of ROCK2 inhibited the activation of NF-κB signaling pathway and NLRP3 inflammasome. Conclusion Therefore, ROCK2 could be a potential target to treat alcohol-induced astrocytic injury and the downregulation of ROCK2 might be a promising approach to protect against alcohol-induced astrocytic injury.

Cyclical amyloid beta-astrocyte activity induces oxidative stress in Alzheimer’s disease

Glial cell involvement in Alzheimer’s disease (AD) is multi-faceted. The role of astrocytes in AD pathology, both as a causative agent of amyloid-beta (Aβ) production as well as a casualty of dysfunction resulting from the presence of Aβ has been well-delineated. In this review, we explore the influence of oxidative stress in astrocytes and the subsequent effect on Aβ levels in the brain from a perspective of intracellular calcium homeostasis and NADPH oxidase activity. The response of astrocytes to the presence of Aβ, as well astrocytic and microglial interaction and inflammatory cytokine release is also discussed, highlighting a cyclical behaviour of these cells in contributing to AD pathogenesis.

Titanium dioxide nanoparticles promote oxidative stress, autophagy and reduce NLRP3 in primary rat astrocytes

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in the food industry, cosmetics, personal care and paints among others. Through occupational exposure and daily consumption, and because of their small size, TiO2-NPs can enter the body through different routes such as oral, dermal and inhalation, and accumulate in multiple organs including the brain. TiO2-NPs cause severe damage to many cell types, however their effects in the central nervous system remain largely unexplored. Therefore, in the present study we determined the cytotoxic effect of TiO2-NPs on rat astrocytes. We tested the oxidant properties of TiO2-NPs through DTT depletion, and measured oxidative stress-induced damage in mitochondria, through oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) and loss of mitochondrial membrane potential (ΔΨm) with Mitotracker Green FM. We further examined oxidative stress-derived responses such as IκB-α degradation by Western Blot, NF-κB translocation by EMSA, autophagy induction by LC3-II levels, and expression of the inflammasome protein NLRP3. TiO2-NPs showed high oxidant properties and induced strong oxidative stress in astrocytes following their internalization, causing mitochondrial damage detected by ΔΨm loss. Responses against oxidative damage such as NF-κB translocation and autophagy were induced and NLRP3 protein expression was downregulated, indicating lower inflammasome-mediated responses in astrocytes. These results support TiO2-NPs cytotoxicity in astrocytes, cells that play key roles in neuronal homeostasis and their dysfunction can lead to neurological disorders including cognitive impairment and memory loss.

Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity.

BackgroundAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors.MethodsWe used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles.FindingsWe report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs.InterpretationOur results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.

TTB Protects Astrocytes Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Activation of Nrf2/HO-1 Signaling Pathway.

Neonatal hypoxic/ischemic encephalopathy (NHIE) is a severe condition that leads to death or neurological disability in newborns. The underlying pathological mechanisms are unclear, and developing the target neuroprotective strategies are urgent. 2,7,2′-trihydroxy-4,4′7′-trimethoxy-1,1′-biphenanthrene (TTB) is a natural product isolated from Cremastra appendiculata (D. Don) Makino and Liparis nervosa (Thunb.) Lindl. TTB has demonstrated potent cytotoxic activity against stomach (HGC-27) and colon (HT-29) cancer cell lines. However, none of the studies have addressed the effects of TTB in NHIE. In the present study, an oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocyte injury model was established to investigate the effect of TTB and its potential mechanisms. Our results showed that TTB alleviated the OGD/R-induced reactive oxygen species increase and the intracellular antioxidant capacity of superoxide dismutase activity decrease. Moreover, TTB potentially prolonged the activation state of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway and maintained the protection against oxidative stress in OGD/R-induced astrocytes by inducing the nuclear translocation and up-regulation of Nrf2 along with the enhanced expression of the downstream target gene HO-1. Furthermore, TTB treatment diminished the accumulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression induced by OGD/R. We also found TTB-treated astrocytes reversed the inhibition of OGD/R on neurite growth of neurons by the astrocyte-neuron coculture system. In conclusion, TTB inhibited the OGD/R-induced astrocyte oxidative stress at least partially through the inhibition of HIF-1α and VEGF via the Nrf2/HO-1 signaling pathway.

Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response.

Brain calcifications are commonly detected in aged individuals and accompany numerous brain diseases, but their functional importance is not understood. In cases of primary familial brain calcification, an autosomally inherited neuropsychiatric disorder, the presence of bilateral brain calcifications in the absence of secondary causes of brain calcification is a diagnostic criterion. To date, mutations in five genes including solute carrier 20 member 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), myogenesis regulating glycosidase (MYORG), platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor β (PDGFRB), are considered causal. Previously, we have reported that mutations in PDGFB in humans are associated with primary familial brain calcification, and mice hypomorphic for PDGFB (Pdgfbret/ret) present with brain vessel calcifications in the deep regions of the brain that increase with age, mimicking the pathology observed in human mutation carriers. In this study, we characterize the cellular environment surrounding calcifications in Pdgfbret/ret animals and show that cells around vessel-associated calcifications express markers for osteoblasts, osteoclasts and osteocytes, and that bone matrix proteins are present in vessel-associated calcifications. Additionally, we also demonstrate the osteogenic environment around brain calcifications in genetically confirmed primary familial brain calcification cases. We show that calcifications cause oxidative stress in astrocytes and evoke expression of neurotoxic astrocyte markers. Similar to previously reported human primary familial brain calcification cases, we describe high interindividual variation in calcification load in Pdgfbret/ret animals, as assessed by ex vivo and in vivo quantification of calcifications. We also report that serum of Pdgfbret/ret animals does not differ in calcification propensity from control animals and that vessel calcification occurs only in the brains of Pdgfbret/ret animals. Notably, ossification of vessels and astrocytic neurotoxic response is associated with specific behavioural and cognitive alterations, some of which are associated with primary familial brain calcification in a subset of patients.

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple dysfunctional pathways. Therefore, a sophisticated treatment strategy that simultaneously targets multiple brain cell types and disease pathways could be advantageous for effective intervention. To elucidate an effective treatment, we developed an in vitro high-throughput screening (HTS) assay to evaluate candidate drugs for their ability to enhance the integrity of the blood-brain barrier (BBB) and improve clearance of amyloid-β (Aβ) using a cell-based BBB model. Results from HTS identified etodolac and α-tocopherol as promising drugs for further investigation. Both drugs were tested separately and in combination for the purpose of targeting multiple pathways including neuroinflammation and oxidative stress. In vitro studies assessed the effects of etodolac and α-tocopherol individually and collectively for BBB integrity and Aβ transport, synaptic markers and Aβ production in APP-transfected neuronal cells, as well as effects on inflammation and oxidative stress in astrocytes. Transgenic 5XFAD mice were used to translate in vitro results of etodolac and α-tocopherol independently and with concurrent administration. Compared to either drug alone, the combination significantly enhanced the BBB function, decreased total Aβ load correlated with increased expression of major transport proteins, promoted APP processing towards the neuroprotective and non-amyloidogenic pathway, induced synaptic markers expression, and significantly reduced neuroinflammation and oxidative stress both in vitro and in vivo. Collective findings demonstrated the combination produced mixed interaction showing additive, less than additive or synergistic effects on the evaluated markers. In conclusion, this study highlights the significance of combination therapy to simultaneously target multiple disease pathways, and suggest the repurposing and combination of etodolac and α-tocopherol as a novel therapeutic strategy against AD.