Mn Inhibits GSH Synthesis via Downregulation of Neuronal EAAC1 and Astrocytic xCT to Cause Oxidative Damage in the Striatum of Mice

Excessive manganese (Mn) can accumulate in the striatum of the brain following overexposure. Oxidative stress is a well-recognized mechanism in Mn-induced neurotoxicity. It has been proven that glutathione (GSH) depletion is a key factor in oxidative damage during Mn exposure. However, no study has focused on the dysfunction of GSH synthesis-induced oxidative stress in the brain during Mn exposure. The objective of the present study was to explore the mechanism of Mn disruption of GSH synthesis via EAAC1 and xCT in vitro and in vivo. Primary neurons and astrocytes were cultured and treated with different doses of Mn to observe the state of cells and levels of GSH and reactive oxygen species (ROS) and measure mRNA and protein expression of EAAC1 and xCT. Mice were randomly divided into seven groups, which received saline, 12.5, 25, and 50 mg/kg MnCl2, 500 mg/kg AAH (EAAC1 inhibitor) + 50 mg/kg MnCl2, 75 mg/kg SSZ (xCT inhibitor) + 50 mg/kg MnCl2, and 100 mg/kg NAC (GSH rescuer) + 50 mg/kg MnCl2 once daily for two weeks. Then, levels of EAAC1, xCT, ROS, GSH, malondialdehyde (MDA), protein sulfhydryl, carbonyl, 8-hydroxy-2-deoxyguanosine (8-OHdG), and morphological and ultrastructural features in the striatum of mice were measured. Mn reduced protein levels, mRNA expression, and immunofluorescence intensity of EAAC1 and xCT. Mn also decreased the level of GSH, sulfhydryl, and increased ROS, MDA, 8-OHdG, and carbonyl in a dose-dependent manner. Injury-related pathological and ultrastructure changes in the striatum of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GSH synthesis through inhibition of EAAC1 and xCT to trigger oxidative damage in the striatum.

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NSun2 promotes cell migration through methylating autotaxin mRNA

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012009 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18134-18147

Author(s):

Xin Xu ◽

Yihua Zhang ◽

Junjie Zhang ◽

Xiaotian Zhang

Keyword(s):

Cell Migration ◽

Biological Effects ◽

Mrna Translation ◽

Glioma Cell Line ◽

Mrna Levels ◽

Dependent Manner ◽

Protein Levels ◽

Key Factor

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.

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Abstract 3653: Akt3 Offers Stronger Protection than Akt1 Against Brain Injury by Differentially Promoting Mtor Protein Levels In Both In Vitro and In Vivo Stroke Models

Stroke ◽

10.1161/str.43.suppl_1.a3653 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Rong Xie ◽

Michelle Cheng ◽

Mei Li ◽

Robert Sapolsky ◽

Heng Zhao

Keyword(s):

Gene Transfer ◽

Western Blotting ◽

Ischemic Injury ◽

Akt Pathway ◽

Over Expression ◽

Protein Levels ◽

Akt Isoforms ◽

The Brain

Background and Objective: Akt is a serine-threonine kinase that plays critical role in promoting cell survival. Akt consists of three isoforms (Akt1, 2, 3), with Akt3 predominantly expressed in the brain. Although Akt pathway has been shown to mediate neuronal survival in cerebral ischemic injury, it is unclear how these Akt isoforms contribute to neuronal protection, and whether exogenous Akt can protect the brain against ischemic injury or not. In this study, we over-expressed Akt isoforms and its downstream signaling proteins such as FKHR and PRAS40 to investigate the role of the Akt pathway along with its potential relationship with the mTOR pathway in stroke. Methods: Sprauge Dawley rats (250∼280g) were used for all studies. A lentiviral vector consists of a CMV promoter driving IRES-eGFP was used to clone an active Akt 1 and 3 (cAKt 1 and 3), dominant-negative Akt (AktDN), active FKHR (AAA FKHR), and PRAS40. Lentivirus expressing these genes were added to primary mixed cortical cultures for two days prior to oxygen glucose deprivation (OGD) (MOI=1:5). Neuronal survival was measured by LDH release. Lentivirus were stereotaxically injected into the cortex, and rats were subjected to focal ischemia induced by distal MCA occlusion combined with bilateral CCA occlusion. Western blotting and immunofluorescent confocal microscopy were used to detect the expression of Akt isoforms and other proteins in both the Akt and mTOR pathways. Results: Western blotting analysis showed that both endogenous Akt1 and 3 proteins degraded as early as 1 h after stroke, while Akt2 protein remained unchanged until 24 h after stroke. In vitro studies showed that over-expression of both constitutively active cAkt1 and cAkt3 decreased LDH release after OGD, while AktDN worsened neuronal death ( P <0.05). In vivo over-expression of cAkt1, cAkt3 and PRAS40 reduced infarct size after stroke ( P <0.01). Gene transfer of cAkt1 and 3 also promoted protein levels of pAkt (phosphorylated Akt), pPRAS40, pFKHR, pPTEN, pmTOR, but not pGSK3β. Both in vitro and in vivo studies showed that over-expression of cAkt3 resulted in a stronger protection than cAkt1 ( P <0.05). Interestingly, cAkt3 gene transfer preserved both endogenous protein levels of Akt1 and 3, whereas cAkt1 gene transfer only preserved endogenous Akt1. Furthermore, cAkt3 promoted higher pmTOR levels than cAkt1. Treatment of rapamycin, an mTOR inhibitor, blocked the protective effects of both cAkt1 and cAkt3 both in vitro and in vivo. Conclusion: Lentiviral-mediated overexpression of cAkt3 confers stronger protection than that of cAkt1, by maintaining both endogenous Akt1 and Akt3, as well as promoting higher mTOR activities after stroke.

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Vascular Dysfunction Induced by Mercury Exposure

International Journal of Molecular Sciences ◽

10.3390/ijms20102435 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2435 ◽

Cited By ~ 4

Author(s):

Tetsuya Takahashi ◽

Takayoshi Shimohata

Keyword(s):

Oxidative Stress ◽

Vascular Dysfunction ◽

Brain Parenchyma ◽

Transport Systems ◽

Mehg Exposure ◽

In Vivo Models ◽

The Central Nervous System ◽

The Brain

Methylmercury (MeHg) causes severe damage to the central nervous system, and there is increasing evidence of the association between MeHg exposure and vascular dysfunction, hemorrhage, and edema in the brain, but not in other organs of patients with acute MeHg intoxication. These observations suggest that MeHg possibly causes blood–brain barrier (BBB) damage. MeHg penetrates the BBB into the brain parenchyma via active transport systems, mainly the l-type amino acid transporter 1, on endothelial cell membranes. Recently, exposure to mercury has significantly increased. Numerous reports suggest that long-term low-level MeHg exposure can impair endothelial function and increase the risks of cardiovascular disease. The most widely reported mechanism of MeHg toxicity is oxidative stress and related pathways, such as neuroinflammation. BBB dysfunction has been suggested by both in vitro and in vivo models of MeHg intoxication. Therapy targeted at both maintaining the BBB and suppressing oxidative stress may represent a promising therapeutic strategy for MeHg intoxication. This paper reviews studies on the relationship between MeHg exposure and vascular dysfunction, with a special emphasis on the BBB.

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The Antiviral Effect of Novel Steroidal Derivatives on Flaviviruses

Frontiers in Microbiology ◽

10.3389/fmicb.2021.727236 ◽

2021 ◽

Vol 12 ◽

Author(s):

Luping Zhang ◽

Dengyuan Zhou ◽

Qiuyan Li ◽

Shuo Zhu ◽

Muhammad Imran ◽

...

Keyword(s):

Antiviral Effect ◽

Therapeutic Drug ◽

Dependent Manner ◽

Design And Synthesis ◽

Invasion Stage ◽

Synthetic Derivatives ◽

Dose Dependent ◽

The Brain

Flaviviruses are the major emerging arthropod-borne pathogens globally. However, there is still no practical anti-flavivirus approach. Therefore, existing and emerging flaviviruses desperately need active broad-spectrum drugs. In the present study, the antiviral effect of steroidal dehydroepiandrosterone (DHEA) and 23 synthetic derivatives against flaviviruses such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), and Dengue virus (DENV) were appraised by examining the characteristics of virus infection both in vitro and in vivo. Our results revealed that AV1003, AV1004 and AV1017 were the most potent inhibitors of flavivirus propagation in cells. They mainly suppress the viral infection in the post-invasion stage in a dose-dependent manner. Furthermore, orally administered compound AV1004 protected mice from lethal JEV infection by increasing the survival rate and reducing the viral load in the brain of infected mice. These results indicate that the compound AV1004 might be a potential therapeutic drug against JEV infection. These DHEA derivatives may provide lead scaffolds for further design and synthesis of potential anti-flavivirus potential drugs.

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Synthesis and preclinical evaluation of [11C]MTP38 as a novel PET ligand for phosphodiesterase 7 in the brain

10.1101/2020.10.29.354696 ◽

2020 ◽

Author(s):

Naoyuki Obokata ◽

Chie Seki ◽

Takeshi Hirata ◽

Jun Maeda ◽

Hideki Ishii ◽

...

Keyword(s):

Arterial Input Function ◽

Inflammatory Diseases ◽

Input Function ◽

Binding Potential ◽

Dependent Manner ◽

Reference Tissue ◽

Molar Activity ◽

The Brain

AbstractPurposePhosphodiesterase (PDE) 7 is a potential therapeutic target for neurological and inflammatory diseases, although in-vivo visualization of PDE7 has not been successful. In this study, we aimed to develop [11C]MTP38 as a novel positron emission tomography (PET) ligand for PDE7.Methods[11C]MTP38 was radiosynthesized by 11C-cyanation of a bromo precursor with [11C]HCN. PET scans of rat and rhesus monkey brains and in-vitro autoradiography of brain sections derived from these species were conducted with [11C]MTP38. In monkeys, dynamic PET data were analyzed with an arterial input function to calculate the total distribution volume (VT). The non-displaceable binding potential (BPND) in the striatum was also determined by a reference tissue model with cerebellar reference. Finally, striatal occupancy of PDE7 by an inhibitor was calculated in monkeys according to changes in BPND.Results[11C]MTP38 was synthesized with radiochemical purity ≥ 99.4% and molar activity of 38.6 ± 12.6 GBq/μmol. Autoradiography revealed high radioactivity in the striatum and its reduction by non-radiolabeled ligands, in contrast with unaltered autoradiographic signals in other regions. In-vivo PET after radioligand injection to rats and monkeys demonstrated that radioactivity was rapidly distributed to the brain and intensely accumulated in the striatum relative to the cerebellum. Correspondingly, estimated VT values in the monkey striatum and cerebellum were 3.59 and 2.69 mL/cm3, respectively. The cerebellar VT value was unchanged by pretreatment with unlabeled MTP38. Striatal BPND was reduced in a dose-dependent manner after pretreatment with MTP-X, a PDE7 inhibitor. Relationships between PDE7 occupancy by MTP-X and plasma MTP-X concentration could be described by Hill’s sigmoidal function.ConclusionWe have provided the first successful preclinical demonstration of in-vivo PDE7 imaging with a specific PET radioligand. [11C]MTP38 is a feasible radioligand for evaluating PDE7 in the brain and is currently being applied to a first-in-human PET study.

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Mechanism of KLF4 Protection against Acute Liver Injury via Inhibition of Apelin Signaling

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/6140360 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Weitao Ji ◽

Hongyun Shi ◽

Hailin Shen ◽

Jing Kong ◽

Jiayi Song ◽

...

Keyword(s):

Liver Injury ◽

Signaling Pathway ◽

Acute Liver Injury ◽

Control Group ◽

Necrosis Factor Alpha ◽

Protein Levels ◽

Klf4 Expression ◽

Mrna And Protein Expression

Krüppel-like factor 4 (KLF4) is a key transcription factor that regulates genes involved in the proliferation or differentiation in different tissues. Apelin plays roles in cardiovascular functions, metabolic disease, and homeostatic disorder. However, the biological function of apelin in liver disease is still ongoing. In this study, we investigated the mechanism of KLF4-mediated protection against acute liver injury via the inhibition of the apelin signaling pathway. Mice were intraperitoneally injected with carbon tetrachloride (CCl4; 0.2 mL dissolved in 100 mL olive oil, 10 mL/kg) to establish an acute liver injury model. A KLF4 expression plasmid was injected through the tail vein 48 h before CCl4 treatment. In cultured LX-2 cells, pAd-KLF4 or siRNA KLF4 was overexpressed or knockdown, and the mRNA and protein levels of apelin were determined. The results showed that the apelin serum level in the CCl4-injected group was higher than that of control group, and the expression of apelin in the liver tissues was elevated while KLF4 expression was decreased in the CCl4-injected group compared to the KLF4-plasmid-injected group. HE staining revealed serious hepatocellular steatosis in the CCl4-injected mice, and KLF4 alleviated this steatosis in the mice injected with KLF4 plasmid. In vitro experiments showed that tumor necrosis factor-alpha (TNF-α) could downregulate the transcription and translation levels of apelin in LX-2 cells and also upregulate KLF4 mRNA and protein expression. RT-PCR and Western blotting showed that the overexpression of KLF4 markedly decreased basal apelin expression, but knockdown of KLF4 restored apelin expression in TNF-α-treated LX-2 cells. These in vivo and in vitro experiments suggest that KLF4 plays a key role in inhibiting hepatocellular steatosis in acute liver injury, and that its mechanism might be the inhibition of the apelin signaling pathway.

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Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts

Rheumatology ◽

10.1093/rheumatology/kez583 ◽

2019 ◽

Vol 59 (9) ◽

pp. 2258-2263 ◽

Cited By ~ 3

Author(s):

Tiago Carvalheiro ◽

Beatriz Malvar Fernández ◽

Andrea Ottria ◽

Barbara Giovannone ◽

Wioleta Marut ◽

...

Keyword(s):

Protein Expression ◽

Therapeutic Target ◽

Dermal Fibroblasts ◽

Dependent Manner ◽

Healthy Controls ◽

Multiple Organs ◽

Extracellular Matrix Components ◽

Mrna And Protein Expression

Abstract Objectives SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. Methods Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. Results Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling. Conclusion These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

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An Antioxidant Phytotherapy to Rescue Neuronal Oxidative Stress

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nen053 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Zhihong Lin ◽

Danni Zhu ◽

Yongqing Yan ◽

Boyang Yu ◽

Qiujuan Wang ◽

...

Keyword(s):

Oxidative Stress ◽

Aqueous Extract ◽

Ischemia Reperfusion ◽

Brain Infarction ◽

Reaction System ◽

Artery Occlusion ◽

Dependent Manner ◽

Endogenous Antioxidant

Oxidative stress is involved in the pathogenesis of ischemic neuronal injury. A Chinese herbal formula composed ofPoria cocos(Chinese name:Fu Ling),Atractylodes macrocephala(Chinese name:Bai Zhu) andAngelica sinensis(Chinese names:Danggui, Dong quai, Donggui; Korean name:Danggwi) (FBD), has been proved to be beneficial in the treatment of cerebral ischemia/reperfusion (I/R).This study was carried out to evaluate the protective effect of FBD against neuronal oxidative stressin vivoandin vitro. Rat I/R were established by middle cerebral artery occlusion (MCAO) for 1 h, followed by 24 h reperfusion. MCAO led to significant depletion in superoxide dismutase and glutathione and rise in lipid peroxidation (LPO) and nitric oxide in brain. The neurological deficit and brain infarction were also significantly elevated by MCAO as compared with sham-operated group. All the brain oxidative stress and damage were significantly attenuated by 7 days pretreatment with the aqueous extract of FBD (250 mg kg−1, p.o.). Moreover, cerebrospinal fluid sampled from FBD-pretreated rats protected PC12 cells against oxidative insult induced by 0.2 mM hydrogen peroxide, in a concentration and time-dependent manner (IC5010.6%, ET501.2 h). However, aqueous extract of FBD just slightly scavenged superoxide anion radical generated in xanthine–xanthine oxidase system (IC502.4 mg ml−1) and hydroxyl radical generated in Fenton reaction system (IC503.6 mg ml−1). In conclusion, FBD was a distinct antioxidant phytotherapy to rescue neuronal oxidative stress, through blocking LPO, restoring endogenous antioxidant system, but not scavenging free radicals.

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Honokiol Alleviates Methionine-Choline Deficient Diet-Induced Hepatic Steatosis and Oxidative Stress in C57BL/6 Mice by Regulating CFLAR-JNK Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/2313641 ◽

2020 ◽

Vol 2020 ◽

pp. 1-14

Author(s):

Ting Zhai ◽

Wei Xu ◽

Yayun Liu ◽

Kun Qian ◽

Yanling Xiong ◽

...

Keyword(s):

Oxidative Stress ◽

Insulin Resistance ◽

Regulatory Gene ◽

Deficient Diet ◽

Jnk Pathway ◽

Beneficial Effects ◽

Protein Levels ◽

And Oxidative Stress

Background. Honokiol (HNK) has been reported to possess various beneficial effects in the context of metabolic disorders, including fatty liver, insulin resistance, and oxidative stress which are closely related to nonalcoholic steatohepatitis (NASH), however with no particular reference to CFLAR or JNK. Methods. C57BL/6 mice were fed methionine-choline-deficient (MCD) diet and administered simultaneously with HNK (10 and 20 mg/kg once a day, ig) for 6 weeks, and NCTC1469 cells were pretreated, respectively, by oleic acid (OA, 0.5 mmol/L) plus palmitic acid (PA, 0.25 mmol/L) for 24 h, and adenovirus-down Cflar for 24 h, then exposed to HNK (10 and 20 μmol/L) for 24 h. Commercial kits, H&E, MT, ORO staining, RT-qPCR, and Western blotting were used to detect the biomarkers, hepatic histological changes, and the expression of key genes involved in NASH. Results. The in vivo results showed that HNK suppressed the phosphorylation of JNK (pJNK) by activating CFLAR; enhanced the mRNA expression of lipid metabolism-related genes Acox, Cpt1α, Fabp5, Gpat, Mttp, Pparα, and Scd-1; and decreased the levels of hepatic TG, TC, and MDA, as well as the levels of serum ALT and AST. Additionally, HNK enhanced the protein expression of oxidative stress-related key regulatory gene NRF2 and the activities of antioxidases HO-1, CAT, and GSH-Px and decreased the protein levels of prooxidases CYP4A and CYP2E1. The in vivo effects of HNK on the expression of CLFAR, pJNK, and NRF2 were proved by the in vitro experiments. Moreover, HNK promoted the phosphorylation of IRS1 (pIRS1) in both tested cells and increased the uptake of fluorescent glucose 2-NBDG in OA- and PA-pretreated cells. Conclusions. HNK ameliorated NASH mainly by activating the CFLAR-JNK pathway, which not only alleviated fat deposition by promoting the efflux and β-oxidation of fatty acids in the liver but also attenuated hepatic oxidative damage and insulin resistance by upregulating the expression of NRF2 and pIRS1.

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ROS-Dependent Neuroprotective Effects of NaHS in Ischemia Brain Injury Involves the PARP/AIF Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000430317 ◽

2015 ◽

Vol 36 (4) ◽

pp. 1539-1551 ◽

Cited By ~ 35

Author(s):

Qian Yu ◽

Zhihong Lu ◽

Lei Tao ◽

Lu Yang ◽

Yu Guo ◽

...

Keyword(s):

Brain Injury ◽

Focal Cerebral Ischemia ◽

Ischemia Reperfusion ◽

Dependent Manner ◽

Neuroprotective Effects ◽

Ht22 Cells ◽

In Vivo Models ◽

The Brain

Background/Aims: Stroke is among the top causes of death worldwide. Neuroprotective agents are thus considered as potentially powerful treatment of stroke. Methods: Using both HT22 cells and male Sprague-Dawley rats as in vitro and in vivo models, we investigated the effect of NaHS, an exogenous donor of H2S, on the focal cerebral ischemia-reperfusion (I/R) induced brain injury. Results: Administration of NaHS significantly decreased the brain infarcted area as compared to the I/R group in a dose-dependent manner. Mechanistic studies demonstrated that NaHS-treated rats displayed significant reduction of malondialdehyde content, and strikingly increased activity of superoxide dismutases and glutathione peroxidase in the brain tissues compared with I/R group. The enhanced antioxidant capacity as well as restored mitochondrial function are NaHS-treatment correlated with decreased cellular reactive oxygen species level and compromised apoptosis in vitro or in vivo in the presence of NaHS compared with control. Further analysis revealed that the inhibition of PARP-1 cleavage and AIF translocation are involved in the neuroprotective effects of NaHS. Conclusion: Collectively, our results suggest that NaHS has potent protective effects against the brain injury induced by I/R. NaHS is possibly effective through inhibition of oxidative stress and apoptosis.

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