Nanoliposomes Reduce Stroke Injury Following Middle Cerebral Artery Occlusion in Mice.

BACKGROUND AND PURPOSE: Neuroprotective strategies for stroke remain inadequate. Nanoliposomes comprised of phosphatidylcholine, cholesterol, and monosialogangliosides (nanoliposomes) induced an antioxidant protective response in endothelial cells exposed to amyloid insults. We tested the hypotheses that nanoliposomes will preserve human neuroblastoma (SH-SY5Y) and human brain microvascular endothelial cells viability following oxygen-glucose deprivation (OGD)-reoxygenation and will reduce injury in mice following middle cerebral artery occlusion. METHODS: SH-SY5Y and human brain microvascular endothelial cells were exposed to oxygen-glucose deprivation-reoxygenation (3 hours 0.5%-1% oxygen and glucose-free media followed by 20-hour ambient air/regular media) without or with nanoliposomes (300 µg/mL). Viability was measured (calcein-acetoxymethyl fluorescence) and protein expression of antioxidant proteins HO-1 (heme oxygenase-1), NQO1 (NAD[P]H quinone dehydrogenase 1), and SOD1 (superoxide dismutase 1) were measured by Western blot. C57BL/6J mice were treated with saline (n=8) or nanoliposomes (10 mg/mL lipid, 200 µL, n=7) while undergoing 60-minute middle cerebral artery occlusion followed by reperfusion. Day 2 postinjury neurological impairment score and infarction size were compared. RESULTS: SH-SY5Y and human brain microvascular endothelial cells showed reduced viability post-oxygen-glucose deprivation-reoxygenation that was reversed by nanoliposomes. Nanoliposomes increased protein expressions of HO-1, NQO1 in both cell types and SOD1 in human brain microvascular endothelial cells. Nanoliposomes-treated mice showed reduced neurological impairment and brain infarct size (18.8±2% versus 27.3±2.3%, P=0.017) versus controls. CONCLUSIONS: Nanoliposomes reduced stroke injury in mice subjected to middle cerebral artery occlusion likely through induction of an antioxidant protective response. Nanoliposome is a candidate novel agent for stroke.

Survivin in breast cancer-derived exosomes activates fibroblasts by up-regulating SOD1, whose feedback promotes cancer proliferation and metastasis.

Cancer-associated fibroblasts (CAFs) play a critical role in the coevolution of breast tumor cells and their microenvironment by modifying cellular compartments and regulating cancer cell functions via stromal-epithelial dialogue. However, the relationship and interaction between stromal and epithelial cells is still poorly understood. Herein, we revealed that breast cancer cells have a stronger ability to activate fibroblasts and transform them into myofibroblasts (CAF-like) than normal breast epithelial cells, and this stronger ability occurs through paracrine signaling. In turn, myofibroblasts promote the proliferation, epithelial-to-mesenchymal transition (EMT), and stemness of breast cancer cells. Detailed regulatory mechanisms showed that, compared with normal cells, Survivin is overexpressed in breast cancer cells and secreted extracellularly in the form of exosomes, which are then internalized by fibroblasts. Breast cancer cell-derived survivin up-regulates SOD1 expression in fibroblasts and then converts them into myofibroblasts, conversely inducing breast cancer progression in vitro and in vivo Thus, our results indicate that survivin acts as an activator of the tumor microenvironment and that SOD1 up-regulation in fibroblasts can promote breast cancer progression. These results suggest that targeting survivin and SOD1 may be a potential therapeutic strategy for breast cancer.

Reduction of Superoxide Dismutase 1 Delays Regeneration of Cardiotoxin-Injured Skeletal Muscle in KK/Ta-Ins2Akita Mice with Progressive Diabetic Nephropathy.

Superoxide dismutase (SOD) is a major antioxidant enzyme for superoxide removal, and cytoplasmic SOD (SOD1) is expressed as a predominant isoform in all cells. We previously reported that renal SOD1 deficiency accelerates the progression of diabetic nephropathy (DN) via increasing renal oxidative stress. To evaluate whether the degree of SOD1 expression determines regeneration capacity and sarcopenic phenotypes of skeletal muscles under incipient and advanced DN conditions, we investigated the alterations of SOD1 expression, oxidative stress marker, inflammation, fibrosis, and regeneration capacity in cardiotoxin (CTX)-injured tibialis anterior (TA) muscles of two Akita diabetic mouse models with different susceptibility to DN, DN-resistant C57BL/6-Ins2Akita and DN-prone KK/Ta-Ins2Akita mice. Here, we report that KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, exhibit delayed muscle regeneration after CTX injection, as demonstrated by the finding indicating significantly smaller average cross-sectional areas of regenerating TA muscle myofibers relative to KK/Ta-wild-type mice. Furthermore, we observed markedly reduced SOD1 expression in CTX-injected TA muscles of KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, along with increased inflammatory cell infiltration, prominent fibrosis and superoxide overproduction. Our study provides the first evidence that SOD1 reduction and the following superoxide overproduction delay skeletal muscle regeneration through induction of overt inflammation and fibrosis in a mouse model of progressive DN.

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft-bodied tick vector.

The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft-bodied tick vector, Ornithodoros turicata. Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata. A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase (gpx), thioredoxin peroxidase (tpx), manganese superoxide dismutase (sod-1), copper-zinc superoxide dismutase (sod-2), and catalase (cat) by reverse-transcriptase droplet digital polymerase chain reaction (RT-ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi, suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.

microRNA-378a Regulates the Reactive Oxygen Species (ROS)/Phosphatidylinositol 3-Kinases (PI3K)/AKT Signaling Pathway in Human Lens Epithelial Cells and Cataract.

BACKGROUND Cataract is associated with increased apoptosis of the epithelial cells of the ocular lens. Previous studies have shown that microRNA-378a (miR-378a) has a role in the development of cataract, but the molecular mechanisms remain unclear. This study aimed to investigate the effects of miR-378a in human lens epithelial cells (HLECs) in vitro and normal lens tissues and cataract tissues. MATERIAL AND METHODS HLECs were grown in culture. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot were used to examine gene expression levels. The MTT and TUNEL assay measured cell growth and apoptosis. Changes in the fluorescence ratio of ethidium to dihydroethidium (E: DHE) and in 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (C-H2DCFDA) were used to detect superoxide (O2⁻) and hydrogen peroxide (H2O2). The expression levels of miR-378a and the superoxide dismutase 1 gene (SOD1) were measured in normal human lens tissues and cataract tissues. RESULTS Upregulation of miR-378a reduced the expression of SOD1. Levels of O2⁻ were upregulated and H2O2 was slightly down-regulated by miR-378a. The use of a miR-378a mimic suppressed cell growth and enhanced apoptosis of HLECs, which were reversed by the use of a miR-378a inhibitor. SOD1 overexpression rescued the miR-378a-induced phenotypes of HLEC cells. Treatment with the PI3K inhibitor, LY294002, reversed miR-378a and ROS-regulated proliferation and apoptosis of HLEC cells. Also, miR-378a was upregulated, and SOD1 was down-regulated in human cataract tissues. CONCLUSIONS In HLECs, expression of miR-378a regulated ROS and PI3K/AKT signaling, and miR-378a was upregulated, and SOD1 was down-regulated in human cataract tissue.

Recombinant human islet amyloid polypeptide forms shorter fibrils and mediates ß-cell apoptosis via generation of oxidative stress.

Protein misfolding and aggregation play an important role in many human diseases including Alzheimer's, Parkinson's and type 2 diabetes mellitus (T2DM). The human islet amyloid polypeptide (hIAPP) forms amyloid plaques in the pancreas of T2DM subjects (>95%) that are involved in deteriorating islet function and in mediating ß-cell apoptosis. However, the detailed mechanism of action, structure and nature of toxic hIAPP species responsible for this effect remains elusive to date mainly due to the high cost associated with the chemical synthesis of pure peptide required for these studies. In the present work, we attempted to obtain structural and mechanistic insights into the hIAPP aggregation process using recombinant hIAPP (rhIAPP) isolated from Escherichia coli Results from biophysical and structural studies indicate that the rhIAPP self-assembled into highly pure, ß-sheet-rich amyloid fibrils with uniform morphology. rhIAPP-mediated apoptosis in INS-1E cells was associated with increased oxidative stress and changes in mitochondrial membrane potential. The transcript levels of apoptotic genes - Caspase-3 and Bax were found to be up-regulated, while the levels of the anti-apoptotic gene - Bcl2 were down-regulated in rhIAPP-treated cells. Additionally, the expression levels of genes involved in combating oxidative stress namely Catalase, SOD1 and GPx were down-regulated. rhIAPP exposure also affected glucose-stimulated insulin secretion from isolated pancreatic islets. The aggregation of rhIAPP also occurred significantly faster when compared with that of the chemically synthesized peptide. We also show that the rhIAPP fibrils were shorter and more cytotoxic. In summary, our study is one among the few to provide comprehensive evaluation of structural, biophysical and cytotoxic properties of rhIAPP.

Relationship between mutant Cu/Zn superoxide dismutase 1 maturation and inclusion formation in cell models.

A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In this study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time stamp that distinguishes pre-existing and newly made proteins. In cells that transiently over-express the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu co-factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly made protein, are prone to misfolding and aggregation.

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines.

CONTEXT: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen. OBJECTIVE: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines. MATERIALS AND METHODS: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco's modified Eagle's medium for 5 days, and next cultured in Hypoxic Chamber in 1% O2, 10% O2, 21% O2. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software. RESULTS: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line. CONCLUSION: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.

The role of Melatonin receptor MTNR1A in the action of Melatonin on bovine granulosa cells.

Granulosa cells (GCs) play an important role in ovarian follicle growth, development, and follicular atresia. In the present study, we investigated the effects of Melatonin on bovine GCs, and asked if MTNR1A was involved in their response to this indole hormone. Our results indicated that Melatonin inhibited GC apoptosis by up-regulating the expression of BCL2, BCL-XL, GPX4, and SOD1, and down-regulating the expression of BAX, CASP3, and TP53. Moreover, Melatonin modulated bovine GC function by decreasing the expression of INHA, INHBB, FSHR, and TGFBR3, and the abundance of Inhibin ß and Activin B, while increasing the expression of LHR, INHBA, and secretion of progesterone by GCs. In contrast, knockdown of MTNR1A significantly increased the expression of BAX, CASP3, TP53, INHA, FSHR, and TGFBR3, as well as Inhibin ß abundance, while decreasing the expression of BCL2, GPX4, SOD1, and LHR, and production of progesterone and estradiol; no effect was observed on the expression of BCL-XL, INHBA, or INHBB. These results suggest that Melatonin and MTNR1A play an important role in modulating bovine GC function by regulating cellular progression, apoptosis, hormones secretion, and reproduction-related genes. Furthermore, altered expression of MTNR1A could affect how bovine GCs respond to Melatonin.

The effect of n-3/n-6 polyunsaturated fatty acids on acute reflux esophagitis in rats.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) play various roles in inflammation. However, the effect of PUFAs in the development of reflux esophagitis (RE) is unclear. This study is to investigate the potential effect of n-3/n-6 PUFAs on acute RE in rats along with the underlying protective mechanisms. METHODS: Forty Sprague Dawley rats were randomly divided into four groups (n = 10 in each group). RE model was established by pyloric clip and section ligation. Fish oil- and soybean oil-based fatty emulsion (n-3 and n-6 groups), or normal saline (control and sham operation groups) was injected intraperitoneally 2 h prior to surgery and 24 h postoperatively (2 mL/kg, respectively). The expressions of interleukin (IL)-1ß, IL-8, IL-6 and myeloid differentiation primary response gene 88 (MyD88) in esophageal tissues were evaluated by Western blot and immunohistochemistry after 72 h. The malondialdehyde (MDA) and superoxide dismutase (SOD) expression in the esophageal tissues were determined to assess the oxidative stress. RESULTS: The mildest macroscopic/microscopic esophagitis was found in the n-3 group (P < 0.05). The expression of IL-1ß, IL-8, IL-6 and MyD88 were increased in all RE groups, while the lowest and highest expression were found in n-3 and n-6 group, respectively (P < 0.05). The MDA levels were increased in all groups (P < 0.05), in an ascending trend from n-3, n-6 groups to control group. The lowest and highest SOD levels were found in the control and n-3 group, respectively (P < 0.05). CONCLUSION: n-3 PUFAs may reduce acute RE in rats, which may be due to inhibition of the MyD88-NF-kB pathway and limit oxidative damage.

BMP-4 impedes endothelial cell migration in neointimal hyperplasia via FoXO-3 specific modulation of reactive oxygen species.

BACKGROUND AND AIMS: Endothelial cell injury causes vascular barrier dysfunction and leukocyte recruitment to the underlying tissue. Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor that exerts pro-inflammatory effects on the endothelium. Here, we investigated the effects of BMP-4 on endothelial cell (EC) migration following balloon injury in SD rats. METHODS: An intimal hyperplasia model was established using balloon injury. Hematoxylin-eosin staining (HE) and silver staining were used to detect the alteration of endothelial cells recovery after balloon injury. Serum BMP-4 levels were assessed by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured. MTT assay was used to measure cell viability. Protein expression was detected by Western blot. Intracellular reactive oxygen species (ROS) was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA). HUVECs migration was measured via transwell assay and scratch wound assay. RESULTS: The results indicated that BMP-4 inhibition significantly decreased total plasma activity of BMP-4 and reduced neointimal hyperplasia by stimulating endothelial cell migration, but did not affect the medial area following balloon injury. BMP-4 suppressed the formation of ROS via forkhead box O3 (FoXO-3)/superoxide dismutase 1 (SOD-1). In vitro, a high level of ROS induced by BMP-4 impeded HUVECs migration. CONCLUSIONS: The results suggest that BMP-4 inhibition is a potential means of preventing intimal hyperplasia formation after balloon injury.

Variation in the vulnerability of mice expressing human superoxide dismutase 1 to prion-like seeding: a study of the influence of primary amino acid sequence.

Misfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease.

Human superoxide dismutase 1 attenuates quinoneimine metabolite formation from mefenamic acid.

Mefenamic acid (MFA), one of the nonsteroidal anti-inflammatory drugs (NSAIDs), sometimes causes liver injury. Quinoneimines formed by cytochrome P450 (CYP)-mediated oxidation of MFA are considered to be causal metabolites of the toxicity and are detoxified by glutathione conjugation. A previous study reported that NAD(P)H:quinone oxidoreductase 1 (NQO1) can reduce the quinoneimines, but NQO1 is scarcely expressed in the human liver. The purpose is to identify enzyme(s) responsible for the decrease in MFA-quinoneimine formation in the human liver. The formation of MFA-quinoneimine by recombinant CYP1A2 and CYP2C9 was significantly decreased by the addition of human liver cytosol, and the extent of the decrease in the metabolite formed by CYP1A2 was larger than that by CYP2C9. By column chromatography, superoxide dismutase 1 (SOD1) was identified from the human liver cytosol as an enzyme decreasing MFA-quinoneimine formation. Addition of recombinant SOD1 into the reaction mixture decreased the formation of MFA-quinoneimine from MFA by recombinant CYP1A2. By a structure-activity relationship study, we found that SOD1 decreased the formation of quinoneimines from flufenamic acid and tolfenamic acid, but did not affect those produced from acetaminophen, amodiaquine, diclofenac, and lapatinib. Thus, SOD1 may selectively decrease the quinoneimine formation from fenamate-class NSAIDs. To examine whether SOD1 can attenuate cytotoxicity caused by MFA, siRNA for SOD1 was transfected into CYP1A2-overexpressed HepG2 cells. The leakage of lactate dehydrogenase caused by MFA treatment was significantly increased by knockdown of SOD1. In conclusion, we found that SOD1 can serve as a detoxification enzyme for quinoneimines to protect from drug-induced toxicity.

Nmnat1 Modulates Mitochondrial Oxidative Stress by Inhibiting Caspase-3 Signaling in Alzheimer's Disease.

Nigrostriatal pathway disturbance is one of the major pathogenic factors in Alzheimer's disease (AD). Dopaminergic neuron dysfunction results in bradykinesia and akinesia (inability to initiate movement), indicating a significant risk factor for substantia nigra pars compacta lesions. Furthermore, the nicotinamide adenine dinucleotide (NAD+) is associated with Aß toxicity decline in AD therapy. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) is an essential enzyme that preserves normal neuronal function and protects neurons from insult. This study aimed to investigate the potential therapeutic effects of Nmnat1 and its underlying mechanisms in a triple-transgenic mouse model of AD (3xTgAD). Results showed that Nmnat1 improved the substantial behavioral measures of cognitive impairments compared with the 3xTgAD control. Additionally, Nmnat1 overexpression significantly increased tyrosine hydroxylase-positive neurons and anti-apoptotic protein Bcl2 and caspase-3 expression levels in 3xTgAD mice. Nmnat1 also effectively controlled SOD1 activation. In conclusion, Nmnat1 substantially decreases multiple AD-associated pathological characteristics at least partially by the increase of caspase-3 activation.

Superoxide dismutase 1 (SOD1) and cadmium: A three models approach to the comprehension of its neurotoxic effects.

Cadmium (Cd) is a widespread toxic environmental contaminant, released by anthropogenic activities. It interferes with essential metal ions homeostasis and affects protein structures and functions by substituting zinc, copper and iron. In this study, the effect of cadmium on SOD1, a CuZn metalloenzyme catalyzing superoxide conversion into hydrogen peroxide, has been investigated in three different biological models. We first evaluated the effects of cadmium combined with copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21. The enzyme activity and expression were investigated in the presence of fixed copper and/or zinc doses with different cadmium concentrations, in the cellular medium. Cadmium caused a dose-dependent reduction in SOD1 activity, while the expression remains constant. Similar results were obtained in the cellular model represented by the human SH-SY5Y neuronal cell line. After cadmium treatment for 24 and 48 h, SOD1 enzymatic activity decreased in a dose- and time-dependent way, while the protein expression remained constant. Finally, a 16 h cadmium treatment caused a 25 % reduction of CuZn-SOD activity without affecting the protein expression in the Caenorhabditis elegans model. Taken together our results show an inhibitory effect of cadmium on SOD1 enzymatic activity, without affecting the protein expression, in all the biological models used, suggesting that cadmium can displace zinc from the enzyme catalytic site.

Exosome-mediated radiosensitizing effect on neighboring cancer cells via increase in intracellular levels of reactive oxygen species.

The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membrane­enclosed small vesicles comprising lipid bilayers and are mediators of intercellular communication that transport a variety of intracellular components, including microRNAs (miRNAs or miRs). The present study aimed to identify novel roles of exosomes released from irradiated cells to neighboring cancer cells. In order to confirm the presence of exosomes in the human pancreatic cancer cell line MIAPaCa­2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome­specific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival following irradiation and the addition of exosomes was evaluated by colony forming assay. Expression levels of miRNAs in exosomes were then quantified by microarray analysis, while protein expression levels of Cu/Zn­ and Mn­superoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa­2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non­irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa­2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up­ and five downregulated miRNAs were identified in 5 and 8 Gy­irradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcription­quantitative PCR identified miR­6823­5p as a potential candidate to inhibit SOD1, leading to increased intracellular ROS levels and DNA damage. To the best of our knowledge, the present study is the first to demonstrate that irradiated exosomes enhance the radiation effect via increasing intracellular ROS levels in cancer cells. This contributes to improved understanding of the bystander effect of neighboring cancer cells.

Tetramethylpyrazine nitrone improves motor dysfunction and pathological manifestations by activating the PGC-1α/Nrf2/HO-1 pathway in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons that results in skeletal muscle atrophy, weakness and paralysis. Oxidative stress plays a key role in the pathogenesis of ALS, including familial forms of the disease arising from mutation of the gene coding for superoxide dismutase (SOD1). We have used the SOD1G93A ALS mouse model to investigate the efficacy of 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel tetramethylpyrazine derivative armed with a powerful free-radical scavenging nitrone moiety. TBN was administered to mice by intraperitoneal or intragastric injection after the onset of motor deficits. TBN slowed the progression of motor neuron disease as evidenced by improved motor performance, reduced spinal motor neuron loss and the associated glial response, and decreased skeletal muscle fiber denervation and fibrosis. TBN treatment activated mitochondrial antioxidant activity through the PGC-1α/Nrf2/HO-1 pathway and decreased the expression of human SOD1. These findings suggest that TBN holds promise as a therapeutic agent for ALS.

Inhibition of heat shock proteins increases autophagosome formation, and reduces the expression of APP, Tau, SOD1 G93A and TDP-43.

Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer's Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.

The protective effect of hydroxylated fullerene pretreatment on pilocarpine-induced status epilepticus.

Status epilepticus (SE) is a neurological emergency. The pathological hallmark of neuronal damage after epileptic seizures could be the chain reaction of oxygen free radicals. Hydroxylated fullerenes (HFs) are novel and effective free radical scavengers, which play an important role in various neurological diseases. However, whether they have a protective effect against epileptic seizures remains elusive. Our study explores the effect of pretreatment with HFs in different doses (0.5, 5, and 10 mg/kg) on SEmodels induced by pilocarpine (PILO). The results suggest that HFs have a protective effect on SE in a dose-dependent manner. HFs significantly reduce the incidence of SE, prolong the latency to SE, reduce the malondialdehyde (MDA) levels, and increase the glutathione (GSH) and superoxide dismutase (SOD) levels. In addition, HFs significantly raise the expression of B-cell lymphoma-2 (Bcl-2) and reduce the expression of Bcl-2-associated X protein (Bax). We found that expressions of nuclear NF-E2-related factor 2 (nNrf2), heme oxygenase-1 (HO-1) and NADPH: quinone oxidoreductase-1 (NQO1) were upregulated 24 h after the onset of SE, but the increase was not enough to combat oxidative stress damage, nor to attenuate lipid peroxidation and apoptosis. The expressions of these proteins in HFs pretreatment groups increased more significantly than those in the epilepsy (EP) group, which effectively reduced lipid peroxidation and apoptosis in the hippocampus. In summary, these findings highlight that HFs pretreatment has a protective effect against PILO-induced SE in rats. It may relieve oxidative stress damage by activating the Nrf2-ARE signaling pathway. It provides evidence that fullerene derivatives may have therapeutic potential for epileptic seizures.

Pubertal LPS treatment selectively alters PSD-95 expression in male CD-1 mice.

Puberty includes a highly stress-sensitive period with significant sex differences in the neurophysiological and behavioural outcomes of a peripheral immune challenge. Sex differences in the pubertal neuroimmune network's responses to systemic LPS may explain some of these enduring sex-specific outcomes of a pubertal immune challenge. However, the functional implications of these sex-specific neuroimmune responses on the local microenvironment are unclear. Western blots were used to examine treatment- and sex-related changes in expression of regulatory proteins in inflammation (NFκB), cell death (AIF), oxidative stress (SOD-1), and synaptic plasticity (PSD-95) following symptomatic recovery (i.e., one week post-treatment) from pubertal immune challenge. Across the four examined brain regions (i.e., hippocampus, PFC, hypothalamus, and cerebellum), only PSD-95 levels were altered one week post-treatment by the pubertal LPS treatment. Unlike their female counterparts, seven-week-old males showed increased PSD-95 expression in the hippocampus (p < .05). AIF, SOD-1, and NFκB levels in both sexes were unaffected by treatment (all p > .05), which suggests appropriate resolution of NFκB-mediated immune responses to pubertal LPS without stimulating AIF-mediated apoptosis and oxidative stress. We also report a significant male-biased sex difference in PSD-95 levels in the PFC and in cerebellar expression of SOD-1 during puberty (all p < .05). These findings highlight the sex-specific vulnerability of the pubertal hippocampus to systemic LPS and suggest that a pubertal immune challenge may expedite neurodevelopment in the hippocampus in a sex-specific manner.