Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy

Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43) is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation) induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.     

Cocoa extract protects against early alcohol-induced liver injury in the rat

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.     

Progranulin protects against cerebral ischemia-reperfusion (I/R) injury by inhibiting necroptosis and oxidative stress

Ischemic stroke is a leading cause of mortality and disability worldwide. Nevertheless, its molecular mechanisms have not yet been adequately illustrated. Progranulin (PGRN) is a secreted glycoprotein with pleiotropic functions. In the present study, we found that PGRN expression was markedly reduced in mice after stroke onset through middle cerebral artery occlusion (MCAO). We also showed that necroptosis was a mechanism underlying cerebral I/R injury. Importantly, PGRN knockdown in vivo significantly promoted the infarction volume and neurological deficits scores in mice after MCAO surgery. Necroptosis induced by MCAO was further accelerated by PGRN knockdown, as evidenced by the promoted expression of phosphorylated receptor-interacting protein (RIP) 1 kinase (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), which was accompanied with increased expression of cleaved Caspase-8 and Caspase-3. However, PGRN over-expression was neuroprotective. Additionally, PGRN-regulated ischemic stroke was related to ROS accumulation that MCAO-mice with PGRN knockdown exhibited severe oxidative stress, as proved by the aggravated malondialdehyde (MDA) and lipid peroxidation (LPO) contents, and the decreased superoxide dismutase (SOD) activity. However, PGRN over-expression in mice with cerebral ischemia showed anti-oxidative effects. Finally, PGRN was found to attenuate oxidative damage partly via its regulatory effects on necroptosis. Therefore, promoting PGRN expression could reduced cerebral I/R-induced brain injury by suppressing neroptosis and associated reactive oxygen species (ROS) production. These data elucidated that PGRN might provide an effective therapeutic treatment for ischemic stroke.     

Inosine protects against the development of diabetes in multiple-low-dose streptozotocin and nonobese diabetic mouse models of type 1 diabetes

Inosine, a naturally occurring purine, was long considered to be an inactive metabolite of adenosine. However, recently inosine has been shown to be an immunomodulator and anti-inflammatory agent. The aim of this study was to determine whether inosine influences anti-inflammatory effects and affects the development of type 1 diabetes in murine models. Type 1 diabetes was induced either chemically by streptozotocin or genetically using the nonobese diabetic mouse (NOD) model. Mice were treated with inosine (100 or 200 mg kg(-1)d(-1)d) and diabetes incidence was monitored. The effect of inosine on pancreas immune cell infiltration, oxidative stress, and cytokine profile also was determined. For the transplantation model islets were placed under the renal capsule of NOD mice and inosine (200 mg kg(-1)d d(-1)d) treatment started the day of islet transplantation. Graft rejection was diagnosed by return of hyperglycemia accompanied by glucosuria and ketonuria. Inosine reduced the incidence of diabetes in both streptozotocin-induced diabetes and spontaneous diabetes in NOD mice. Inosine decreased pancreatic leukocyte infiltration and oxidative stress in addition to switching the cytokine profile from a Th1 to a Th2 profile. Inosine prolonged pancreatic islet graft survival, increased the number of surviving beta cells, and reduced the number of infiltrating leukocytes. Inosine protects against both the development of diabetes and against the rejection of transplanted islets. The purine exerts anti-inflammatory effects in the pancreas, which is its likely mode of action. The use of inosine should be considered as a potential preventative therapy in humans susceptible to developing Type 1 diabetes and as a possible antirejection therapy for islet transplant recipients.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Combining ursodeoxycholic acid or its NO-releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.     

Caffeine protects against MPTP-induced blood-brain barrier dysfunction in mouse striatum

The blood-brain barrier (BBB) is important physiologically. Pathologically, BBB disruption has been implicated in a wide spectrum of neurological disorders including Parkinson's disease (PD). Recent studies indicate that caffeine is protective against PD, but by poorly understood mechanisms. Using a MPTP neurotoxin model of PD we tested the hypothesis that the protective actions of caffeine were because of, at least in part, preventing MPTP-induced BBB dysfunction. FVB mice were pre-treated with caffeine (10 mg/kg, i.p.) or saline for 7 days prior to initiation of neurotoxin treatments; during the 7 days of neurotoxin treatment, caffeine or saline continued to be administered 10 min before each dose of MPTP (20 mg/kg, i.p.). Striatum (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, tight junction protein expression levels, integrity of dopaminergic neurons, and activation of astrocytes and microglia using immunostaining, immunoblotting and real-time PCR techniques. We found that caffeine blocked MPTP-induced decreases in numbers of tyrosine hydroxylase-positive dopaminergic neurons, increases in leakage of Evan's blue dye and FITC-albumin in striatum but not in cerebral cortex or hippocampus, decreases in levels of the tight junction proteins occludin and ZO-1, and increases in reactive gliosis. Our results suggest that caffeine might protect against PD and PD-like features in animal models, in part, by stabilizing the BBB.     

Quercetin protects mouse oocytes against chromium-induced damage in vitro and in vivo

BackgroundChromium (Cr) is a naturally-occurring element that is used in various fields of industry. Humans may be exposed to hexavalent chromium [Cr(VI)], which is one of the stable valence states of the chromium through contaminated soil, air, and water. Exposure to Cr(VI) through contaminated drinking water, soil and air causes various cancers and also fertility problems in animals and humans. Quercetin (QCT), a common flavonoid compound, has numerous biological effects as an antioxidant and free radical scavenger, but its function and mechanisms in reproductive processes in various species remain unclear. This study aims to determine the chromium effects on mice oocyte quality and the ameliorative effect of QCT in both in vitro and in vivo experimental models.MethodsFor the in vitro experiment, oocytes were collected and divided into the control, sham, QCT-treated, Cr(VI) (potassium dichromate), and treatment [Cr(VI)+QCT] groups. Collected oocytes were cultured in maturation medium with or without 10 µM quercetin and 10 µM Cr(VI) for 14 h based on the defined experimental design. For the in vivo experiment, the mice were randomly divided into the control, sham, QCT-treated, Cr(VI), and Cr(VI) + QCT groups. Control and sham mice received regular drinking water and diet. Cr(VI) group received Cr(VI) (50 ppm in drinking water) and Cr(VI) + QCT group received 50 ppm Cr(VI) with QCT (20 mg/kg body wt, through i.p) for a period of 21 days and then oocytes were collected and cultured for 14 h for in vitro maturation. For both experiments, at the end of the culture period, we examined the ameliorative effect of QCT on oocyte maturation, spindle formation, ROS production, mitochondrial function, and apoptosis.ResultsOur in vitro and in vivo results showed that Cr(VI) disrupt the oocyte maturation and spindle formation (P < 0.001). Furthermore, we found that exposure to Cr(VI) significantly increased ROS levels and decreased mitochondrial membrane potential (P < 0.001). In addition, exposure to Cr(VI) induced early apoptosis and downregulated the Bcl-2 mRNA expression and upregulated the Caspase-3 and Bax mRNAs expression (P < 0.01). Finally, quercetin significantly restored the detrimental effects of Cr(VI).ConclusionThe results indicated that quercetin protects the oocytes against Cr(VI) toxicity through the suppression of oxidative stress and apoptosis. The conclusions drawn from our study's findings suggest that quercetin might be useful agent for oocyte maturation in case of possible exposure to toxic substances such as chromium.     

Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity

Background

Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8 ^−/−), in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system.

Methodology/Principal Findings

Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml) or TRAIL (250 ng/mL) plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI) model of traumatic brain injury (TBI) and seizure-induced brain injury caused by kainic acid (KA). Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8 ^−/− mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.

Conclusions

Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma.     

A lipophilic heparin derivative protects elastin against degradation by leucocyte elastase.

An oleolylated derivative (I) of partially N-desulphated heparin was prepared containing an average number of three oleoyl residues for one molecule of heparin. The inhibitory capacity of I (IC50 = 0.55 microM) for leucocyte elastase resembles that of heparin (IC50 = 0.2 microM). In contrast to heparin, I is also an inhibitor of porcine pancreatic elastase (IC50 = 0.68 microM) and it also has the capacity to protect elastin fibres against the degradation by leucocyte elastase. When insoluble elastin is pretreated with I its degradation by leucocyte elastase is inhibited by almost 90% while pretreatment of elastin with heparin exhibited only a moderate effect on elastolysis (10% inhibition).     

High-selenium lentil diet protects against arsenic-induced atherosclerosis in a mouse model

Background: Cardiovascular disease (CVD) is a major cause of death worldwide, and arsenic (As) intake, mainly through drinking water, is a well-known risk factor for CVD as well as other health problems. Selenium (Se) is a known antagonist to As toxicity. Objective: We tested the potential of high-Se lentils from the Canadian prairies as a therapeutic food to alter the outcome of As-enhanced atherosclerosis. Materials and Methods: Male ApoE^−/− mice exposed to a moderate level of As (200 ppb) in their drinking water, and control mice on tap water received one of three lentil diets: Se-deficient (0.009 mg/kg), Se-adequate (0.16 mg/kg) or Se-high (0.3 mg/kg). After 13 weeks, lesion formation in the aortic arch and sinus were assessed. Intralesional cellular composition, serum lipid levels and hepatic oxidative stress were assessed as well. Results: Arsenic-exacerbated plaque formation was reduced in the sinus and completely abolished in the aortic arch of mice on the Se-fortified lentil diet, whereas lesions were increased in As-exposed mice on both the Se-deficient and Se-adequate diets. Notably, Se deficiency contributed to proatherogenic composition of serum lipids in As-exposed mice as indicated by high-density lipoprotein:low-density lipoprotein. At least adequate Se status was crucial for counteracting As-induced oxidative stress. Conclusion: This study is the first to show the potential of high-Se lentils to protect against As-triggered atherosclerosis, and this invites further investigations in human populations at risk from As contamination of their drinking water.     

Manganese superoxide dismutase protects against 6-hydroxydopamine injury in mouse brains

Dopaminergic neurons of the substantia nigra are susceptible to toxin-based insults. Intrastriatal injection of 6-hydroxydopamine results in selective toxicity to these neurons. A mechanistic role for reactive oxygen species is supported by observations that antioxidants confer protection from 6-hydroxydopamine. Although cell culture studies have suggested extracellular or nonmitochondrial mechanisms in 6-hydroxydopamine toxicity, the compartmentalization of oxidative injury mechanisms is incompletely defined in vivo. Transgenic mice overexpressing mitochondrial manganese superoxide dismutase or extracellular superoxide dismutase received unilateral intrastriatal injections of 6-hydroxydopamine. Mice that overexpress manganese superoxide dismutase showed significantly smaller striatal lesions than littermate controls. There were no differences in nonspecific striatal injury associated with contralateral vehicle injection. Manganese superoxide dismutase overexpression also protected against loss of neuronal cell bodies in the substantia nigra. In contrast, mice overexpressing extracellular superoxide dismutase showed no protection from 6-hydroxydopamine toxicity in either brain region. Protection of the nigrostriatal system by overexpression of manganese superoxide dismutase supports a role for mitochondrially derived superoxide in 6-hydroxydopamine toxicity. Mitochondrial oxidative stress appears to be a common mechanism among diverse models of Parkinson disease, whether involving toxins, mutated genes, or cybrid cells containing patient mitochondria. Antioxidant therapies that target this subcellular compartment may prove promising.     

A dibenzoylmethane derivative protects dopaminergic neurons against both oxidative stress and endoplasmic reticulum stress

The enhancement of intracellular stresses such as oxidative stress and endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative disorders including Parkinson's disease (PD). During a search for compounds that regulate ER stress, a dibenzoylmethane (DBM) derivative 14-26 (2,2'-dimethoxydibenzoylmethane) was identified as a novel neuroprotective agent. Analysis in SH-SY5Y cells and in PC12 cells revealed that the regulation of ER stress by 14-26 was associated with its anti-oxidative property. 14-26 prevented the production of reactive oxygen species (ROS) when the cells were exposed to oxidants such as hydrogen peroxide and 6-hydroxydopamine (6-OHDA) or an ER stressor brefeldin A (BFA). 14-26 also prevented ROS-induced damage in both the ER and the mitochondria, including the protein carbonylation in the microsome and the reduction of the mitochondrial membrane potential. Further examination disclosed the presence of the iron-chelating activity in 14-26. In vivo, 14-26 suppressed both oxidative stress and ER stress and prevented neuronal death in the substantia nigra pars compacta (SNpc) after injection of 6-OHDA in mice. These results suggest that 14-26 is an antioxidant that protects dopaminergic neurons against both oxidative stress and ER stress and could be a therapeutic candidate for the treatment of PD.     

Protective effect of isoflavone derivative against photocarcinogenesis in a mouse model

Abstract

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic and antioxidant activities. In this study, we describe novel photoprotective effects of one isoflavone derivative from red clover (NV07), following its application topically in Skh:HR-1 hairless mice. We found that in mice irradiated in the short-term (3 days) with minimally erythemal solar simulated UV radiation, topical lotions containing NV07 dose-responsively reduced the erythema-associated oedema, the induction of ornithine decarboxylase, and the suppression of contact hypersensitivity. In mice irradiated chronically (50 days), daily application of topical NV07-lotion reduced photocarcinogenesis significantly, and appeared to be actively protective during both the initiation phase and the later promotion phase of tumour induction.     

Adenosine protects against angiotensin II-induced apoptosis in rat cardiocyte cultures

Adenosine has been found to be cardioprotective during episodes of cardiac ischemia/reperfusion through activation of the A₁ and possibly A₃ receptors. Therefore, we have investigated whether activation of these receptors can protect also against apoptotic death induced by angiotensin II (Ang II) in neonatal rat cardiomyocyte cultures. Exposure to Ang II (10 nM) resulted in a 3-fold increase in programmed cell death (p < 0.05). Pretreatment with the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA, 1 M), abolished the effects of Ang II on programmed cardiomyocyte death. Moreover, exposure of cells to the A₁ adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) before pretreatment with CCPA, prevented the protective effect of the latter. Pretreatment with the A₃ adenosine receptor agonist N⁶-(3-iodobenzyl) adenosine-5-N-methyluronamide (IB-MECA, 0.1 M), led to a partial decrease in apoptotic rate induced by Ang II. Exposure of myocytes to Ang II caused an immediate increase in the concentration of intracellular free Ca²⁺ that lasted 40–60 sec. Pre-treatment of cells with CCPA or IB-MECA did not block Ang II-induced Ca²⁺ elevation. In conclusion, activation of adenosine A₁ receptors can protect the cardiac cells from apoptosis induced by Ang II, while activation of the adenosine A₃ receptors confers partial cardioprotection.     

Green tea extract protects against early alcohol-induced liver injury in rats

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g kg(-1) day(-1)) and green tea (300 mg kg(-1) day(-1)) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (approximately 4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0-550 mg dl(-1) day(-1)). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35+/-3 IU/l) by enteral ethanol (114+/-18); inclusion of green tea extract in the diet significantly blunted this increase (65+/-10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFalpha protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcohol-induced liver injury, most likely by preventing oxidative stress.