[Show abstract][Hide abstract] ABSTRACT: Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and OPEN ACCESS Biomolecules 2015, 5 680 the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.
[Show abstract][Hide abstract] ABSTRACT: Macroautophagy is a highly regulated intracellular process that, under certain circumstances, delivers cytoplasmic components to the lysosomes for degradation. It consists of several sequential steps including initiation and nucleation, double membrane formation and elongation, formation and maturation of autophagosomes and finally autophagosomes/lysosomes fusion and degradation of intra-autophagosomal contents by lysosomal enzymes. After decades of considering autophagy as a cell death pathway, it has recently been shown to have a survival function through clearing of protein aggregates and damaged cytoplasmic organelles in response to a variety of stress conditions. Most recently, there is increasing evidence from literature revealing that autophagy induction may combat neurodegeneration. In the light of this, our current review tried to address the recent advances in the role of induced autophagy in neuroprotection with a particular focus on its contribution in the most common neurodegenerative disorders like Alzheimer's disease, Parkinson's disease and Huntington's disease.
[Show abstract][Hide abstract] ABSTRACT: Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regula-tion of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical re-construction microscopy) to visualize several mitochondrial pro-teins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various ex-pression levels in different mitochondria, supporting the hypoth-esis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from act-ing as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production.
Proceedings of the National Academy of Sciences 12/2014; 112(1). DOI:10.1073/pnas.1415261112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phytocannabinoids are potentially candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [3H]CP-55,940 and the phytocannabinoids. Resulting Ki values to CB1 range from 23.5 μM (THCA) to 14711 μM (CBDV), whereas Ki values to CB2 range from 8.5 μM (THC) to 574.2 μM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas also CBC and THCA displayed slightly positive activities. These findings are neither linked to structural characteristics nor to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one.
Neurotoxicology and Teratology 10/2014; 46. DOI:10.1016/j.ntt.2014.09.003 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apart from the first family member, uncoupling protein 1 (UCP1), the functions of other UCPs (UCP2-UCP5) are still unknown. In analyzing our own results and those previously published by others, we have assumed that UCP's cellular expression pattern coincides with a specific cell metabolism and changes if the latter is altered. To verify this hypothesis, we analyzed the expression of UCP1-5 in mouse embryonic stem cells before and after their differentiation to neurons. We have shown that only UCP2 is present in undifferentiated stem cells and it disappears simultaneously with the initiation of neuronal differentiation. In contrast, UCP4 is simultaneously up-regulated together with typical neuronal marker proteins TUJ-1 and NeuN during mESC differentiation in vitro as well as during murine brain development in vivo. Notably, several tested cell lines express UCP2, but not UCP4. In line with this finding, neuroblastoma cells that display metabolic features of tumor cells express UCP2, but not UCP4. UCP2's occurrence in cancer, immunological and stem cells indicates that UCP2 is present in cells with highly proliferative potential, which have a glycolytic type of metabolism as a common feature, whereas UCP4 is strongly associated with non-proliferative highly differentiated neuronal cells.
PLoS ONE 02/2014; 9(2):e88474. DOI:10.1371/journal.pone.0088474 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tocopherols (TOH) are lipophilic antioxidants which require the phenolic OH group for their redox activity. In contrast, non-redox active esters of α-TOH with succinate (α-TOS) were shown to possess proapoptotic activity in cancer cells. It was suggested that this activity is mediated via mitochondrial inhibition with subsequent O2(-) production triggering apoptosis and that the modification of the linker between the succinate and the lipophilic chroman may modulate this activity. However, the specific mechanism and the influence of the linker are not clear yet on the level of the mitochondrial respiratory chain. Therefore, this study systematically compared the effects of α-TOH acetate (α-TOA), α-TOS and α-tocopheramine succinate (α-TNS) in cells and submitochondrial particles (SMP). The results showed that not all cancer cell lines are highly sensitive to α-TOS and α-TNS. In HeLa cells α-TNS did more effectively reduce cell viability than α-TOS. The complex I activity of SMP was little affected by α-TNS and α-TOS while the complex II activity was much more inhibited (IC50=42±8μM α-TOS, 106±8μM α-TNS, respectively) than by α-TOA (IC50 >1000μM). Also the complex III activity was inhibited by α-TNS (IC50=137±6μM) and α-TOS (IC50=315±23μM). Oxygen consumption of NADH- or succinate-respiring SMP, involving the whole electron transfer machinery, was dose-dependently decreased by α-TOS and α-TNS, but only marginal effects were observed in the presence of α-TOA. In contrast to the similar inhibition pattern of α-TOS and α-TNS, only α-TOS triggered O2(-) formation in succinate- and NADH-respiring SMP. Inhibitor studies excluded complex I as O2(-) source and suggested an involvement of complex III in O2(-) production. In cancer cells only α-TOS was reproducibly able to increase O2(-) levels above the background level but neither α-TNS nor α-TOA. Furthermore, the stability of α-TNS in liver homogenates was significantly lower than that of α-TOS. In conclusion, this suggests that α-TNS although it has a structure similar to α-TOS is not acting via the same mechanism and that for α-TOS not only complex II but also complex III interactions are involved.
[Show abstract][Hide abstract] ABSTRACT: The present study aims to investigate the protective effects of thymoquinone, the major active ingredient of Nigella sativa seeds, against lead-induced brain damage in Sprague-Dawley rats. In which, 40 rats were divided into four groups (10 rats each). The first group served as control. The second, third and fourth groups received lead acetate, lead acetate and thymoquinone, and thymoquinone only, respectively, for one month. Lead acetate was given in drinking water at a concentration of 0.5g/l (500ppm). Thymoquinone was given daily at a dose of 20mg/kg b.w. in corn oil by gastric tube. Control and thymoquinone-treated rats showed normal brain histology. Treatment of rats with lead acetate was shown to produce degeneration of endothelial lining of brain blood vessels with peri-vascular cuffing of mononuclear cells consistent to lymphocytes, congestion of choroid plexus blood vessels, ischemic brain infarction, chromatolysis and neuronal degeneration, microglial reaction and neuronophagia, degeneration of hippocampal and cerebellar neurons, and axonal demyelination. On the other hand, co-administration of thymoquinone with lead acetate markedly decreased the incidence of lead acetate-induced pathological lesions. Thus the current study shed some light on the beneficial effects of thymoquinone against neurotoxic effects of lead in rats.
Experimental and toxicologic pathology: official journal of the Gesellschaft fur Toxikologische Pathologie 07/2013; 66(1). DOI:10.1016/j.etp.2013.07.002 · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Resveratrol interacts with the complex III of the respiratory chain, is a radical scavenger and also suppressor of radical formation in the mitochondria. It reduces the intracellular calcium levels in pre- and postsynaptic neurons and also may inhibit the pro-apoptotic factors in glutamate overflow that occurs, e.g. in excitotoxicity. In cell cultures, glutamate overflow leads to formation of free radicals and results in apoptosis. This increase of radical concentration is enhanced by influx of cations like iron or copper ions into the cell. In present study, the beneficial action of resveratrol was investigated in glutamate-affected dissociated cultures of mice mesencephalic primary cultures. On the 10th day in vitro, 5 mM of glutamate was administered for 15 min and the cultures were further maintained in medium containing 0, 0.01, 0.1 or 1 μM of resveratrol. Resveratrol reduced glutamate-induced damages. The number of dopaminergic neurons was increased and their morphology ameliorated when resveratrol followed glutamate treatment. A significant reduction of glutamate-induced radical formation in cultures treated with resveratrol corresponded with a considerable high antioxidative potential of this stilbene determined using the DPPH assay. In addition, ICP-OES was set up to measure the tissues' copper and iron contents in organotypic cortical cultures of glutamate treated (0 or 30 μM) slices and those in which resveratrol (0, 0.01, 0.1 or 1 μM) was co-administered. Levels of copper were dose-dependently increased, and also the concentration of iron was higher in resveratrol-treated organotypic cultures. The hypothesis that resveratrol has beneficial actions against glutamate damages was verified.
[Show abstract][Hide abstract] ABSTRACT: Over recent decades, engineered nanoparticles are increasingly produced as the result of the rapid development in nanotechnology. They are currently used in a wide range of industrial and public sectors including healthcare, agriculture, transport, energy, materials, and information and communication technologies. As the result, an increasing concern has been raised over the potential impacts of engineered nanoparticles to human health. In the light of this, it is the purpose of the present review to discuss: (1) novel properties of engineered nanoparticles particularly in biomedical sciences, (2) most recently reported adverse effects of manufactured nanoparticles on human health and (3) different aspects of toxicological risk assessment of these nanoparticles.
[Show abstract][Hide abstract] ABSTRACT: Swainsonine (SW) is an indolizidine triol plant alkaloid isolated from the species Astragalus, colloquially termed locoweed. When chronically ingested by livestock and wildlife, symptoms include severe neuronal disturbance. Toxicity to the central and peripheral nervous system is caused by inhibition of lysosomal α-mannosidase (AMA) and accumulation of intracellular oligosaccharide. Consequently, SW has been used as a model substance in investigations of lysosomal storage diseases. Involvement of the basal ganglia has been postulated due to the neuronal symptoms of affected animals. Therefore, primary midbrain cultures from embryonic mice containing dopaminergic neurons were utilized in this study. Neural cells were exposed to SW (0.01-100 μM) for 72 h. AMA activity was 50 % inhibited at 1 μM SW. Cytotoxic changes in cultures were observed above 25 μM SW by increases in lactate dehydrogenase activity and nitric oxide content. Neurotoxicity to dopaminergic cells was visualized by tyrosine hydroxylase immunohistochemistry. Structural degeneration scored as dendritic shortening and shrinkage of cell bodies was dose-dependent and resulted in nerve loss above 25 μM. SW exposure caused progression from reversible to irreversible cytotoxicity. Partial regeneration of AMA-activity in culture was observed on removal of SW. The antioxidative vitamins ascorbic acid and tocopherol (both 100 μM) partially reversed the toxic effect on dopaminergic cells and ascorbic acid decreased AMA inhibition. Thus, neuronal midbrain cell cultures can demonstrate the neurotoxic action of SW and cytoprotective strategies may be tested at a single nerve cell level.
[Show abstract][Hide abstract] ABSTRACT: Cannabinoids derived from Cannabis sativa demonstrate neuroprotective properties in various cellular and animal models. Mitochondrial impairment and consecutive oxidative stress appear to be major molecular mechanisms of neurodegeneration. Therefore we studied some major cannabinoids, i.e. delta-9-tetrahydrocannabinolic acid (THCA), delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in mice mesencephalic cultures for their protective capacities against 1-methyl-4-phenyl pyridinium (MPP(+)) toxicity. MPP(+) is an established model compound in the research of parkinsonism that acts as a complex I inhibitor of the mitochondrial respiratory chain, resulting in excessive radical formation and cell degeneration. MPP(+) (10 μM) was administered for 48 h at the 9th DIV with or without concomitant cannabinoid treatment at concentrations ranging from 0.01 to 10 μM. All cannabinoids exhibited in vitro antioxidative action ranging from 669 ± 11.1 (THC), 16 ± 3.2 (THCA) to 356 ± 29.5 (CBD) μg Trolox (a vitamin E derivative)/mg substance in the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Cannabinoids were without effect on the morphology of dopaminergic cells stained by tyrosine hydroxylase (TH) immunoreaction. THC caused a dose-dependent increase of cell count up to 17.3% at 10 μM, whereas CBD only had an effect at highest concentrations (decrease of cell count by 10.1-20% at concentrations of 0.01-10 μM). It influenced the viability of the TH immunoreactive neurons significantly, whereas THCA exerts no influence on dopaminergic cell count. Exposure of cultures to 10 μM of MPP(+) for 48 h significantly decreased the number of TH immunoreactive neurons by 44.7%, and shrunken cell bodies and reduced neurite lengths could be observed. Concomitant treatment of cultures with cannabinoids rescued dopaminergic cells. Compared to MPP(+) treated cultures, THC counteracted toxic effects in a dose-dependent manner. THCA and CBD treatment at a concentration of 10 μM lead to significantly increased cell counts to 123% and 117%, respectively. Even though no significant preservation or recovery of neurite outgrowth to control values could be observed, our data show that cannabinoids THC and THCA protect dopaminergic neurons against MPP(+) induced cell death.
Phytomedicine: international journal of phytotherapy and phytopharmacology 05/2012; 19(8-9):819-24. DOI:10.1016/j.phymed.2012.04.002 · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemorrhagic-traumatic shock (HTS) followed by reperfusion induces heme oxygenase (HO) 1. Free iron (Fe2+) may cause oxidative stress, if not adequately sequestered. We aimed to characterize HO-1-mediated effects on Fe2+ levels in liver and transferrin-bound iron (TFBI) in plasma following HTS, including laparotomy, bleeding, and inadequate and adequate reperfusion. Anesthetized rats showed upregulated HO-1 mRNA at 40 min after HTS, which was followed by increased HO activity at 3 h after shock. Fe2+ levels were transiently increased at 40 min after shock, a time point when HO activity was not affected yet. Levels of plasma TFBI were higher in HTS animals, showing the highest levels at 40 min after shock, and decreased thereafter. In addition, we modulated HO activity 6 h before HTS by administering an inhibitor (zinc-protoporphyrin IX) or an activator (hemin) of HO. At 18 h after HTS in all shock groups, HO activity was increased, the highest being in the hemin-pretreated group. The zinc-protoporphyrin IX-treated HTS animals showed increased HO-1 mRNA and Fe2+ levels in the liver compared with the untreated HTS animals. Transferrin-bound iron levels were affected by pharmacological modulation before shock. All animals undergoing HTS displayed increased TFBI levels after reperfusion; however, in animals pretreated with hemin, TFBI levels increased less. Our data indicate that increase in Fe2+ levels in liver and plasma early after HTS is not mediated by HO-1 upregulation, but possibly reflects an increased mobilization from internal iron stores or increased cell damage. Thus, upregulation of HO activity by hemin does not increase Fe2+ levels following HTS and reperfusion.
[Show abstract][Hide abstract] ABSTRACT: Even though rotenone has been used extensively in recent years to produce a model of Parkinson disease in rats, its systemic effects either on neurons apart from dopaminergic structures or non-neuronal tissues have not been elucidated well. In our present study, 30 adult Sprague-Dawley rats were divided into three even groups. A short-term rotenone-treated group received 10mg/kg b.w. rotenone daily for 7 days. The long-term rotenone-treated group received 3mg/kg b.w. rotenone daily for 30 days. The control group received vehicle only and were kept 5 rats each in parallel to both short- and long-term rotenone treated groups. It was found that short-term rotenone treatment produced marked vascular damages associated with ischemic neuronal degeneration particularly in the thalamus, cerebellum and nucleus dentatus. In long-term rotenone-treated group, vascular changes were less severe and neuronal degeneration was associated with mild microglial proliferation and astrocytosis. Non-neuronal pathology as the result of short-term rotenone exposure consisted of degeneration and necrosis of seminiferous tubular epithelia with formation of spermatide multinucleate giant cells. On the other hand, long-term rotenone treatment did not affect testicles and only caused sinusoidal dilatation in the liver, myocardial degeneration in the heart and interstitial hemorrhages in the kidneys and lungs. In conclusions, damage to blood vasculature by rotenone appeared mediating neuronal and non-neuronal pathology in Sprague-Dawley rats. This effect might provide new insights for ethiopathogenesis of neurodegenerative diseases and contributes to the understanding of hemorrhagic stroke.
Experimental and toxicologic pathology: official journal of the Gesellschaft fur Toxikologische Pathologie 06/2011; 65(1-2). DOI:10.1016/j.etp.2011.05.008 · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ginsenosides are a special group of triterpenoid saponins attributed to medical effects of ginseng. Therefore, they have been research targets over the last three decades to explain ginseng actions and a wealth of literature has been presented reporting on ginsenosides' effects on the human body. Recently, there is increasing evidence on beneficial effects of ginsenosides to the central nervous system (CNS). Using a wide range of in vitro and in vivo models, researchers have attributed these effects to specific pharmacological actions of ginsenosides on cerebral metabolism, oxidative stress and radical formation, neurotransmitter imbalance and membrane stabilizing effects, and even antiapoptotic effects. Modulating these particular mechanisms by ginsenosides has thus been reported to exert either general stimulatory effects on the brain functions or protecting the CNS against various disease conditions. In this review, we try to address the recently reported ginsenosides' actions on different CNS targets particularly those supporting possible therapeutic efficacies in CNS disorders and neurodegenerative diseases.