Lavinia Cantoni

Mitochondrial dysfunction and death in motor neurons exposed to the glutathione-depleting agent ethacrynic acid

This study investigated the mechanisms of toxicity of glutathione (GSH) depletion in one cell type, the motor neuron. Ethacrynic acid (EA) (100 microM) was added to immortalized mouse motor neurons (NSC-34) to deplete both cytosolic and mitochondrial glutathione rapidly. This caused a drop in GSH to 25% of the initial level in 1 h and complete loss in 4 h. This effect was accompanied by enhanced generation of reactive oxygen species (ROS) with a peak after 2 h of exposure, and by signs of mitochondrial dysfunction such as a decrease in 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) (30% less after 4 h). The increase in ROS and the MTT reduction were both EA concentration-dependent. Expression of heme oxygenase-1 (HO-1), a marker of oxidative stress, also increased. The mitochondrial damage was monitored by measuring the mitochondrial membrane potential (MMP) from the uptake of rhodamine 123 into mitochondria. MMP dropped (20%) after only 1 h exposure to EA, and slowly continued to decline until 3 h, with a steep drop at 5 h (50% decrease), i.e. after the complete GSH loss. Quantification of DNA fragmentation by the TUNEL technique showed that the proportion of cells with fragmented nuclei rose from 10% after 5 h EA exposure to about 65% at 18 h. These results indicate that EA-induced GSH depletion rapidly impairs the mitochondrial function of motor neurons, and this precedes cell death. This experimental model of oxidative toxicity could be useful to study mechanisms of diseases like spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS), where motor neurons are the vulnerable population and oxidative stress has a pathogenic role.

Characterization of detergent-insoluble proteins in ALS indicates a causal link between nitrative stress and aggregation in pathogenesis.

Background Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. Methodology/Principal Findings We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. Conclusion/Significance Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.

Cell culture models to investigate the selective vulnerability of motoneuronal mitochondria to familial ALS-linked G93ASOD1

Mitochondrial damage induced by superoxide dismutase (SOD1) mutants has been proposed to have a causative role in the selective degeneration of motoneurons in amyotrophic lateral sclerosis (ALS). In order to investigate the basis of the tissue specificity of mutant SOD1 we compared the effect of the continuous expression of wild‐type or mutant (G93A) human SOD1 on mitochondrial morphology in the NSC‐34 motoneuronal‐like, the N18TG2 neuroblastoma and the non‐neuronal Madin–Darby Canine Kidney (MDCK) cell lines. Morphological alterations of mitochondria were observed in NSC‐34 expressing the G93A mutant (NSC‐G93A) but not the wild‐type SOD1, whereas a ten‐fold greater level of total expression of the mutant had no effect on mitochondria of non‐motoneuronal cell lines. Fragmented network, swelling and cristae remodelling but not vacuolization of mitochondria or other intracellular organelles were observed only in NSC‐G93A cells. The mitochondrial alterations were not explained by a preferential localization of the mutant within NSC‐G93A mitochondria, as a higher amount of the mutant SOD1 was found in mitochondria of MDCK‐G93A cells. Our results suggest that mitochondrial vulnerability of motoneurons to G93ASOD1 is recapitulated in NSC‐34 cells, and that peculiar features in network dynamics may account for the selective alterations of motoneuronal mitochondria.

Low levels of ALS-linked Cu/Zn superoxide dismutase increase the production of reactive oxygen species and cause mitochondrial damage and death in motor neuron-like cells

Mutations of Cu/Zn superoxide dismutase (SOD1) are found in patients with familial amyotrophic lateral sclerosis (FALS). A cellular model of FALS was developed by stably transfecting the motor neuron-like cell line NSC-34 with human wild type (wt) or mutant (G93A) SOD1. Expression levels of G93ASOD1 were close to those seen in the human disease. The presence of G93ASOD1 did not alter cell proliferation but toxicity was evident when the cells were in the growth plateau phase. Flow cytometry analysis indicated that, in this phase, G93ASOD1 significantly lowered viability and that the level of reactive oxygen species was significantly higher in living G93ASOD1 cells compared to wt SOD1 cells. Biparametric analysis of mitochondrial membrane potential and viability of transfected cells highlighted a peculiar population of damaged cells with strong mitochondrial depolarization in the G93ASOD1 cells. Mitochondrial function seemed related to the level of the mutant protein since MTT conversion decreased when expression of G93ASOD1 doubled after treating cells with sodium butyrate. The mutant protein rendered G93ASOD1 cells more sensitive to mitochondrial dysfunction induced by stimuli that alter cellular free radical homeostasis, like serum withdrawal, depletion of glutathione by ethacrynic acid or rotenone-mediated inhibition of complex I of the mitochondrial electron transport chain. In conclusion, even a small amount of mutant SOD1 put motor neurons in a condition of oxidative stress and mitochondrial damage that causes cell vulnerability and death.

Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity

BACKGROUND/AIMSnThe role of oxidative stress in diclofenac hepatotoxicity is still not clear. This study examined whether the drug induced heme oxygenase-1 (HO-1), a stress protein.nnnMETHODSnHO-1 mRNA and HO activity were measured in mouse liver and in rat hepatocytes after treatment with diclofenac parallel to release of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) as a marker of hepatic damage.nnnRESULTSnHO-1 was transcriptionally and dose-dependently induced by diclofenac in mouse liver and rat hepatocytes. HO-1 mRNA, ALT and SDH peaked at the same time. Mechanistic studies revealed that the drug synergized with buthionine sulfoximine (BSO) in lowering hepatic glutathione, increased the formation of reactive oxygen intermediates and activated NF-kappaB and AP-1 in rat hepatocytes. HO-1 induction and hepatic damage were increased by BSO and only HO-1 induction was attenuated by the antioxidant N-acetylcysteine. HO-1 induction was also reduced by the cytochrome P-450 inhibitors ketoconazole and tranylcypromine, concomitantly with a significant decrease in the formation of diclofenac oxidative metabolites, which may give rise to reactive compounds.nnnCONCLUSIONSnAcute treatment with diclofenac induces HO-1 in rodent hepatocytes. Induction is influenced by changes in the cellular redox states and by cytochrome P-450 activity and gives a new insight into the response of the liver to diclofenac.

In vitro inhibitory effect on porphyrinogen carboxylyase of liver extracts from tcdd treated mice

Marked inhibition of porphyrinogen carboxylyase was produced in vitro by cytosol fractions, deproteinized and free of porphyrins, obtained from livers of mice made porphyric by 9 weeks i.p. treatment with 25 micrograms/kg/week of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition was proportional to the amount of the fraction added, was increased by preincubation in the absence of the substrate and, once established, could not be reversed by dialysis. TCDD itself, added to the control enzyme in the incubation mixture, did not affect enzyme activity up to a concentration of 77 nM, which is 10 times higher than the liver TCDD concentration found after in vivo TCDD treatment.

Glutamate and glutathione interplay in a motor neuronal model of amyotrophic lateral sclerosis reveals altered energy metabolism.

Impairment of mitochondrial function might contribute to oxidative stress associated with neurodegeneration in amyotrophic lateral sclerosis (ALS). Glutamate levels in tissues of ALS patients are sometimes altered. In neurons, mitochondrial metabolism of exogenous glutamine is mainly responsible for the net synthesis of glutamate, which is a neurotransmitter, but it is also necessary for the synthesis of glutathione, the main endogenous antioxidant. We investigated glutathione synthesis and glutamine/glutamate metabolism in a motor neuronal model of familial ALS. In standard culture conditions (with glutamine) or restricting glutamine or cystine, the level of glutathione was always lower in the cell line expressing the mutant (G93A) human Cu, Zn superoxide dismutase (G93ASOD1) than in the line expressing wild-type SOD1. With glutamine the difference in glutathione was associated with a lower glutamate and impairment of the glutamine/glutamate metabolism as evidenced by lower glutaminase and cytosolic malate dehydrogenase activity. d-β-hydroxybutyrate, as an alternative to glutamine as energy substrate in addition to glucose, reversed the decreases of cytosolic malate dehydrogenase activity and glutamate and glutathione. However, in the G93ASOD1 cell line, in all culture conditions the expression of pyruvate dehydrogenase kinase l protein, which down-regulates pyruvate dehydrogenase activity, was induced, together with an increase in lactate release in the medium. These findings suggest that the glutathione decrease associated with mutant SOD1 expression is due to mitochondrial dysfunction caused by the reduction of the flow of glucose-derived pyruvate through the TCA cycle; it implies altered glutamate metabolism and depends on the different mitochondrial energy substrates.

Neurodegeneration induced by complex I inhibition in a cellular model of familial amyotrophic lateral sclerosis

G93A Cu/Zn superoxide dismutase (SOD1), a human mutant SOD1 associated with familial amyotrophic lateral sclerosis, increased the toxicity of the mitochondrial toxin rotenone in the NSC-34 motoneuronal cell line. G93ASOD1 cells died more than untransfected and wild-type SOD1 cells after 6 and 24h exposure to 12.5 microM rotenone. Biparametric flow cytometry showed that rotenone induced rapid hyperpolarization of mitochondrial membrane potential (deltapsi(m)) in all the cell lines, followed by depolarization, and then by cell death. However, G93ASOD1 mitochondria were significantly more likely to shift from a hyperpolarized to a depolarized condition, and within the still viable cell population there was a higher proportion with depolarized mitochondria, a condition that can be envisaged as a commitment to cell death. ATP, which is needed to prevent loss of deltapsi(m), decreased more rapidly and to a greater extent in rotenone-treated G93ASOD1 cells than in the untransfected and wtSOD1cells. In all the cell lines, 1h after rotenone exposure, mitochondrial hyperpolarization was accompanied by the formation of a comparable amount of reactive oxygen species. However, G93ASOD1 cells reached the highest reactive oxygen species level since their basal level was higher than in untransfected and wild-type SOD1 cells. Our findings indicate that the mutant protein G93ASOD1 enhances the vulnerability of motor neurons to rotenone by mechanism(s) involving oxidative stress and perturbed mitochondrial homeostasis. This suggests that motor neurons from individuals carrying the mutant G93ASOD1 are at greater risk of death after inhibition of the electron transport chain.

Intercalation with DNA is a prerequisite for daunomycin, adriamycin and its congeners in inhibiting DNAase I.

DNAase I from bovine pancreas is inhibited by Daunomycin, Adriamycin, Adriamycin-14-acetate and Adriamycin-14-octanoate, whereas it is not inhibited by N-trifluoroacetyladriamycin-14-valerate or N-trifluoroacetyl-adriamycin. The present study suggests that these inhibitors act not directly on the enzyme, but on DNA, forming stable complexes and thus interfering with enzyme activity. The correlation between DNA binding and enzyme inhibition is demonstrated by the fact that the compounds forming complexes with DNA inhibit DNAase I activity, whereas those which do not form complexes with DNA cause no inhibition.

Effects of Phlebotomy on Urinary Porphyrin Pattern and Liver Histology in Patients with Porphyria Cutanea Tarda

Urinary porphyrin profiles and liver histology have been investigated in a group of adult alcoholics with porphyria cutanea tarda (PCT) before and after one year phlebotomy. Both parameters were evaluated during the same period in a group of patients who did not undergo specific therapy for PCT. All patients were advised to abstain from alcohol. At the end of the one year observation period there was a significant fall of urinary total porphyrins and in the uro/coproporphyrin ratio in treated patients compared to basal values whereas no changes were found in controls. Liver biopsy findings revealed a significant reduction of hepatic fatty degeneration and siderosis with no changes in inflammatory infiltrates and fibrosis in treated patients, so the progression of liver disease was similar to controls. These results show that clinical and biochemical remission of PCT can occur independently of the evolution of the concomitant liver disease.