Alessandra Mangolini

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.

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/AIMS The 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. METHODS HO-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. RESULTS HO-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. CONCLUSIONS Acute 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.

Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR

BackgroundBreast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15–3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique.ResultsWe investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis.ConclusionsThe study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs.

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.

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

Abstract.  Objectives: Polycystin‐1 (PC1), a signalling receptor regulating Ca2+‐permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca2+ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non‐capacitative Ca2+ entry (NCCE) and Ca2+ oscillations, with downstream effects on cell proliferation. Results and discussion: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum‐induced Ca2+ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca2+ oscillations and cell proliferation are increased in PC1‐mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca2+ oscillations and a molecular mechanism to explain the association between abnormal Ca2+ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.

Polycystin-1 regulates amphiregulin expression through CREB and AP1 signalling: implications in ADPKD cell proliferation.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst development and enlargement, as well as cell growth, are associated with alterations in several pathways, including cAMP and activator protein 1 (AP1) signalling. However, the precise mechanism by which these molecules stimulate cell proliferation is not yet fully understood. We now show by microarray analysis, luciferase assay, mutagenesis, and chromatin immunoprecipitation that CREB and AP1 contribute to increased expression of the amphiregulin gene, which codifies for an epidermal growth factor-like peptide, in ADPKD cystic cells, thereby promoting their cell growth. Increased amphiregulin (AR) expression was associated with abnormal cell proliferation in both PKD1-depleted and -mutated epithelial cells, as well as primary cystic cell lines isolated from ADPKD kidney tissues. Consistently, normal AR expression and proliferation were re-established in cystic cells by the expression of a mouse full-length PC1. Finally, we show that anti-AR antibodies and inhibitors of AP1 are able to reduce cell proliferation in cystic cells by reducing AR expression and EGFR activity. AR can therefore be considered as one of the key activators of the growth of human ADPKD cystic cells and thus a new potential therapeutic target.

Deficiency of polycystic kidney disease-1 gene (PKD1) expression increases A3 adenosine receptors in human renal cells: Implications for cAMP-dependent signalling and proliferation of PKD1-mutated cystic cells

Cyst growth and expansion in autosomal dominant polycystic kidney disease (ADPKD) has been attributed to numerous factors, including ATP, cAMP and adenosine signalling. Although the role of ATP and cAMP has been widely investigated in PKD1-deficient cells, no information is currently available on adenosine-mediated signalling. Here we investigate for the first time the impact of abnormalities of polycystin-1 (PC1) on the expression and functional activity of adenosine receptors, members of the G-protein-coupled receptor superfamily. Pharmacological, molecular and biochemical findings show that a siRNA-dependent PC1-depletion in HEK293 cells and a PKD1-nonsense mutation in cyst-derived cell lines result in increased expression of the A(3) adenosine receptor via an NFkB-dependent mechanism. Interestingly, A(3) adenosine receptor levels result higher in ADPKD than in normal renal tissues. Furthermore, the stimulation of this receptor subtype with the selective agonist Cl-IB-MECA causes a reduction in both cytosolic cAMP and cell proliferation in both PC1-deficient HEK293 cells and cystic cells. This reduction is associated with increased expression of p21(waf) and reduced activation not only of ERK1/2, but also of S6 kinase, the main target of mTOR signalling. In the light of these findings, the ability of Cl-IB-MECA to reduce disease progression in ADPKD should be further investigated. Moreover, our results suggest that NFkB, which is markedly activated in PC1-deficient and cystic cells, plays an important role in modulating A(3)AR expression in cystic cells.

NF-κB activation is required for apoptosis in fibrocystin/polyductin-depleted kidney epithelial cells

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in PKHD1, a gene encoding fibrocystin/polyductin (FC1), a membrane-associated receptor-like protein involved in the regulation of tubular cell adhesion, proliferation and apoptosis. Although it is generally accepted that apoptosis is implicated in ARPKD, the question of whether increased apoptosis is a normal response to abnormal cell proliferation or, instead, it is a primary event, is still subject to debate. In support of the latter hypothesis, we hereby provide evidence that apoptosis occurs in the absence of hyper-proliferation of FC1-depleted kidney cells. In fact, a decrease in cell proliferation, with a concomitant increase in apoptotic index and caspase-3 activity was observed in response to FC1-depletion by PKHD1 siRNA silencing in HEK293 and 4/5 tubular cells. FC1-depletion also induced reduction in ERK1/2 kinase activation, upregulation of the pro-apoptotic protein p53 and activation of NF-κB, a transcription factor which reduces apoptosis in many organs and tissues. Interestingly, selective inactivation of NF-κB using either an NF-κB decoy or parthenolide, a blocker of IKK-dependent NF-κB activation, reduced, rather then increased, apoptosis and p53 levels in FC1-depleted cells. Therefore, the proapoptotic function of NF-κB during cell death by FC1-depletion in kidney cells is evident.