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Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems – glutathione (GSH) and thioredoxin (Trx) systems – are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5 μM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells.

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

Alzheimer's disease (AD) is a neurodegenerative disease hallmarked by extracellular Aβ1–42 containing plaques, and intracellular neurofibrillary tangles (NFT) containing hyperphosphorylated tau protein. Progressively, memory deficits and cognitive disabilities start to occur as these hallmarks affect hippocampus and frontal cortex, regions highly involved in memory. Connective tissue growth factor (CTGF) expression, which is high in the vicinity of Aβ plaques and NFTs, was found to influence γ-secretase activity, the molecular crux in Aβ1–42 production. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that downregulates CTGF expression in hepatocytes and has been shown to possess therapeutic efficacy in neurodegenerative models. To investigate the possible in vivo therapeutic effects of TUDCA, we provided 0.4% TUDCA-supplemented food to APP/PS1 mice, a well-established AD mouse model. Six months of TUDCA supplementation prevented the spatial, recognition and contextual memory defects observed in APP/PS1 mice at 8 months of age. Furthermore, TUDCA-supplemented APP/PS1 mice displayed reduced hippocampal and prefrontal amyloid deposition. These effects of TUDCA supplementation suggest a novel mechanistic route for Alzheimer therapeutics.

Bile acids are a group of molecular species of acidic steroids with peculiar physical-chemical and biological characteristics. At high concentrations they become toxic to mammalian cells, and their presence is pertinent in the pathogenesis of several liver diseases and colon cancer. Bile acid cytoxicity has been related to membrane damage, but also to nondetergent effects, such as oxidative stress and apoptosis. Strikingly, hydrophilic ursodeoxycholic acid (UDCA), and its taurine-conjugated form (TUDCA), show profound cytoprotective properties. Indeed, these molecules have been described as potent inhibitors of classic pathways of apoptosis, although their precise mode of action remains to be clarified. UDCA, originally used for cholesterol gallstone dissolution, is currently considered the first choice therapy for several forms of cholestatic syndromes. However, the beneficial effects of both UDCA and TUDCA have been tested in other experimental pathological conditions with deregulated levels of apoptosis, including neurological disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. Here, we review the role of bile acids in modulating the apoptosis process, emphasizing the anti-apoptotic effects of UDCA and TUDCA, as well as their potential use as novel and alternate therapeutic agents for the treatment of apoptosis-related diseases.

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status.These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

Summary: Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/β-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.