Showing papers by "Cecília M. P. Rodrigues published in 2002"

Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease

Abstract: Huntington's disease (HD) is an untreatable neurological disorder caused by selective and progressive degeneration of the caudate nucleus and putamen of the basal ganglia. Although the etiology of HD pathology is not fully understood, the observed loss of neuronal cells is thought to occur primarily through apoptosis. Furthermore, there is evidence in HD that cell death is mediated through mitochondrial pathways, and mitochondrial deficits are commonly associated with HD. We have previously reported that treatment with tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, prevented neuropathology and associated behavioral deficits in the 3-nitropropionic acid rat model of HD. We therefore examined whether TUDCA would also be neuroprotective in a genetic mouse model of HD. Our results showed that systemically administered TUDCA led to a significant reduction in striatal neuropathology of the R6/2 transgenic HD mouse. Specifically, R6/2 mice began receiving TUDCA at 6 weeks of age and exhibited reduced striatal atrophy, decreased striatal apoptosis, as well as fewer and smaller size ubiquitinated neuronal intranuclear huntingtin inclusions. Moreover, locomotor and sensorimotor deficits were significantly improved in the TUDCA-treated mice. In conclusion, TUDCA is a nontoxic, endogenously produced hydrophilic bile acid that is neuroprotective in a transgenic mouse model of HD and, therefore, may provide a novel and effective treatment in patients with HD.

Neuroprotection by a Bile Acid in an Acute Stroke Model in the Rat

Abstract: Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane per...

Rat Cultured Neuronal and Glial Cells Respond Differently to Toxicity of Unconjugated Bilirubin

Abstract: High levels of unconjugated bilirubin (UCB) can be neurotoxic. Nevertheless, the mechanism of UCB interaction with neural cells is still unknown. This study investigates whether cultured rat neurons and astrocytes respond differently to UCB exposure. UCB toxicity was evaluated by lactate dehydrogenase release, induction of apoptosis, cytoskeleton degeneration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and glutamate uptake. Primary cultures of rat brain astrocytes and neurons were incubated at 37 degrees C with 85.5 microM UCB plus 28.5 microM albumin for 4 h. In assays of glutamate uptake, cells were exposed to 80-120 microM UCB plus 100 microM albumin for 15 min. The results showed that after incubation with 85.5 microM UCB, lactate dehydrogenase release was greater in neurons than in astrocytes (38% versus 14%, p < 0.05). Also, levels of apoptosis were markedly enhanced in neurons (29% versus 19%, p < 0.01). In accordance, neuronal cytoskeleton disassembly was evident during incubation with 85.5 microM UCB, whereas equivalent effects on astrocytes required as much as 171 microM. Conversely, inhibition of MTT metabolism and glutamate uptake by UCB was more pronounced in astrocytes than in neurons (74% versus 60%, p < 0.05 and 41% to 56% versus 25% to 33%, p < 0.05, respectively). In conclusion, the study demonstrates that astrocytes are more susceptible to inhibition of glutamate uptake and MTT reduction by UCB, whereas neurons are more sensitive to cell death by necrosis or apoptosis. These results suggest that UCB is toxic to both astrocytes and neurons, although through distinct pathways.

Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes.

Abstract: The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.

Aging confers different sensitivity to the neurotoxic properties of unconjugated bilirubin.

Abstract: The pathogenesis of bilirubin encephalopathy appears to result from accumulation of unconjugated bilirubin (UCB), which, in turn, may cause mitochondrial perturbation, release of intermembrane proteins, and, ultimately, cell death. Aging imparts to cells a different susceptibility to this toxic stimulus, as neonates are particularly vulnerable to the accumulation of UCB in the CNS. In this paper, we further characterize UCB-induced toxicity in isolated neuronal and glial cells according to age in culture. In addition, we investigate sensitivity of mitochondria derived from young and old rats to UCB-induced membrane permeabilization and, finally, evaluate whether age-dependent changes in UCB toxicity are accompanied by alterations in the mitochondrial content of cytochrome c. The results showed that UCB is more toxic to immature neural cells after 4-5 d in culture (p < 0.001), whereas neurons were more sensitive than astrocytes (p < 0.05). In fact, approximately 40% of cells were apoptotic in immature cultures compared with 20% in mature cultures. Unexpectedly, mitochondrial swelling and subsequent efflux of cytochrome c induced by UCB were 2-fold greater in organelles derived from older rats (p < 0.01). In conclusion, UCB toxicity of isolated rat neuronal and glial cells is modulated by age in culture in that immature cells are more susceptible. Mitochondria derived from younger rats are nevertheless more resistant to membrane permeabilization and cytochrome c release induced by UCB. The data indicate that the cells of young animals are relatively resistant to UCB toxicity, through a protective mechanism at the mitochondrial level; however, this is not sufficient to prevent apoptosis of cells in the young animal. Thus, although playing a role, direct mitochondrial injury may not be the sole mechanism of UCB cytotoxicity.

[Release of cytochrome C with the interaction of bilirubin, amyloid beta-peptide and glycochenodeoxycholate from isolated mitochondria].

Abstract: The pathogenesis of cholestasis, bilirubin encephalopathy, and Alzheimer's disease appears to result from accumulation of diverse cytotoxic agents, which in turn may cause apoptotic cell death. In addition, mitochondria has lately been considered as a central executioner of programmed cell death, through the release of caspase activating factors. The aims of this study were to: (a) investigate mitochondrial perturbation during incubation of isolated mitochondria with unconjugated bilirubin (Bb), amyloid beta-peptide (A beta), and glycochenodeoxycholate (GCDC); (b) characterize membrane perturbation in isolated mitochondria induced by each toxic agent, and determine whether the mitochondrial permeabilization is required for cytochrome c redistribution. Mitochondria were isolated from rat liver and brain. Swelling and cytochrome c release were evaluated by spectrophotometry and western blot, respectively. The results showed that Bb as well as A beta and GCDC act directly at the mitochondrial level causing increased organelle volume, permeabilization, as well as cytochrome c release from the intermembrane space in a dose-dependent manner (P < 0.01). Moreover, cyclosporine A inhibited mitochondrial permeability, particularly after Bb- and GCDC-induced swelling (P < 0.01). Cytochrome c efflux was invariably prevented by cyclosporine A (P < 0.05). In conclusion, the results indicate that Bb-, A beta-, and GCDC-induced toxicity, culminating in apoptosis, may result from enhanced mitochondrial permeability, followed by cytochrome c efflux, which can be explained at least in part by the megapore opening.