Jose M. Moran

Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: Modulation by the Nrf2/Trx axis

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases.

Paraquat-induced apoptotic cell death in cerebellar granule cells

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinsons disease (PD). Paraquat is structurally similar to the neurotoxin MPP+ that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 microM) produces apoptotic cell death with a reduction in mitochondrial cytochrome c content, proteolytic activation and caspase-3 activity increase and DNA fragmentation. Paraquat-induced apoptosis was significantly attenuated by co-treatment of cerebellar granule cells with the radical scavenger vitamin E, suggesting that paraquat-induced free radicals serve as important signal in initiation of cell death. As a decrease in mitochondrial cytochrome c content is also prevented by allopurinol, we suggest that xanthine oxidase plays an important role in the free radical production that precedes the apoptotic cascade and cell death after paraquat exposition.

Silencing DJ‐1 reveals its contribution in paraquat‐induced autophagy

J. Neurochem. (2009) 109, 889–898.

Protection against MPP+ neurotoxicity in cerebellar granule cells by antioxidants

The neuropathology associated with Parkinsons disease (PD) is thought to involve excessive production of free radicals, dopamine autoxidation, defects in glutathione peroxidase expression, attenuated levels of reduced glutathione, altered calcium homeostasis, excitotoxicity and genetic defects in mitochondrial complex I activity. While the neurotoxic mechanisms are vastly different for excitotoxins and 1‐methyl‐4‐phenylpyridinium ion (MPP+), both are thought to involve free radical production, compromised mitochondrial activity and excessive lipid peroxidation. We show here that the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) increased significantly after treatment of cultured cerebellar granule cells (CGCs) with 50 μM MPP+. Co‐treatment with antioxidants such as ascorbate (ASC), catalase, α‐tocopherol (α‐TOH), coenzyme Q10 (CoQ10) or superoxide dismutase (SOD) rescued the cells from MPP+‐induced death. MPP+‐induced cell death was also abolished by co‐treatment with nitric oxide synthase (NOS) inhibitors such as 7‐nitroindazole (7‐NI), 2‐ethyl‐2‐thiopseudourea hydrobromide (EPTU) or S‐methylisothiourea sulphate (MPTU). We also tested the protective effects of an iron chelator (deferoxamine mesylate, DFx) and a peroxynitrite scavenger (FeTTPS) and the results lend further support to the view that the free radical cytotoxicity plays an essential role in MPP+‐induced death in primary cultures of CGC.

Nitric oxide in paraquat-mediated toxicity: A review.

Paraquat, a cationic herbicide, produces degenerative lesions in the lung and in the nervous system after systemic administration to man and animals. Many cases of acute poisoning and death have been reported over the past few decades. Although a definitive mechanism of toxicity of paraquat has not been delineated, a cyclic single electron reduction/oxidation is a critical mechanistic event. The redox cycling of paraquat has two potentially important consequences relevant to the development of toxicity: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species which are highly reactive to cellular macromolecules; and the oxidation of reducing equivalents (e.g., NADPH, reduced glutathione), which results in the disruption of important NADPH‐requiring biochemical processes necessary for normal cell function. Nitric oxide is an important signaling molecule that reacts with superoxide derived from the paraquat redox cycle, to form the potent oxidant peroxynitrite, which causes serious cell damage. Although nitric oxide has been involved in the mechanism of paraquat‐mediated toxicity, the role of nitric oxide has been controversial as both protective and harmful effects have been described. The present review summarizes recent findings in the field and describes new knowledge on the role of nitric oxide in the paraquat‐mediated toxicity.

Relationship between Autophagy and Apoptotic Cell Death in Human Neuroblastoma Cells Treated with Paraquat: Could Autophagy be a “Brake” in Paraquat-Induced Apoptotic Death?

Paraquat (PQ) (1, 1’-dimethyl-4, 4’-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson’s disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD. Addendum to Inhibition of Paraquat-Induced Autophagy Accelerates the Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells R.A. González-Polo, M. Niso-Santano, M.A. Ortíz-Ortíz, A. Gómez-Martín, J.M. Morán, L. García-Rubio, J. Francisco-Morcillo, C. Zaragoza , G. Soler and J.M. Fuentes Toxicological Science 2007; In press

Identification of genes associated with paraquat-induced toxicity in SH-SY5Y cells by PCR array focused on apoptotic pathways.

Paraquat (PQ) (1,1-dimethyl-4,4′-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinsons disease (PD). In this sense, understanding of the molecular mechanism underlying PQ-induced toxicity to neural cells is important for optimal use as well as for the development of new drugs. To gain insights into PQ-induced neurotoxicity, polymerase chain reaction (PCR) array analysis focused on a panel of apoptosis-related genes was performed using neuroblastoma SH-SY5Y cells. Up to 65 apoptosis-related genes were monitored. Our analysis of apoptotic process through microarray technology showed that in PQ-induced neuroblastoma SH-SY5Y cells, there is a different expression of BIK, CASP3, CASP7, CRADD, DAPK, FAS, and other related genes, in comparison to unstimulated cells. Evaluation of genes regulated differentially is essential for the development of therapeutic approaches in multifactorial diseases as PD. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in PQ-mediated toxicity of neural cells.

Paraquat Exposure Induces Nuclear Translocation of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and the Activation of the Nitric Oxide-GAPDH-Siah Cell Death Cascade

Paraquat (PQ) is a well-known herbicide that exerts its effects by elevating intracellular levels of superoxide. It has been previously demonstrated that oxidative and nitrosative stress participate to PQ-induced cell death. Here, we document that PQ increases the levels of nitric oxide (NO) in rat mesencephalic cells and causes nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to activate the NO/GAPDH/Siah cell death cascade. PQ exposure increases expression of the p300/CREB-binding protein (p300/CBP) and phosphorylation of p53 at Ser 15, which stimulates p53-dependent transactivation through increased binding with p300. Although this cascade could be inhibited by preincubation with the monoamine oxidase B inhibitor deprenyl, cell death was not prevented. Pretreatment of cells with the neuronal nitric oxide synthase inhibitor 7-nitroindazole efficiently prevented the activation of the GAPDH/NO/Siah cell death cascade, thereby protecting cells against PQ-induced toxicity. The results suggest that PQ induces this novel cell death cascade in rat mesencephalic cells, but inhibition of the pathway does not impede cell death because of an oxidative burst generated by the pesticide.

High resolution mapping and mutation analyses of candidate genes in the urofacial syndrome (UFS) critical region

The urofacial (Ochoa) syndrome (UFS) characterized by congenital obstructive uropathy and abnormal facial expression is a rare disorder caused by a single recessive disease gene. Our previous studies using homozygosity mapping have located the UFS gene to a genomic interval of approximately 360 kb on chromosome 10q23‐10q24. In this study, we have constructed a genomic sequence map covering the entire UFS interval and narrowed the disease interval to a genomic region of 220 kb that harbor the newly identified ACDP1 gene in addition to part of the GOT1 gene which has already been excluded as a candidate for UFS. Extensive search for mutations in the coding region, the 5′ and 3′ untranslated regions, the promoter region, and the exon/intron junctions failed to identify a pathogenic mutation in UFS patients. Furthermore, our analyses indicated that the same gene on chromosome 10q is responsible for all UFS patients from multiple ethnic groups.

Dietary Habits, Nutrients and Bone Mass in Spanish Premenopausal Women: The Contribution of Fish to Better Bone Health

The moderate consumption of fish is recommended for a healthy diet and is also a feature of the Mediterranean diet. Fish is a major food group in diets throughout the world, and studies show that fish consumption is associated with a lower risk of a number of conditions. Spain has one of the highest annual per capita consumptions of fish worldwide. As fish is a source of high quality protein; n-3 polyunsaturated fatty acids; vitamins, such as A and D; and minerals, such as selenium, calcium, iodine, magnesium, copper and zinc, nutrients that have positive effects on bone characteristics, it has been proposed that its consumption could improve bone health. In this cross-sectional study, we have investigated the relationship between dietary habits and nutrient intake of 151 Spanish premenopausal women and analyzed the association of fish consumption on bone mass measured by quantitative ultrasound of the phalanges. A higher (P < 0.05) bone mass and vitamin D intake (P < 0.05) was observed in the group with a fish intake of 5–7 servings/week. We conclude that increased fish consumption is helpful in maintaining an adequate bone mass in Spanish premenopausal women.