Involvement of superoxide in malaoxon-induced toxicity in primary cultures of cortical neurons.

Organophosphorus compounds (OP) represent a class of insecticides that are used most globally. The neurotoxic effects are attributed mainly to acetylcholinesterase (AChE) enzyme inhibition, which is responsible for cholinergic manifestations in individuals acutely exposed to OP. However, AChE inhibition alone cannot account for the wide range of symptoms that were reported following OP exposures. In agreement with this, evidence shows that non-cholinergic events may be mechanistically linked to OP-induced neurotoxicity. The aim of this study was to investigate the potential occurrence of oxidative stress as a critical step in the toxicity induced by the OP malaoxon(MAL) using primary cultures of mouse cortical neurons, as well as to distinguish MAL-induced oxidative stress and cell toxicity from an action on AChE blockade. Primary cultures of mouse cortical neurons were treated with MAL (0.01; 0.1; 1; 10; or 100 µM) at varying time points (1, 3, 6, 24, 48, or 144 hr) and the following biochemical parameters determined including cell viability, AChE activity, and superoxide production. MAL significantly reduced cell viability in a concentration- and time-dependent manner. Of note, 1 µM MAL significantly inhibited (approximately 75%) AChE activity after 48 hr incubation. Pralidoxime (PRAL) (600 µM), a classical AChE reactivator, significantly protected against MAL-induced AChE blockade; however, PRAL did not affect MAL-mediated fall in cellular viability, indicating that AChE inhibition is not necessarily correlated with insecticide-induced decrease in cell survival. MAL-induced diminished cell viability was preceded by a significant increase in superoxide anion production. The antioxidant agent ascorbic acid (AA) (200 µM), which significantly protected against MAL-induced superoxide anion production, did not alter MAL-induced AChE inhibition and significantly prevented insecticide-mediated fall in cell survival. Data show that increased superoxide anion production is an event that precedes MAL-induced cell toxicity in primary cultures of mouse cortical neurons. Based on the preventative effects of AA against MAL-mediated superoxide anion production and reduced cell viability, evidence indicates that oxidative stress represents an important step mediating MAL-induced toxicity in neurons and that AChE inhibition is not necessarily correlated with lowered cell survival noted in insecticide-exposed cells.

ConBr, A Lectin Purified from the Seeds of Canavalia brasiliensis, Protects Against Ischemia in Organotypic Culture of Rat Hippocampus: Potential Implication of Voltage-Gated Calcium Channels.

Lectins are proteins that bind cellular glycans and can modulate various neuronal functions. We have evaluated the neuroprotective effect of ConBr, a lectin purified from the seeds of Canavalia brasiliensis in a model of rat organotypic hippocampal cultures (OHCs) exposed to oxygen and glucose deprivation (OGD). OGD for 15 min followed by 24 h re-oxygenation significantly increased cell death, caused mitochondrial depolarization and increased reactive oxygen species (ROS) in CA1 region of OHCs. ConBr (0.1 µg/mL) added during the re-oxygenation period counteracted cell death, mitochondrial depolarization and overproduction of ROS induced by OGD. Moreover, ConBr restored the levels of Akt and ERK1 phosphorylation that were reduced by OGD. Modulation of intracellular Ca2+ by ConBr was evaluated in isolated hippocampal neurons loaded with the fluorescent calcium dye Fluo-4/AM. ConBr (0.1 and 1 µg/mL) reduced by 25-30 % the Ca2+ increment induced by 70 mM K+. A sub effective concentration of ConBr (0.01 µg/mL) together with a sub effective concentration of the L-type calcium channel antagonist nifedipine (0.3 µM) conferred a synergic neuroprotective effect in OHCs subjected to OGD. In conclusion, ConBr provides OHCs neuroprotection against OGD. The mechanism was not fully addressed but it may involve modulation of L-type voltage-gated Ca2+ channels by ConBr.

Atorvastatin Prevents Glutamate Uptake Reduction Induced by Quinolinic Acid Via MAPKs Signaling.

Statins have been shown to promote neuroprotection in a wide range of neurological disorders. However, the mechanisms involved in such effects of statins are not fully understood. Quinolinic acid (QA) is a neurotoxin that induces seizures when infused in vivo and promotes glutamatergic excitotoxicity in the central nervous system. The aim of this study was to evaluate the putative glutamatergic mechanisms and the intracellular signaling pathways involved in the atorvastatin neuroprotective effects against QA toxicity. Atorvastatin (10 mg/kg) treatment for 7 days prevented the QA-induced decrease in glutamate uptake, but had no effect on increased glutamate release induced by QA. Moreover, atorvastatin treatment increased the phosphorylation of ERK1 and prevented the decrease in Akt phosphorylation induced by QA. Neither atorvastatin treatment nor QA infusion altered glutamine synthetase activity or the levels of phosphorylation of p38(MAPK) or JNK1/2 during the evaluation. Inhibition of MEK/ERK signaling pathway, but not PI3K/Akt signaling, abolished the neuroprotective effect of atorvastatin against QA-induced decrease in glutamate uptake. Our data suggest that atorvastatin protective effects against QA toxicity are related to modulation of glutamate transporters via MAPK/ERK signaling pathway.

Highly palatable diet consumption increases protein oxidation in rat frontal cortex and anxiety-like behavior.

Obesity is frequently associated with consumption of high amounts of sugar and/or fat. Studies have demonstrated a high prevalence of overweight and obesity associated or not with increase rates of psychiatry disorders, in particular mood and anxiety disorders. Recent works have demonstrated an association between specific genes involved in oxidative stress metabolism and anxiety-like behavior. The aim of this study was to investigate the effect of a highly palatable diet enriched with sucrose in body fat mass composition, anxiety behavior and brain oxidative status. Twenty male Wistar rats received two different diets during four months: standard chow (SC) and highly palatable (HP). Metabolic parameters, behavioral tests and oxidative stress status were evaluated. Body fat mass, insulin sensitivity and glucose tolerance were altered in the HP group (p<0.01). The same group spends less time in light compartment and had a lower risk assessment behavior (p<0.05) but no differences were observed in the open field test habituation (p>0.05). Protein degradation, DCF and TBARS levels were not different in the hippocampus between groups; however, there were higher levels of protein degration in frontal cortex of HP groups (p<0.05), although DCF and TBARS levels don't differ from the SC group (p>0.05). In conclusion, our data suggest that the consumption of HP diet leads to an obese phenotype, increases protein oxidation in frontal cortex and appears to induce anxiety-like behavior in rats.

A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice.

The recessive aphakia (ak) mouse mutant is characterized by bilateral microphthalmia due to a failure of lens morphogenesis. We fine-mapped the ak locus to the interval between D19Umi1 and D19Mit9, developed new polymorphic markers, and mapped candidate genes by construction of a BAC contig. The Pitx3 gene, known to be expressed in lens primordia, shows zero recombination with the ak mutation on our intersubspecific intercross panel representing 1170 meioses. A recent report described a deletion in the intergenic region between Gbf1 and Pitx3 as the possible ak mutation. Our results differ in that we find not only the distant intergenic deletion, but also a much larger deletion directly in the Pitx3 gene, eliminating exon 1 and extending into intron 1 and the promoter region. Pitx3 transcript levels are severely reduced in ak/ak mice from E11.5 to newborn (5 +/- 1% of the wildtype levels at E13.5), while an involvement of the flanking Gbf1 and Cig30 genes in the aberrant lens development is highly unlikely based on expression analysis. We conclude that the ak mutation consists of two deletions, the larger of which removes part of Pitx3, indicating a crucial role of this gene in early lens development.