Identification of small molecule inhibitors of neurite loss induced by Aß peptide using high content screening.

Multiple lines of evidence indicate a strong relationship between Αß peptide-induced neurite degeneration and the progressive loss of cognitive functions in Alzheimer disease (AD) patients and in AD animal models. This prompted us to develop a high content screening assay (HCS) and Neurite Image Quantitator (NeuriteIQ) software to quantify the loss of neuronal projections induced by Aß peptide neurons and enable us to identify new classes of neurite-protective small molecules, which may represent new leads for AD drug discovery. We identified thirty-six inhibitors of Aß-induced neurite loss in the 1,040-compound National Institute of Neurological Disorders and Stroke (NINDS) custom collection of known bioactives and FDA approved drugs. Activity clustering showed that non-steroidal anti-inflammatory drugs (NSAIDs) were significantly enriched among the hits. Notably, NSAIDs have previously attracted significant attention as potential drugs for AD; however their mechanism of action remains controversial. Our data revealed that cyclooxygenase-2 (COX-2) expression was increased following Aß treatment. Furthermore, multiple distinct classes of COX inhibitors efficiently blocked neurite loss in primary neurons, suggesting that increased COX activity contributes to Aß peptide-induced neurite loss. Finally, we discovered that the detrimental effect of COX activity on neurite integrity may be mediated through the inhibition of peroxisome proliferator-activated receptor γ (PPARγ) activity. Overall, our work establishes the feasibility of identifying small molecule inhibitors of Aß-induced neurite loss using the NeuriteIQ pipeline and provides novel insights into the mechanisms of neuroprotection by NSAIDs.

Effects of maternal protein malnutrition on oxidative markers in the young rat cortex and cerebellum.

Malnutrition affects a large number of children worldwide. Inadequate nutrition during pre- and postnatal period may alter brain development resulting in biochemical, physiological and anatomical changes which in turn could cause behavioral abnormalities. The impairment of the central nervous system following protein deficit have been extensively studied and this deprivation produces deleterious effects upon cerebral structures. The aim of this study was to identify oxidative parameters present in the developing brain as consequence of maternal protein malnutrition. Female Wistar rats were fed a normal protein diet (25% casein) or low protein diet (8% casein) from the time of conception up to 21 days after the parturition. In addition, the diets were supplemented or not with l-methionine. Cortex and cerebellum were removed from offspring to determine the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and the levels of lipoperoxidation (TBARS). Our findings demonstrated heterogeneity in response to protein restriction. The levels of lipoperoxidation were increased in the cerebellum of malnourished offspring. Methionine supplementation caused an increase in lipoperoxidation in both brain structures. CAT activity was decreased in the cerebellum of the offspring supplemented with methionine whereas the cerebellum of malnourished pups with or not methionine supplementation showed a decrease in SOD activity. The activity of SOD in the cortex did not differ among groups. CAT activity, however, was increased in the cortex of malnourished pups supplemented or not with methionine. Thus, these results provide clues to the knowledge of malnutrition effects upon the brain.

Morphological and oxidative alterations on Sertoli cells cytoskeleton due to retinol-induced reactive oxygen species.

Retinol (vitamin A) is involved in several cellular processes, like cell division, differentiation, transformation and apoptosis. Although it has been shown that retinol is a limitant factor for all these processes, the precise mechanisms by which retinol acts are still unknown. In the present study we hypothesised that alterations in the cytoskeleton of Sertoli cells induced by retinol supplementation could indicate an adaptive maintenance of its functions, since it plays an important role in the transformation process that we observed. Previous results demonstrated that Sertoli cells treated with retinol showed an oxidative imbalance, that leads the cell to two phenotypes: apoptosis or transformation. Our group has identified characteristics of Sertoli cells transformed by retinol which results in normal cell functions modification. In the present study the actin filament fluorescence assay and the deformation coefficient showed a modification in the morphology induced by retinol. We also observed an oxidative alteration in isolated cytoskeleton proteins and did not show alterations when these proteins are analyzed by electrophoreses. Our results showed an increase in mitochondria superoxide production and a decrease in nitric oxide levels. All results were partially or completely reverted by co-treatment of the antioxidant Trolox. These findings suggest that the cytoskeleton components suffer individual alterations in different levels and that these alterations generate a global phenotype modification and that these processes are probably ROS dependent. We believe that the results from this study indicate an adaptation of the cytoskeleton to oxidative imbalance since there was not a loss of its function.

Effect of protein malnutrition on redox state of the hippocampus of rat.

The protein malnutrition is a worldwide problem, affecting mainly newborns and children of developing countries. This deficiency reaches the brain in the most critical period of the development. Various consequences are related to this insult, such as memory disturbance, learning, and behavioral impairment. Protein content of the diet plays an important role on antioxidant mechanisms. This study observed the effects of protein malnutrition on rat hippocampus redox state. Wistar rats were separate in four groups, receiving different diets: first group with 25% casein, protein deficient group with 8% casein, and the same two groups supplemented with methionine (0.15%). Diets were isocaloric and were administered since the prenatal period up to the sacrifice. Rats were decapitated at 21 or 75 days old and hippocampus were isolated for measuring the lipoperoxidation by TBARS method, protein oxidative damage by carbonyl (DNPH) levels, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). There was significant alterations in the activities of the enzyme SOD, lipoperoxidation, and protein oxidation in hippocampus of 21 and 75 day-old rats fed with 25% of protein with methionine and the groups fed with low levels of protein (8%) both supplemented or not with methionine. Our data suggest that both the content of protein in the diet and the essential amino acid methionine may alter the antioxidant system and the redox state of the brain.