Aspectos do envelhecimento cerebral e função cognitiva em modelo experimental animal e estudo de mecanismos de neurodegeneração em cultura celular

Abstract

The two phenomena, biological and behavioural, approached in thepresent work are brain aging and spatial memory. Several studies,comparative and experimental, have shown that aged subjects presentsignificantly impaired performances in different types of cognitive tasks.It is also known that contradictions in literature exist in relation toconsequences of the interaction between the effects of aging with theeffects of chronic ethanol consumption and food restriction, in relation tothe biological as well as the behavioural effects. Despite the advancesand several studies carried out in an attempt to understand theneurobiological mechanisms responsible for these dysfunctions, little isknown about the biological basis correlated with specific aspects ofcognitive dysfunction in the aged. Within the different neurochemicalsystems, one relatively less studied, with relation to the deficits inlearning and spatial memory in the aged, is the serotoninergic centralsystem. Another relevant question in this biological approach is relatedto the mechanisms responsible for neurodegeneration that possiblyresults in dysfunctions neurochemical circuit activity. In this case, earlyidentification of molecular alterations, associated to this process, hasbeen even more important for the understanding of biological basisrelated to behavioral deficits normally associated to aging and/or toseveral neuropathologies. A common mechanism that neuronal cellssuffer due to acute injuries is unchained by the excitotocity, aphenomenon that becomes unchained when there is an excess of activityof glutamate receptors, which induce a series of intracellular processespossibly associated with neurodegeneration. There is also evidence thatreactive species of oxygen (ex. hydrogen peroxide) as much in aging asin chronic alcoholism and in various neurodegenerative cerebral diseases (ex. Parkinsons and Alzheimers disease, etc) seem to be involved with cell death. On the other hand, some works indicate that food restriction (FD), that consist in reduction of the habitual caloric quantity, has beneficial effects on the organism, probably through a mecanism that results in decrease of the reactive species of oxigen. It has already been reported that it acts by increasing the time of life of the animals andretards the development of degenerative diseases. Protective effects ofFD over central nervous system were shown as a prevention in/or thereduction of oxidative stress, excitotoxic or metabolic insults, sensorialand motor deficits and cognitive damage. Among the intracellularsystems involved with the mechanisms of cell death, induced by highconcenrtrations of glutamate and/or reactive species of oxygen, theprocess of protein phosphorylation stands out. The reactions ofphosphorylation and subsequently dephosphorylation are catalyzed bykinase proteins and fosfatases, respectively. They have an importantrole in the translation of signs, responsible for a series of cellular events,such as proliferation and death cell. Alterations in the kinase proteinsand fosfatases are intimately related to various cellular dysfunctions.The kinase proteins not only phosphorylate their proteic substrates, butthey are also phosphorylated and dephosphorylated being, however,regulated by phosphorylation, too. The present work is divided into twoparts, which correspond to two different levels of studies. (i) The fiststage, the independent variables are age, chronic consumption ofethanol and caloric food restriction. In this stage, adult rats (5 months)and aged rats (16 months) were used as experimental animalmodels, as well as methods of experimental psychological andbiochemistry, to assess aspects of learning and spatial memory and todetermine functional parameters of the central serotoninergic centralsystem, respectively. In addition, the correlation between behavioral andbiological data was assessed. (ii) In the second stage, the independentvariables are different conditions of neurodegenerative stimuli. Cellculture of neurons and biochemical methods were used for the studyof the components involved in the mechanisms of cell death. In the firststage, using wistar rats we observed changes related to age in theaspects of learning, memory and behavioral extinction in the Morriswater maze. Using the chromatographic method (HPLC= High Performance Liquid Chromatography), we verified also the significanteffect of age in the endogenous levels of serotonin (5-HT) and of 5-hydroxyindole acetic acid (5-HIAA) in the neocortex, hippocampus,thalamus and dorsal raphe nucleus (DRN). The middle-aged subjectssucceeded in learning the behavioural task, albeit with significantlyworse performance when compared to adult animals. Aging also hadsignificant main effects on memory and extinction. An age-dependentdecrease in 5-HIAA levels was observed in both hippocampus and dorsal raphe nucleus (DRN). The decrease in DRN 5-HIAA was paralleled by a decrease in 5-HIAA/5-HT ratio in this brain area, which was significantly correlated to the animals spatial memory performance and behavioural extinction. In addition, using middle-aged rats, a 2×2 factorial study was carried out to examine the effects of food restriction and chronic ethanol consumption on rats performance in a spatial behavioural task and on central serotoninergic parameters. None of these two treatments had a significant effect on the behavioural and biochemical parameters assessed, with the exception of extinction index, which was significantly affected by ethanol consumption. Long-term ethanol ameliorated the impairment in behavioural flexibility caused by aging. The data of the first stage were published in the journal Behavioral Brain Research in 2007. In the second stage of the study, using primary culture of cortical neurons, we investigated the involvement of molecular components of cytoskeletal elements, tau protein, and the system of translation of signs, MAPKs and GSK3, mechanism of two neurotoxic factors: i) high concentration of glutamate and, ii) oxidative stress, provoked by hydrogen peroxide. The phosphoproteins were separated by gel electrophoresis and identified with antibodies, by immunofluorescence, through the Western blot technique. The quantitative analyses were done by densitometer. As first step, we confirmed data of literature which showed that a high concentration of glutamate as well as hydrogen peroxide caused significantly loss of cortical neurons. In addition, we added information, showing that these effects are dependent on incubation time. The next step was to verify if the high concentration of glutamate or hydrogen peroxide had effects on the level of phosphorylation and/or total concentration of the following classes of phosphoproteins: tyrosine phosphoproteins, isoforms of phosphoproteins tau, and kinase phosphoproteins. Hydrogen peroxide as well as glutamate provoked an increase in the dephosphorelated form of tau protein. However, the effects of glutamate and of hydrogen peroxidediffered and the affected aims, tau isoforms changed and besides that, ifwe consider the curve of time of the effects caused by the twotreatments (glutamate and hydrogen prpxide), we verified that theglutamate induces an increase of the dephosphorolated form in atemporarily different way from hydrogen peroxide. Both stimuli provokespecification alterations, these being dependent effects of time in subclasses of MAPKs. Some of these effects are dysfunctions in components of the phosphorylation systems relation only with conformation changes, in other words, the active for inactive form or vice verse and, others; result in alterations in the expression of important phosphoproteins of the cellular signal systems, possibly, relate with cell death.