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

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

The two phenomena, biological and behavioural, approached in the present work are brain aging and spatial memory. Several studies, comparative and experimental, have shown that aged subjects present significantly impaired performances in different types of cognitive tasks. It is also known that contradictions in literature exist in relation to consequences of the interaction between the effects of aging with the effects of chronic ethanol consumption and food restriction, in relation to the biological as well as the behavioural effects. Despite the advances and several studies carried out in an attempt to understand the neurobiological mechanisms responsible for these dysfunctions, little is known about the biological basis correlated with specific aspects of cognitive dysfunction in the aged. Within the different neurochemical systems, one relatively less studied, with relation to the deficits in learning and spatial memory in the aged, is the serotoninergic central system. Another relevant question in this biological approach is related to the mechanisms responsible for neurodegeneration that possibly results in dysfunctions neurochemical circuit activity. In this case, early identification of molecular alterations, associated to this process, has been even more important for the understanding of biological basis related to behavioral deficits normally associated to aging and/or to several neuropathologies. A common mechanism that neuronal cells suffer due to acute injuries is unchained by the excitotocity, a phenomenon that becomes unchained when there is an excess of activity of glutamate receptors, which induce a series of intracellular processes possibly associated with neurodegeneration. There is also evidence that reactive species of oxygen (ex. hydrogen peroxide) as much in aging as in 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 and retards the development of degenerative diseases. Protective effects of FD over central nervous system were shown as a prevention in/or the reduction of oxidative stress, excitotoxic or metabolic insults, sensorial and motor deficits and cognitive damage. Among the intracellular systems involved with the mechanisms of cell death, induced by high concenrtrations of glutamate and/or reactive species of oxygen, the process of protein phosphorylation stands out. The reactions of phosphorylation and subsequently dephosphorylation are catalyzed by kinase proteins and fosfatases, respectively. They have an important role in the translation of signs, responsible for a series of cellular events, such as proliferation and death cell. Alterations in the kinase proteins and fosfatases are intimately related to various cellular dysfunctions. The kinase proteins not only phosphorylate their proteic substrates, but they are also phosphorylated and dephosphorylated being, however, regulated by phosphorylation, too. The present work is divided into two parts, which correspond to two different levels of studies. (i) The fist stage, the independent variables are age, chronic consumption of ethanol and caloric food restriction. In this stage, adult rats (5 months) and aged rats (16 months) were used as experimental animal models, as well as methods of experimental psychological and biochemistry, to assess aspects of learning and spatial memory and to determine functional parameters of the central serotoninergic central system, respectively. In addition, the correlation between behavioral and biological data was assessed. (ii) In the second stage, the independent variables are different conditions of neurodegenerative stimuli. Cell culture of neurons and biochemical methods were used for the study of the components involved in the mechanisms of cell death. In the first stage, using wistar rats we observed changes related to age in the aspects of learning, memory and behavioral extinction in the Morris water maze. Using the chromatographic method (HPLC= High Performance Liquid Chromatography), we verified also the significant effect 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 subjects succeeded in learning the behavioural task, albeit with significantly worse performance when compared to adult animals. Aging also had significant main effects on memory and extinction. An age-dependent decrease 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 peroxide differed and the affected aims, tau isoforms changed and besides that, if we consider the curve of time of the effects caused by the two treatments (glutamate and hydrogen prpxide), we verified that the glutamate induces an increase of the dephosphorolated form in a temporarily different way from hydrogen peroxide. Both stimuli provoke specification alterations, these being dependent effects of time in sub classes 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.

ASSUNTO(S)

modelos animais decs idoso decs memória decs cultura decs cognição decs envelhecimento teses. tese da faculdade de medicina. ufmg doenças neurodegenerativas decs cérebro teses. envelhecimento decs dissertações acadêmicas decs

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