Apoptotic cell death may play a prominent role in the loss of neurons during nervous system development and in neurologic diseases. The tumor suppressor gen, p53, a key regulator of cellular proliferation, has recently been associated with the induction of cell death in response to some forms of cellular damage. Although the function of this gene has been studied primarily within the context of oncological issues, a possible role for p53-related modulation of neuronal viability has been suggested by the finding that p53 induction may initiate or participate in an intracellular pathway that culminates in neuronal cell death. Our long range objective is to assess the pattern and function of p53 gene expression in the CNS. In the present application, we will test the hypothesis that p53 expression regulates neuronal viability after injury. We will accomplish this goal by: (1) Evaluating regional and cell specific activation of p53 in the CNS in response to neuronal injury; 2) Evaluating the relationship between p53 expression and irreversible cell damage; 3) Evaluating the potential mechanisms by which p53 promotes irreversible neuronal cell injury in response to kainic acid; 4) Evaluating the effects of p53 expression on CNS-mediated response to toxic challenge.
Aim 1 will be accomplished by monitoring p53 mRNA and protein expression using in situ hybridization and immunocytochemistry.
In aim 2, the specific contribution of p53 to neuronal vulnerability following a particular injury will be assessed using both animal and cultured neurons containing p53 alleles (+/+, +/-) and those deficient in p53 (-/-, knockout) Particular attention will be paid to the requirement for p53 transcription vs. protein activation following injury. Antisense oligonucleotide strategies will be employed to block p53 transcription in order to enhance neuronal survival., Aim 3 will be accomplished by measuring short term and long term changes in calcium mobilization and free radical generation following kainate treatment in (-/+) and (-/-) mine and cultured neurons, and by immunocytochemical, Western blot and Northern blot analysis to assess changes in the expression of other genes known to modulate cell death (such as bcl-2, bcl-x, and bax).
Aim 4 will be accomplished by comparing the excitable properties of neurons that are vulnerable or resistant (e.g. in p53-deficient (-/-) mice) to kainate treatment and analyzing the reversibility of any functional abnormality. Elucidating the function of p53 in the nervous system may shed light on the regulation of neuronal survival in response to injury in neurologic disease.
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