Several factors contribute to the degeneration and ultimately loss of neurons during aging and especially during Alzheimer's Disease (AD). One potentially critical factor is an increase in neuronal Ca2+ influx through L-type Ca2+ channels. The activity of L-type channels increases with age in rats, and an inhibitor of these channels impressively improves the learning capabilities of aged rabbits when given over several weeks. These findings suggest that L-type channels may be involved in aging-related neurological changes and the etiology of AD. Our objectives are to determine molecular changes of L-type channels that may contribute to the upregulation of L-type channel activity during aging. Based on our preliminary data, we hypothesize that: (A) an increase in protein kinase A (PKA)-mediated phosphorylation of L-type channels is responsible for their upregulation during aging; (B) beta-adrenergic receptors can control the elevation in phosphorylation of L-type channels by PKA. We will test these hypotheses by determining the phosphorylation status of L-type channels in brain samples from adult rats (some treated with beta-adrenergic agonists) and aging rats as a model for senile symptoms. Brain samples from adult and aging rats will be solubilized and L-type channels immunoprecipitated for further biochemical analysis of parameters that might change during aging, e.g., phosphorylation by PKA and other kinases. To test whether the number of L-type channels present in neurons is changed during aging, relative amounts of L-type channel proteins will be determined by immunoblotting and compared to the level of various pre- and postsynaptic marker proteins. PKA is associated with L-type channels. We will measure the relative amounts of PKA subunits and PKA anchoring proteins in the L-type channel immunocomplexes to test potential changes during aging that could explain the increase in PKA-mediated phosphorylation. Finally, we will investigate whether injection of the beta-adrenergic agonist isoproterenol upregulates PKA-mediated phosphorylation of L-type channels in adult rats.
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