The objective of this application is to identify the parameters leading to and protecting against the Na+ independent, delayed neurotoxic consequences of kainic acid (KA) receptor activation. When injected in vivo, KA produces a pattern of neuronal degeneration and neurochemical alterations resembling those seen in a number of neurodegenerative disorders. This investigator hypothesized that the developmental changes in the subunit composition or characteristics of KA gated Ca2+ ion channels account for the susceptibility to KA. This study will use cerebellar granule cells (CGC) primary culture under conditions that selects specifically for delayed KA neurotoxicity. A notable feature of the CGC system is that vulnerability to degeneration requires mature cultures (14 days in culture), which is 9 days after KA receptors achieve their maximum density. Thus, initial studies will identify the molecular characteristics of the receptor during development by examining the expression of the receptor subunits and their permeability to Ca2+ fluxes. The hypothesis is that temporal disparities result from changes in gene expression of the subunits comprising the KA receptor, including changes in mRNA editing. Since increase in intracellular Ca2+ has been linked to neuronal death after glutamate exposure, studies for specific aim #2 will determine whether delayed neurotoxicity after KA stimulation is linked to increases in 45Ca2+ influx, developmental changes in Ca2+ permeability, and whether blocking of (a) voltage sensitive Ca2+ channels or (b) intracellular Ca2+ stores can prevent delayed neurotoxicity in CGC. Based on the evidence of oxidative stress, they hypothesized that elevated intraneuronal Ca2+ activates enzymes such as phospholipase A2 and/or phospholipase C, as well as downstream enzymes such as lipoxygenases.
Specific aim #3 will examine whether KA stimulated phospholipid metabolism is a source of oxyradicals. Enhanced phospholipid hydrolysis resulting in an accumulation of free radicals may lead to inactivation of critical antioxidant enzymes such as quinone reductase (QR), glutathione peroxidase (GSH-PX) and superoxide dismutase (CuZnSOD and MnSOD). KA toxicity will also be evaluated following pharmacologic or molecular manipulations to enhance or reduce the activity of these enzymes. Finally, studies will be carried out to examine whether the delayed KA induces degeneration that has the characteristics of an apototic versus necrotic cell death.
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