Increasing evidence suggests that free radical-mediated oxidative damage to various biologically important macromolecules correlates with the normal aging process. Alzheimer's disease is a devastating disease of elderly people. Epidemiological and clinical studies have shown that Alzheimer's disease is the most common cause of dementia in the elderly. Postmortem histological studies of the brains of former patients with Alzheimer's disease have shown that cerebral neurodegeneration is the primary cause of this condition. The working hypothesis for the present study is that $\beta$-amyloid, a protein associated with neural damage in the brains of Alzheimer's disease patients, produces free radicals which, by leading to a disruption of calcium homeostasis and neuron degeneration, play an important role in the progressive pathologic process of Alzheimer's disease. Consequently, antioxidant intervention should, by scavenging free radicals, prevent or terminate the oxidative cascade and offer protection to neurons from $\beta$-amyloid induced toxicity. In this project, we investigated: (1) the free radical formation in autopsy samples of Alzheimer and control cortex by using the electron paramagnetic resonance spin-trapping method; (2) the mechanism of calcium increase induced by $\beta$-amyloid and the protective effects of antioxidants on $\beta$-amyloid induced calcium disruption and cytotoxicity in cultured neurons; and (3) the effect of hypoxia induced oxidative stress on $\beta$-amyloid induced calcium disruption and cytotoxicity in cultured neurons. In this study, we found that the formation of free radicals in homogenates of frontal cortex from brains taken at autopsy and verified histologically to be from patients with Alzheimer's disease, was 22% higher $\rm (p < 0.05)$ than age matched controls as determined by electron paramagnetic resonance spectroscopy. Following incubation in the presence of ferrous sulfate (200 $\rm\mu M),$ samples of Alzheimer's frontal cortex produced nearly 50% more free radicals than did controls $\rm (p < 0.01).$ We also found that the fragment of $\beta$-amyloid comprised of amino acids 25-35, induces a rapid, concentration-dependent increase in cytosolic free calcium in PC12 neuronal cells. This action of $\beta$-amyloid 25-35 is not altered by pretreatment with the calcium channel blockers nifedipine or cobalt, with the depletor of intracellular calcium stores cyclopiazonic acid, or with the phospholipase C inhibitor neomycin. However, the effects of $\beta$-amyloid 25-35 on cytosolic free calcium are absent in calcium-free buffer, and are blocked by the antioxidant lazaroid U-83836E, by vitamin E and by cholesterol. $\beta$-amyloid 25-35 is also neurotoxic and produces a concentration dependent reduction in the viability of PC12 cells in culture. The neurotoxic action of $\beta$-amyloid is blocked by U-83836E, by vitamin E, and by cholesterol, but not by nifedipine or cobalt. When SH-SY5Y neuronal cells were preincubated in anoxic conditions for one hour, $\beta$-amyloid induced calcium disruption and cytotoxicity were amplified by this hypoxia induced oxidative stress. The interaction of anoxia and $\beta$-amyloid is blocked in a dose dependent manner by U-83836E and vitamin E. These data indicate that both the disruption of calcium homeostasis and the neurotoxicity of $\beta$-amyloid in neurons are mediated by free radical based processes.
Doctor of Philosophy (Ph.D.)