NEUROTOXICITY OF AMMONIA AND SHORT-CHAIN FATTY ACIDS

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Hyperammonemia and organic acidemia are persistent features in many encephalopathies (e.g. hepatic and valproate encephalopathies, Reye's disease, Jamaican vomitting sickness). Although the evidence for ammonia and/or short-chain fatty acids (SCFA) as major toxins in these encephalopathies is not always definitive, the most effective treatments of these diseases are geared toward eliminating the hyperammonemia and organic acidemia. Moreover, administration of ammonia and SCFA induces coma in animals and the resultant neuropathologic changes show striking similarities to the brain structural abnormalities of Reye's disease, and hepatic and valproate encephalopathies. Thus, investigations of the neurotoxicity of ammonia and SCFA should help to elucidate the underlying biochemical and physiologic mechanisms and illuminate the pathophysiology and/or pathogenesis of these encephalopathies. The studies outline in this proposal aim to test our hypothesis that ammonia and SCFA exert their toxic effects by (1) interfering with tricarboxylic acid (TCA) cycle activity, and (2) altering the structure and function of the neuronal plasma membrane. Based on results of our and other studies, we further propose that these toxins could interfere with TCA cycle activity by altering the activities of key, rate-limiting and/or regulated enzymatic steps. We will further test this hypothesis by investigating the effects of ammonia and SCFA on (i) the activities of rate-limiting and/or regulated (pyruvate dehydrogenase complex (PDHC), citrate synthase, isocitrate dehydrogenase, Alpha-ketoglutarate dehydrogenase complex) and non-regulated (fumarase, malate dehydrogenase) enzymes in rat brain mitochondrial extracts, (ii) citrate-, isocitrate- and Alpha-ketoglutarate-supported oxygen uptake by metabolically competent rat brain mitochondria, and (iii) the [1-14C]pyruvate decarboxylation (i.e. flux through PDHC) by and the PDHC activation (i.e. phosphorylation) state in intact brain mitochondria. We will characterize the neuronal plasma membrane monocarboxylic acid carrier (MCC) by studying the substrate and inhibitor specificities of the MCC uptake mechanisms in rat brain synaptosomes. Then, the effects of ammonia and SCFA on synaptosomal pyruvate uptake will be investigated and the dose-effect relationships established. In addition, the toxin's effects on synaptosomal pyruvate metabolism ([1-14C]pyruvate decarboxylation) will be studied. The elucidation of these toxic mechanisms may facilitate the designs of more beneficial therapies for the encephalopathies in which hyperammonemia and organic acidemia are the persistent features.