A1+3.CA+2 AND MITOCHONDRIA IN ALZHEIMER'S DISEASE

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The long range aim of the work is to understand the molecular mechanisms underlying Alzheimer's disease (AD) and age-related changes in the human brain. A number of studies have indicated that alterations observed in aging brains and in some neurodegenerative disorders, including AD, could be due to abnormal levels of free cytosolic CA+2 brought about by high levels of A1+3. The suggestion is that A1+3 or one of its derivatives can compete for Ca+2 binding sites on mitochondria causing release of the Ca+2 into the cytosol. The aim of this pilot study is to obtain enough preliminary data to justify a full scale proposal to study the effects of high levels of A1+3 on mitochondria and intracellular Ca+2 pools. As a test system, human fibroblasts derived from patients with familial and sporadic AD and from normal age and sex matched controls will be used. Fluorescent probes and quantitative video intensification microscopy will be used to determine whether exposure of cells to various concentrations of A1+3 can affect levels of membrane-associated and free intracellular Ca+2 and/or alter mitochondrial function. The probes to be used are: chlorotetracycline, an indicator of membrane-associated Ca+2; quin2, a probe for free cytosolic Ca+2; and rhodamine 123 whose specific staining of mitochondria is thought to be dependent upon mitochondrial membrane potential. A positive result from these experiments would provide a basis for a later extension of this work into the effects of A1+3 on neural cells in culture. Such studies and their extensions should increase our understanding of the neurotoxic effects of A1+3 and may eventually serve to delineate the effects of alterations of mitochondria and Ca+2 regulation on cells of the nervous system. They may also help provide an understanding of the mechanisms behind AD and age related changes in the human brain.