A NEW PROCEDURE TO ISOLATE BRAIN MITOCHONDRIA FROM HUMAN CORTEX AND ITS APPLICATION FOR LIPID ANALYSIS IN PHYSIOLOGICAL AGING

Many studies have revealed the importance of mitochondria as cellular organelles decisively involved in the onset or progression of neurodegenerative diseases, whose main risk factor is aging. Current protocols for brain mitochondria isolation have been developed to preserve viability, sacrificing the purity that is required to perform high-throughput biochemical analyses. My Phd project focused on the development of a new procedure to obtain a highly pure mitochondrial fraction starting from post mortem frozen tissues of human brain cortex of healthy subjects. The evaluation of mitochondrial enrichment and other cellular contaminants has been performed through different enzyme assays, western blot analyses and transmission electron microscopy. These validation experiments demonstrated the purity of mitochondria and their integrity, as well as the preservation of mitochondria-associated membranes. The brain aging process is allegedly responsible for chemical modification of lipids and changes in the lipid composition of cell membranes. In this scenario, there are no previous studies on human brain mitochondria lipids. Thus, this new method has been applied to investigate lipid composition of pure mitochondria by means of thin layer chromatography. Furthermore, we investigated if there were aging related changes in the lipid composition of these organelles essentials to cell life and death, since that could produce an impairment of the membrane function.