Regulation of membrane transport by cardiolipin

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Cardiolipin (CL) is a key mitochondrial membrane phospholipid required for the generation of ATP. Upon its synthesis CL is remodeled with linoleic acid to form tetralinoleoyl-cardiolipin (L4-CL) by the enzyme tafazzin. Reduction in CL and/or alteration in its fatty acid composition results in reduced ability to generate ATP. We hypothesize that the appropriate content and fatty acid composition of CL is required for optimal solute transport and transcellular transport across biological membranes. Specific Objective 1: Determine if cardiolipin regulates solute and drug transport across cell membranes in vitro. CL levels in hCMEC/D3 cells will be lowered using siRNA knock down of CL synthase (hCLS1). Mitochondrial energy metabolism in control and cells with reduced CL will be determined and the level and transport ability of solute, drug efflux transporters, fatty acid transporters as well as tight junction proteins and inflammatory markers will be examined. The membrane permeability and uptake of radiolabeled deoxyglucose, radiolabeled fatty acid and rhodamine 800 will be examined. We anticipate that mitochondrial function including basal oxygen consumption, glycolysis rate, respiratory capacity and ATP turnover will be lower in hCMEC/D3 cells with reduced CL levels and that reduction in CL in hCMEC/D3 cells will result in reduced fatty acid, glucose, creatine and rhodamine 800 transport into hCMEC/D3 cells. We will culture hCMEC/D3 cells on Transwell® plates and measure apical to basolateral transport of radiolabeled glucose, creatine, oleate, and transport of rhodamine 800. It is anticipated that reduction in CL in hCMEC/D3 cells cultured on Transwell® plates will result in reduced mitochondrial function, reduced fatty acid, glucose, creatine and rhodamine 800 transport into cells. CL levels will be restored by expressing hCLS1 in cells in which CL levels were reduced by hCLS1 knock down and all of the above parameters examined. In addition, as corollary experiments, CL levels will be elevated by expressing hCLS1 in hCMEC/D3 cells and the above parameters examined. We anticipate that the appropriate content of CL is required for proper function of solute and drug transport processes. Specific Objective 2: Determine if cardiolipin regulates solute and drug transport across the blood brain barrier (BBB) in vivo. Whole body L4-CL levels will be reduced by knock down of tafazzin in mice and the transport and permeability of radiolabeled mannitol, and Evan’s blue dye and rhodamine 800 permeability will be temporally determined from these and control animals. Microvessel capillary segments will be isolated from brains and mitochondrial function determined. In addition, magnetic resonance imaging with Gad-DTPA contrast enhancement will be used to examine transport across the BBB in vivo. L4-CL levels will be restored in mice in which tafazzin was knocked down and the above parameters examined. We anticipate that mitochondrial function in microvessel capillary segments, transport and permeability of radiolabeled mannitol and rhodamine 800 and Gad-DTPA permeability will be reduced by tafazzin knock down and that this can be reversed by restoration of normal L4-CL levels.The proposed studies will identify important transport/modulation pathways that can be regulated by mitochondrial function through changes in CL. These studies will create a new research program with the long-term vision of characterizing solute and drug membrane transport and determining how mitochondrial function mediated through CL can influence this and potentially aid in drug development for regulation of membrane function. These biological studies are of fundamental importance in drug development.