Grant: Discovery Grants Program - Individual

Cardiolipin (CL) is a key mitochondrial membrane phospholipid required for many cellular functions. Reduction in CL mass by inhibition of its de novo biosynthesis or through loss of the CL remodeling enzyme tafazzin results in reduced mitochondrial function. I hypothesize that CL is required for optimal solute transport and for transcellular transport across biological membranes. Specific Objective 1: Determine the mechanism of how CL regulates solute transport across cell membranes in vitro. We previously demonstrated that reduced CL in blood brain barrier hBMEC/D3 cells increases transport of glucose into and across monolayers of these cells. To further explore how CL regulates solute transport across cell membranes we will reduce CL levels in hCMEC/D3 and examine the transport of solutes and the expression of drug efflux and solute transport proteins. We will culture hCMEC/D3 cells on Transwellr plates and measure apical to basolateral transport of solutes. We will examine how reduced CL modulates transport through protein kinase C epsilon (PKCe) in these cells. We anticipate that reduction in CL will result in altered solute transport into and across hBMEC/D3 cells and that PKCe activation participates in this process. Preliminary data indicate that creatine transport and mRNA expression of the drug efflux proteins P-glycoprotein and breast cancer resistance protein are reduced when CL is lowered in hCMEC/D3 cells. Specific Objective 2: Determine how CL regulates transport across the blood brain barrier in vivo. We previously demonstrated that knock down of tafazzin in the brain of mice reduces CL. We will temporally examine transport and permeability of radiolabeled mannitol, Evan's blue dye and rhodamine 800 into brain microvessel capillary segments isolated from control and tafazzin knock down (TazKD) mice. We will also use magnetic resonance imaging with Gad-DTPA contrast enhancement to examine transport across the in vivo blood brain barrier. CL levels will then be restored in TazKD mice and the above parameters examined. We anticipate that transport and permeability of radiolabeled mannitol, Evan's blue dye and rhodamine 800 in microvessel capillary segments and Gad-DTPA in vivo permeability will be altered in TazKD mice compared to control animals and that this can be reversed by restoration of normal CL levels. We will confirm the in vivo mechanism by examining how PKCe activation is altered in the brain of TazKD mice. The short term research program will identify important membrane transport pathways that are regulated by CL and how changes in CL regulate them. The long-term vision of the research program is to determine how changes in CL can potentially aid in the movement of compounds such as drugs across biological membranes. Given the central role of CL in regulating cellular function these salient biological and biochemical studies are fundamental to our understanding of the basics of membrane transport processes.