Mitochondria playa pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca2 + homeostasis and apoptosis. Given this multifactorial role, they regulate cellular Ca2+ metabolism and bioenergetics function as an integrated s~stem. In terms of normal physiology, this integration is reflected in mitochondrion's high capacity to store Ca2 +, which may protect cells like neurons against transient elevation in intracellular Ca2 + during periods of hyperactivity. Mitochondrial Ca2 + homeostasismust be tightly regulated and is based in a series of specific uptake and release systems. Yet, in vitro themitochondrial inner membrane (IMM) can easily undergo a permeability increase to solutes with molecularmasses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), isregulated by the opening of a membrane pore, the mitochondrial permeability transition pore (PTP). The PTPis voltage-dependent, cyclosporin A (CsA)-sensitive, high-conductance channel of the inner mitochondrialmembrane; pore open-closed transitions are highly regulated by multiple effectors that likely converge on asmaller set of regulatory sites. The PTP has long been implicated as a target for mitochondrial dysfunction invivo, particularly in the context of specific human pathological events. These suspicions have been confirmedby examination of mice in which the expression of mitochondrial CyPD (a key regulator of PTP action and thetarget of CsA) has been eliminated. These studies have confirmed a critical role for the PTP in models of ischemia/reperfusion injury both in the heart and the brain, models of muscular dystrophy, in the axonaldamage occurring during MS, and Alzheimer's disease. However, despite detailed functional characterizationover the last 30 years, none of the candidate pore components in traditional models has withstood critical andunambiguous genetic tests. In this light, the PBR remains the only biochemically identified component in traditional molecular models of the PTP that has not been subjected to thorough genetic testing. Consequently, the overall goal of this application is to use biochemical and genetic tools to critically test the role of the peripheral benzodiazepine receptor (PBR) in PTP function using a variety of in vitro and in vivo tests that we have developed to confirm its role. either as core components or regulators of the PTP. Our studies are based in mice that we have now successfully generated in which the wild-type Tspo gene has been replaced with a modified Tspo gene containing /oxP sites. Using these mice, our plan is to test PTP function in mitochondria, cells and tissues lacking the PBR and thereby rigorously evaluate the role of the PBR in PTP function. Importantly. since mice have now been successfully generated containing a modified Tspo gene containing loxP sites. it is reasonable that we will be able to complete these aims in the two years of funding provided.

Tests of the peripheral benzodiazepine receptor in the permeability transition pore will define therapies targeting this complex as treatments for a number of human diseases.