Accumulation of Ca 2+ within mitochondria regulates both cell survival and cell death. Cell survival is mediated by Ca 2+ stimulated ATP production at low levels of mitochondrial Ca 2+ accumulation. In contrast, programmed cell death (apoptosis) can be initiated at high levels of mitochondrial Ca 2+ accumulation. The goal of this research is to understand the regulation of the mitochondrial permeability transition pore (mPTP), which plays a key role in determining whether mitochondria enhance cell survival or initiate cell death.
The Specific Aims are: 1) To investigate the physiological mechanism(s) by which Cyclophilin D (CyPD) regulates the opening of the rnPTP and to examine the modulation of this process by IP3-mediated Ca 2+ release, 2) To examine the role of the adenine nucleotide translocator (ANT) in CyPD oxidant protection and determine whether the sensitivity of mPTP formation to oxidant stress and Ca 2+ resides in the ANT, 3) To examine the role of molecular chaperone activity in CyPD protection of cells against oxidative stress, and 4) To examine how manipulation of CyPD protein levels affects resistance to oxidative stress and lifespan in two whole-animal model systems, C. elegans and mice. Single photon and two-photon fluorescent imaging techniques will be used to measure Ca 2+, membrane potential and respiration in these experiments. Mitochondrial respiration will also be measured by oxygen consumption in intact cells. Recombinant DNA techniques will be used to introduce point mutations and transiently transfect HEK293 cells as well as cultures of primary astrocytes isolated from transgenic and knockout mice. Small interference double-stranded RNA (siRNA) will be used to down-regulate target genes. The significance of these studies is threefold. First, control of intracellular Ca 2+ and ATP production are central to many cell signaling pathways, including cell growth, maintenance and lifespan. Second, mitochondria are known to play key regulatory roles in apoptosis. Finally, dysfunctional mitochondria, which accumulate with age and/or oxidative stress are promising targets for therapeutic intervention in degenerative diseases.
