Parkinson s disease (PD) is a neurodegenerative disorder that affects one million people in North America, which is likely caused by exposure to as yet unknown chemicals in the environment. The hallmark of PD is loss of dopaminergic neurons in the substantia nigra pars compacta. One chemical known to produce PD-like pathology in vivo is 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). My overall goal is to understand the role of the mitochondrial permeability transition in MPP+-induced dopaminergic cell death. In cultured rat pheochromocytoma-12 (PC12) dopaminergic cells, I will evaluate the following hypotheses: 1) MPP+ causes a mitochondrial permeability transition by promoting opening of permeability transition pores in the inner mitochondrial membranes. The mitochondrial permeability transition leads to increased ion permeability, collapse of the mitochondrial membrane potential and uncoupling of oxidative phosphorylation. These changes further cause large amplitude mitochondrial matrix swelling leading to rupture of the mitochondrial outer membrane, release of pro-apoptotic mitochondrial proteins from the intermembrane space, such as cytochrome c, and apoptotic cell death. According to this hypothesis, interventions that block the mitochondrial permeability transition prevent all these MPP+-induced events. To address this hypothesis directly, I will monitor mitochondrial membrane permeability, mitochondrial membrane potential, mitochondrial swelling and cytochrome c release by confocal microscopy of parameter-indicating fluorophores and transfected green fluoroscent protein fusion proteins in relation to onset of apoptotic death during exposure to MPP+ in single living cells in situ. I will also determine whether anti-apoptotic proteins, such as Bc1-2, prevent onset of the mitochondrial permeability transition and subsequent mitochondrial outer membrane breakage, cytochrome c release and apoptosis. 2) Excitotoxicity contributes to the MPP+-induced onset of the mitochondrial permeability transition by increasing mitochondrial Ca2+ and oxygen radical formation. To test this hypothesis, I will transfect PC12 cells with fully functional N-methyl-D-aspartate receptors. I expect that overstimulation of these receptors will increase mitochondrial free Ca2+, leading to Ca2+-dependent formation of reactive oxygen species and acceleration of the onset of the mitochondrial permeability transition, depolarization, cytochrome c release and apoptosis. The findings obtained with PC12 cells will be further translated to dopaminergic neurons isolated from the substantia nigra. These experiments will yield new information to improve therapy of Parkinson s disease and to prevent the disease s progression.
