Cardiac arrest and stroke are leading causes of death and disability in the US. Stopping blood flow to the brain sets in motion cellular events that resut in delayed death of neurons. Mitochondrial dysfunction leads to release of cytochrome c (cyto c) from the mitochondria and is the final committed step of neuronal death. However, the specific molecular events underlying cyto c release remain unclear. Mitochondria undergo a balance of fusion and fission events in response to changes inside the cell. During reperfusion we discovered loss of a key inner mitochondrial membrane (IMM) protein Opa1, which is critical to both maintaining the structural integrity of the IMM and mitochondrial fusion. We hypothesize that, following brain ischemia, mitochondrial dynamics defaults to increased mitochondrial fission as a result of impaired fusion, thus releasing cyto c from mitochondria, and ultimately cel death. In support of our hypothesis, our preliminary data demonstrate: 1) there is a large increase in mitochondrial fission following brain ischemia with associated cyto c and Opa1 release; and 2) IMM integrity is lost during reperfusion which coincides with Opa1 cleavage and Opa1 complex breakdown. We propose to comprehensively study mitochondrial dynamics and loss of IMM integrity first utilizing our cultured primary neuron model of transient ischemia to define molecular interactions and assign causality to these events. We will then pharmacologically modify mitochondrial dynamics as a potential target for therapeutic intervention using a rodent model of global brain ischemia. Specifically, we will: Interrogate the role of increased mitochondrial fission in cell death following ischemia/reperfusion (Aim1). Mitochondrial dynamics will be targeted using genetic and pharmacologic manipulation of key molecular targets and ischemia/reperfusion will be induced in a primary neuron model. Elucidate the causal relationship between Opa1 complex breakdown, inner mitochondrial membrane dysfunction, and loss of mitochondrial apoptogenic factors (Aim 2). We will genetically block Opa1 complex breakdown in primary neurons and subject them to simulated ischemia/reperfusion, we will also examine inner membrane lipid alterations that lead to Opa1 dissociation, Oma1 knockout mice will be subjected to ischemia/reperfusion, and ROS damage will be assessed to elucidate the mechanism of Opa1-mediated mitochondrial dysfunction.
In Aim 3, we will test a therapeutic intervention for global brain ischemia utilizing a pharmacologic agent directed at mitochondrial dynamics. Ischemia-reperfusion will be induced using our established rodent model and mitochondrial fusion will be promoted using a Drp1 inhibitor.
Cardiac arrest and stroke continue to be leading causes of death and disability in the US. Mitochondrial dysfunction and the release of cytochrome c play a central role in the ischemic death of neurons. Our objective is to elucidate the molecular events underlying mitochondrial dysfunction and cytochrome c release and ascertain a targeted-therapy to prevent death of neurons following brain ischemia and reperfusion.
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