Donald J. DeGracia - US grants

DESCRIPTION (provided by applicant): Brain ischemia and reperfusion injury prevents greater than 90% of the 70,000 patients per year resuscitated from cardiac arrest from resuming their normal lives. Our long-term goal is sufficient understanding of the injury mechanisms to formulate clinically effective therapy. Inhibition of protein synthesis during brain reperfusion correlates with regional selective vulnerability and neuronal death, and is due to modification of two translation initiation factors: the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), and the proteolytic fragmentation of eukaryotic initiation factor 4G (eIF4G). eIF2 phosphorylation and eIF4G fragmentation affect not only the overall protein synthesis rate, but also which peptides are synthesized from the available mRNAs. Moreover, the kinase that phosphorylates eIF2alpha immediately after brain ischemia and reperfusion, PERK, is known to be activated only by the endoplasmic reticulum stress signaling system termed the unfolded protein response (UPR). The UPR can signal either an adaptive pro-survival response, or it can trigger cell death. Thus suppression of protein translation is likely to be part of a more comprehensive cellular response that determines the ultimate fate of reperfused neurons. We hypothesize: (1) the UPR is activated during early brain reperfusion, (2) vulnerable, but not resistant, neurons fail to resolve the UPR, and (3) there is synthesis of only a limited number of proteins during early reperfusion, as a consequence of eIF2alpha phosphorylation and eIF4G fragmentation, that may determine the outcome of neuronal recovery or death. Our Specific Aims are the following. Aim I will compare in ischemia and reperfusion vulnerable and resistant brain regions the activation of the UPR by characterizing activation of its three effectors ATF6, IRE1alpha, and PERK. Aim 2 will examine in ischemia and reperfusion vulnerable and resistant brain regions whether the UPR is resolved (by determining if synthesis of the pro-survival proteins GRP78, XBP-1, GADD34 and SERCA2b occurs), or if the UPR fails to resolve (by determining if synthesis of the pro-cell death proteins ATF4 and CHOP occurs). Aim 3 will identify those proteins being synthesized by residual translation during the early hours of reperfusion and compare them between ischemia and reperfusion vulnerable and resistant brain regions. This approach provides an integrated examination during brain ischemia and reperfusion of: (1) the occurrence and the consequences of UPR activation, (2) the consequences of translation initiation factor alterations on residual protein synthesis, and (3) the relationship of these two events to the selective vulnerability of the brain to ischemia and reperfusion injury.

[unreadable] DESCRIPTION (provided by applicant): Ischemia and reperfusion (I/R) injury of the brain occurs following resuscitation from cardiac arrest and stroke, and results in high morbidity and mortality. There is no clinically effective treatment because of an incomplete understanding of the cellular injury cascades leading to cell death. The long- term goal of my laboratory is to investigate the mechanisms of neuronal death caused by brain I/R to allow for the development of effective treatments. There is a striking correlation between protein synthesis inhibition and the selective death of hippocampal CA1 pyramidal neurons following transient global brain I/R. The mechanism of this irreversible translation arrest and its relationship to cell death is unknown. Stress granules are cytoplasmic particles that sequester inactive translational machinery during cellular stress. We present compelling evidence that stress granule alterations are central to persistent translation arrest in ischemic-vulnerable hippocampal CA1 neurons. Our Specific Aims are: 1. To investigate the mechanism of irreversible translation arrest. We will analyze the functional composition of stress granules utilizing complementary microscopic and biochemical approaches. We will compare stress granules in ischemic resistant CA3 and ischemic vulnerable CA1 from early reperfusion to the point of cell death of vulnerable neurons. 2. To identify the effect of ischemic preconditioning (IPC) on stress granule composition and behavior in reperfused neurons. IPC prevents both cell death and persistent translation arrest in vulnerable CA1 neurons. We will assess the effect of IPC on stress granule behavior and composition, protein synthesis rates, and cell death in CA1 neurons. 3. To show that persistent translation arrest is causally related to neuronal death following brain I/R. Antibiotic protein synthesis inhibitors will be used to predictably alter stress granules in reperfused hippocampal neurons, and we will examine the effect on protein synthesis rates, stress granule composition and behavior and cell death in reperfused hippocampal neurons. By providing an integrated examination of the relationship between persistent translation arrest and I/R-induced cell death, our Specific Aims address a problem that has been a barrier to progress in the field: how irreversible inhibition of protein synthesis in reperfused neurons causes cell death. PUBLIC HEATH RELEVANCE: Every year, millions of people are injured or die from brain damage caused by cardiac arrest or stroke. There are no treatments to prevent this brain damage because physicians and scientists do not understand how the cells die following a period when blood has stopped flowing in the brain and subsequently resumed. The work in this proposal seeks to further our understanding of the way in which neurons in the brain die following a period of low or no blood flow (ischemia) followed by resumption of normal blood flow (reperfusion). [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Human brain ischemia and reperfusion (I/R) occurs clinically following stroke and cardiac arrest. Over 100 stroke and cardiac arrest clinical trials f neuroprotection have completely failed. This spectacular failure suggests there may be something fundamentally wrong with the current understanding of brain I/R. The current model of I/R injury, the ischemic cascade, is a list of intracellular pathways induced in neurons by I/. We suggest that the failure of this viewpoint may rest in the assumption that a linear sequence of molecular changes causes outcome. We offer a novel, alternative, systems biology view of cell injury that posits all injury-induced changes in the cell contribute causally to outcome. Causality is not due to the action of linear pathways but to a nonlinear network of interactions. The theory is expressed by nonlinear ordinary differential equations. We seek to operationalize this theory by assessing means of measuring the differential equation variables. We propose the theory variables can be obtained from proteomic and transcriptomic data treated as quantitative deviations in the total amount of molecular damage and molecular stress responses from the control state. We propose to measure these distances in two classes of neurons, CA1 and CA3, using a well-studied model of global brain I/R in the rat.