2007 — 2009 |
Bayir, Hulya |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Role of Cardiolipin Oxidation After Traumatic Brain Injury in Immature Rat @ University of Pittsburgh At Pittsburgh
[unreadable] DESCRIPTION (provided by applicant): This is an exploratory R21 application in response to PA-06-181. Trauma is the leading cause of death in children. Severe traumatic brain injury (TBI) is an important participant in this mortality and associated morbidity. Apoptosis contributes to neuronal death TBI. The release of cytochrome c (cyt c) from inner mitochondrial space of the mitochondria is a critical early event in apoptotic cell death. Cyt c is bound to the inner mitochondrial membrane by its association with cardiolipin (CL), an anionic phospholipid found exclusively in the inner mitochondrial membrane of eukarydtic cells. Recently we have shown novel redox catalytic properties of cyt c realized though its interactions with CL and PS resulting in their selective oxidation. The resulting products, CL and PS hydroperoxides act as important signals in two apoptotic pathways - regulation of release of apoptotic factors from mitochondria into cytosol, and externalization of PS marking apoptotic cells for phagocytosis. Our hypothesis is that TBI initiates excessive production of ROS and oxidation of CL catalyzed by CL/cyt c complex, which is required for the release of pro-apoptotic factors from mitochondria. CL oxidation is catalyzed by a pool of cyt c that is tightly bound to inner mitochondrial membrane by its complex with CL in neurons. As a consequence, we predict that TBI induced CL oxidation and apoptosis can be prevented by antioxidant strategies and treatments decreasing susceptibility of CL to oxidation by dietary manipulation of its fatty acid residues. In this proposal we will use controlled cortical impact (CCI) model of TBI in post-natal day (PND) 17 rats coupled with in vitro studies to test these specific hypotheses and address the following specific aims: 1) Determine the degree, spatial and temporal pattern of ROS production, antioxidant depletion and CL oxidation in immature brain after TBI and in neurons after glutamate exposure. 2) Determine the potential of antioxidants and dietary manipulation to inhibit mitochondrial CL oxidation and protect against apoptosis in immature brain after TBI and in neurons after glutamate exposure. These studies will employ the newly developing technology of oxidative lipidomics to provide important mechanistic information on the role of cyt c -CL interactions in neuronal apoptosis after pediatric TBI in an experimental model. Relevant to the specific aims of this proposal in vivo studies linking overall CL oxidation with cyt c release and apoptosis have been lacking. The ability to selectively modulate Cyt c release could lead to targeted therapies for TBI and ultimately improve outcome for children. Traumatic brain injury remains a major cause of death and disability in infants and children in the US. We have found that a specific lipid molecule is very sensitive to oxidation during brain cell death and it plays an important role in releasing some of the proteins bound to this lipid from the energy producing organelle in the brain cells, which is necessary and sufficient to trigger cell death. We will use antioxidants and dietary manipulations to prevent oxidation of this lipid in rats that are comparable in age to young children and cultured brain cells. These experiments will help us to better understand how to relieve the harmful neurological consequences of traumatic brain injury and design targeted therapies to ultimately improve outcome for children. [unreadable] [unreadable] [unreadable]
|
1 |
2008 — 2021 |
Bayir, Hulya |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Oxidative Lipidomics in Pediatric Traumatic Brain Injury @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Each year in the US, severe TBI in children results in <7400 deaths and 60 000 hospitalizations. Fifty percent of surviving children with severe TBI have poor neurological outcome at six months. Severe TBI in children is thus a critical problem in desperate need of impactful therapies. Free radicals and oxidative stress have been accepted as universal pathogenic mechanisms of TBI prompting the therapeutic use of antioxidants. Invariably, the clinical trials of non-specific free radical scavengers/antioxidants failed. Unless the true sources and mechanisms of TBI redox imbalance are identified, the use of sacrificial antioxidants and free radical scavengers will fail. Thus, a paradigm-shifting approach is required. During prior funding period, we discovered that selective peroxidation of a mitochondria-specific phospholipid, cardiolipin (CL), occurs in severe pediatric TBI and represents a required mitochondrial stage of neuronal apoptosis. We further identified cytochrome c (cyt c) as a catalyst of CL peroxidation occurring via the formation of cyt c/CL complexes with peroxidase activity triggered by H2O2 . Thus cyt c/CL redox interactions and CL peroxidation represent a missing causal link between known reactive oxygen species production and mitochondrial pro-apoptotic responses. Importantly, our preliminary data show that a mitochondria-targeted small molecule inhibitor of CL peroxidation suppressed TBI-induced apoptosis in vivo and preserved cognitive function in postnatal day (PND) 17 rats. In normal mitochondria, CL and cyt c are physically separated: CL is confined almost exclusively to the inner mitochondrial membrane. Binding of cyt c with CL depends on the collapse of CL asymmetry and translocation of CL to the outer mitochondrial membrane. While the mechanisms of collapse of CL asymmetry are poorly understood, preliminary data show that two candidate proteins - nucleoside diphosphate kinase (NDPK-D) and phospholipid scramblase-3 (PLSCR3) -facilitate CL translocation. Our hypothesis is that collapse of CL asymmetry and formation of cyt c/CL peroxidase complexes trigger - in the presence of H2O2 - CL peroxidation and release of proapoptotic factors from mitochondria in immature brain after TBI. Thus, maintaining CL asymmetry and suppression of peroxidase activity will improve neuronal survival and outcome after TBI. To test our hypothesis and its successful translation, we will employ multi-disciplinary approach combining in vitro (mechanical stretch injury of neurons) and in vivo (controlled cortical impact in PND17 rats) TBI models, biochemical (oxidative lipidomics) and biophysical (new protocols for assessment of asymmetry and externalization of CL in mitochondria) methodology, genetic and pharmacological approaches as well as computational modeling and organic chemical synthesis. These studies will provide important mechanistic information on the role of CL asymmetry and oxidation in neuronal apoptosis after TBI. The ability to selectively modulate CL oxidation, a critical early event in apoptosis, could lead to targeted therapies for TBI and ultimately improve outcome for children after brain injury.
|
1 |
2010 — 2014 |
Bayir, Hulya |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Radiation Mitigators Based On Mnsod Responses to Oxidative and Nitrosadative Stre @ University of Pittsburgh At Pittsburgh
Mitochondrial protection against excessive superoxide production involves an elaborate antioxidant defense system, including that associated with manganese superoxide dismutase (MnSOD). Notably, MnSOD confined to mitochondria but not MnSOD genetically manipulated to be in the cytosol attenuates radiation induced cellular damage [2]. There are at least three possible ways to enhance MnSOD activity in the mitochondria: i) increase expression of the enzyme; ii) stabilize the enzyme against inactivation and prolong its life-time; iii) utilize SOD mimetics that are targeted to mitochondria. Increased expression of MnSOD has been shown to be radioprotective and this can be attained by gene therapy [3, 4] or by thiol compounds (such as WR-1065-the active thiol form of amitostine or N-acetly-cysteine) [5-7]. We have demonstrated significant radiation protection in rodent lung, esophagus, oral cavity, and intestine [3, 8-10] as well as after total-body irradiation (TBI) [11] by overexpression of MnSOD transgene prior to or after irradiation. However, gene therapy approaches will be difficult to administer to mass number of victims during a nuclear and radiological attack or accident. Therefore, we here propose to employ stabilized inactivation-resistant MnSOD and mitochondria targeted SOD mimetics as novel optimized mechanism-based radiomitigation strategies.
|
1 |
2012 — 2016 |
Bayir, Hulya Kagan, Valerian E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mapping Lipid Oxidation in Traumatic Brain Injury by Mass Spectrometric Imaging @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Lipid peroxidation contributes to the evolution of secondary damage in traumatic brain injury (TBI) however; essential information on molecular targets of oxidation is largely unknown. We reported that two anionic phospholipids-mitochondrial cardiolipin (CL) and extramitochondrial phosphatidylserine (PS) - are major targets of TBI-induced oxidation in brain. These oxidation reactions, catalyzed by complexes of cytochrome c (cyt c) with CL and PS, are associated with mitochondrial stages of programmed cell death and recognition of damaged cells by professional phagocytes, respectively. Studies in experimental TBI have revealed that the cortex, hippocampus and thalamus are selectively vulnerable to injury. However, information on spatial distribution of phospholipids and their oxidation products in various brain regions is lacking. The goal of this application is to fill thi gap of knowledge by developing and applying a new technology - imaging mass spectrometry (IMS) - for spatial and temporal mapping of diverse molecular species of phospholipids and their oxidation products and superimposing them onto neuropathology of the injured brain. This information will be critical for the design and development of targeted antioxidant therapies and evaluating their efficacy in TBI. We will use the high mass resolving power and measurement accuracy of Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS (Bruker Solarix) for a panoramic snap-shot of thousands of lipid signals simultaneously to obtain lipid maps of the brain. We will complement these studies by novel IMS technologies with improved spatial resolution: i) Matrix Assisted Laser Desorption Ionization-Postionization-Ion Mobility- orthogonal Time of Flight MS (MALDI-POST-IM-oTOFMS) with employment of nano-scale matrices; ii) oversampling-laser stepping MALDI-FTICR, and iii) micro-deposition of matrix. We will merge this information with fluorescent microscopic imaging to reveal structure and metabolic function of the vulnerable brain regions. This will be the first comprehensive lipidomics, oxidative lipidomics and IMS analysis of CL and PS in different brain regions. This enabling technology will resolve issues of spatial confinements of peroxidation reactions in lipids in the brain that cannot otherwise be readily examined. We will also examine brain tissue removed from TBI patients with refractory intracranial hypertension and brain-bank control tissue using oxidative lipidomics and IMS. As lipids and oxidized lipids are vital signaling molecules, the development of such technology and new information on the biochemistry of lipids should be of broad fundamental interest. Our progress with novel mitochondria targeted electron scavengers (gramicidin conjugated nitroxides) and inhibitors of cyt c/ CL peroxidase (triphenylphosphonium conjugated imidazole fatty acids) will facilitate our ability to pharmacologically delineate the roe of intracellular oxidized CL and PS in TBI. IMS used in a metabolomic mode towards these small molecules will reveal critical molecular pharmacologic information on the disposition and efficacy of these putative neuroprotectants against TBI. Overall, IMS technology and the underlying contribution of dyshomeostasis of mitochondrial CL and extramitochondrial PS are likely to be important for TBI studies and have implications for other CNS disorders.
|
1 |
2014 — 2018 |
Bayir, Hulya Clark, Robert S B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mitochondria-Targeted Redox Therapy For Cerebral Ischemia in the Developing Brain @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Brain damage after cerebral hypoxia-ischemia is a major contributor to death and disability in children. In fact, quality survival after brain injuryis the greatest irreversible unmet need in critically ill children, including those with co-morbiditie such as cancer. The most common cause of cerebral hypoxia-ischemia in infants and children is as a consequence of cardiac arrest; although, cerebral hypoxia-ischemia negatively impacts quality of life in many other diseases including traumatic brain injury, stroke, intracerebral hemorrhage, and inflammatory and neurodegenerative diseases. Disheartening morbidity or mortality with survivability directly related to the degree of hypoxic-ischemic encephalopathy (HIE)-and perceived futile care, are the most common outcomes. Robust therapies to prevent and/or treat cerebral hypoxia-ischemia after cardiac arrest and as a consequence of a host of other diseases are urgently needed. At the crux of hypoxia-ischemic injury, are mitochondria. After hypoxia-ischemia damaged mitochondria produce toxic free radicals that directly attack vital cellular constituents; are at the convergence of several critical cell death pathways; and ar powerful mediators of inflammation. Central to all of these potentially pathological mechanisms is the supraphysiologic generation of reactive oxygen species (ROS), making mitochondria-generated ROS a logical and potentially impactful therapeutic target for HIE. To date, strategies targeting ROS have focused on free radical scavengers or replacing endogenous antioxidants to quench these highly reactive compounds. Disappointingly, these strategies have not translated into efficacious treatments. A paradigm-shifting approach is needed, e.g. preventing generation of ROS, rather than attempting to quench them. Novel compounds that target mitochondria include therapeutic payloads conjugated with: i) chemical moieties utilized in antibacterial agents that have a high affinity for mitochondrial membranes, taking advantage of the shared ancestry between mitochondria and bacteria; or ii) a cationic moiety, taking advantage of electrophoretic properties and mitochondrial membrane potential. As a multidisciplinary team, we are in the fortunate position to synthesize and develop a library of promising nitroxide-based, mitochondria-targeting therapeutics that function primarily as electron scavengers-in contrast to traditional antioxidants, thus preventing formation of ROS. Furthermore, we are uniquely poised to test these powerful mitochondria- targeting therapies in our models of hypoxia-ischemia in the developing brain, including our clinically relevant model of pediatric asphyxial cardiac arrest. The aim of this research is to synthesize and develop novel mitochondria-targeting therapeutics, toward meaningfully improving neurological outcome and quality of life in infants and children suffering from cerebral hypoxia-ischemia.
|
1 |
2015 — 2019 |
Bayir, Hulya |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Radiation Mitigators Targeting Regulated Necrosis Pathways of Necroptosis and Ferroptosis @ University of Pittsburgh At Pittsburgh
Summary: There is emerging need in new medical products that can mitigate and/or treat the short- and long-term consequences of radiation exposure after a radiological or nuclear terrorist event that may cause a public health emergency. Ionizing radiation causes radiolysis of water with production of free radicals. While direct effects of ionizing radiation and water radiolysis products are realized primarily via apoptotic cell death pathways in rapidly proliferating cells within the initial 1-2 days after the exposure, the subsequent mechanisms of damage seem to be triggered by secondary responses whereby inflammatory cytokines, chemotactic factors and lipid mediators are the major players. At least two novel regulated necrotic cell death pathways, necroptosis and ferroptosis, are executed in response to inflammatory cytokines and lipid mediators. Necroptosis is dependent on receptor interacting protein kinase (RIPK) 1 and can be inhibited by Necrostatin- 1. Ferroptosis is an iron dependent cell death regulated by glutathione peroxidase 4 (Gpx4). The goal of our project is to decipher RIPK and Gpx4 driven signaling pathways associated with necroptosis and ferroptosis in mechanism-based discovery of radiomitigators based on our exciting Preliminary data: 1) discovery of radiomitigative potency of necrostatins (inhibitors of RIPK1) and a mitochondria-targeted Gpx4 mimic, Mito- Ebselen; and 2) detection of phospholipid oxidation/hydrolysis products, including cardiolipin-derived products ? during execution of ferroptosis in Gpx4 conditional knock out mice. During the last funding cycle we identified a new Ca2+-independent pathway for biosynthesis of lipid mediators after total body irradiation (TBI) with potential signaling roles in necroptosis and ferroptosis. Our central hypothesis is that necroptosis and ferroptosis are important pathogenic mechanisms of acute radiation injury syndrome triggered by pro- inflammatory responses (cytokines, lipid mediators) thus representing new targets for radiomitigation. Specific Aim 1 will perform detailed characterization of irradiation-triggered and pro-inflammatory cytokine- induced necroptosis and ferroptosis, including their association with phospholipid oxidation products and lipid mediators in the context of the design and development of new radiomitigators. Specific Aim 2 will explore the radiomitigative effectiveness of small molecule inhibitors of necroptosis. Specific Aim 3 will establish the contribution of ferroptosis to the pathogenesis of acute radiation injury and determine the effectiveness of several classes of ferroptosis inhibitors as radiomitgators. Experiments described in the above three Specific Aims include in vitro as well as in vivo studies with quantitation of both tissue and plasma signatures of regulated necrotic cell death pathways, cytokine and lipid mediators, and organ specific drug distribution. These studies will establish and optimize mechanism based novel therapies for radiation damage mitigation applicable for use in radiation counter terrorism.
|
1 |
2018 — 2021 |
Bayir, Hulya Kagan, Valerian E (co-PI) [⬀] Winograd, Nicholas (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lipid Imaging in Traumatic Brain Injury by High Resolution Gcib-Secondary Ion Mass Spectrometry @ University of Pittsburgh At Pittsburgh
Since the invention of microscopy and the initial observation of cells more than three hundred years ago, cell biology has been triumphant in detailed structural and functional characterization of intracellular organelles and macromolecular complexes. The realization that specialized bi-functional molecules, lipids, can form the aqueous interfaces of membrane structures has attracted attention to this group of intracellular compounds. Extensive biochemical studies discovered a huge diversification of lipids that could not be accommodated within a simple concept of their role as membrane building blocks. Indeed, numerous signaling functions of different lipid molecules, including membrane lipids, have been discovered. In spite of the very successful analytical work in biochemical characterization of the countless lipids, the exact intracellular topography of individual molecular species of lipids in the context of their signaling functions has not been established. The major reason for this was the lack of adequate technologies for high resolution imaging of small lipid molecules. The most recent developments of Gas Cluster Ion Beams Secondary Ion Mass Spectrometry (GCIB-SIMS) allows, for the first time, to fill this gap of fundamental knowledge in cell biology and develop a new type of microscopy ? biochemical microscopy of lipids ? that will create intracellular maps of individual lipids and their essential for life asymmetric distribution in biomembranes. Achievement of the goals of this innovative and paradigm shifting work will be based on highly interdisciplinary approaches and the leadership position of the three teams in their respective fields of analytical/physical chemistry of SIMS (at Pennsylvania State University, N. Winograd), lipid biochemistry/biology (at the University of Pittsburgh, V.E. Kagan), and traumatic brain injury (TBI) (at University of Pittsburgh, H. Bay?r. Aim 1 will employ high-resolution GCIB-SIMS to explore molecular speciation and construct cell-specific maps of CL and PE in neuronal, glial, and microglial cells in different anatomical regions of normal mouse brain. Aim 2 will identify TBI induced molecular alterations in cardiolipin (CL) and phosphatidylethanolamine (PE) in neuronal, glial, and microglial cells using GCIB-SIMS in mouse controlled cortical impact (CCI) model. We will further identify TBI induced changes in subcellular distribution of individual CL and PE species related to the execution of apoptotic or ferroptotic programs in the respective cells. We will be particularly interested in pro-apoptotic changes in mitochondrial CL and pro-ferroptotic changes in PE. We will also examine brain tissue removed from TBI patients with refractory intracranial hypertension and brain-bank control tissue. Aim 3 will determine the utility of GCIB-SIMS imaging in assessing the effectiveness of select anti-apoptotic and anti-ferroptotic small molecule regulators in preventing cell-specific changes in CL and PE molecular speciation after TBI. Proposed studies will decode specific features of topography of individual types of lipid molecules in cells and tissues and their role in signaling functions in health and disease.
|
1 |
2020 — 2021 |
Bayir, Hulya Clark, Robert S B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Druggable Mitochondrial Targets For Treatment of Cerebral Ischemia @ University of Pittsburgh At Pittsburgh
Quality survival after brain injury is currently the greatest challenge for critically ill or injured infants and children. A universal contributor limiting quality survivorship is the devastating impact of hypoxic-ischemic encephalopathy (HIE), either as a primary consequence in cases of cardiac arrest, stroke, or intracranial hemorrhage or as secondary sequelae in cases of status epilepticus, circulatory or septic shock, neuroinflammation, or traumatic brain injury (TBI); with the principal cause of HIE spanning from infancy through adolescence a consequence of cardiac arrest. As to-date a cure for HIE has not been discovered, a paradigm-shifting strategy is likely necessary to improve neurological outcome for victims of HIE. Accordingly, we have developed a new class of therapeutics to treat HIE via preservation of critical cellular energy stores by selectively targeting poly(ADP-ribose) polymerase (PARP) in mitochondria (mtPARP), linking the mitochondria-targeting moieties hemi-gramicidin S (XJB) or triphenylphosphonium (TPP) to PARP inhibitors used clinically. Ischemia-induced PARP overactivation triggered by DNA damage consumes NAD+, generating branch chain poly(ADP-ribose) polymers (PARylation) resulting in ATP depletion, energy failure, and cell death by necrosis and/or apoptosis-inducing factor (AIF)-mediated parthanatos. As mitochondria are the major source of ATP and NAD+ in aerobic organisms, preservation of mitochondrial energy stores represents a logical ?druggable? target for mitigation of HIE. We recently reported that the mitochondria- targeting PARP1 inhibitor XJB-veliparib preserves NAD+ stores and prevents neuronal death after oxygen- glucose deprivation (OGD) in vitro at nanomolar concentrations. Importantly, XJB-veliparib selectively targets mitochondria and thereby does not impede nuclear DNA repair in vitro. We present provocative pilot data suggesting that XJB-veliparib and the readily translatable mitochondria-targeting compound TPP-veliparib may be efficacious after cardiac arrest in post-natal day (PND) 17 rats, a developmental age equivalent to a young child and a time associated with peak cerebral metabolism. This new class of therapeutics has the advantage of preventing PARP-mediated energy failure and cell death by selectively targeting mtPARP while sparing PARP1-facilitated nuclear DNA repair and provide a tool to definitively establish (or refute) a role for mtPARP in the pathogenesis of HIE. If proven effective, mtPARP1 inhibitors would represent novel, safe (in terms of nuclear DNA repair), and translatable therapies to mitigate HIE, with special potential in the highly vulnerable, developing brain where metabolic rate is at its peak.
|
1 |
2020 — 2021 |
Bayir, Hulya |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Radiation Mitigators Targeting Regulated Necrosis Pathways of Parthanatos Pyroptosis @ University of Pittsburgh At Pittsburgh
Necrosis; Pathway interactions; radiation mitigator;
|
1 |