Patrick G. Sullivan, Ph.D. - US grants

DESCRIPTION (provided by applicant): This application is for a bi-institutional collaborative Exploratory/Developmental Award in Epilepsy Research for Junior Investigators. The PI is an Assistant Professor at the University of Kentucky, and although published extensively in the field of mitochondrial bioenergetics and mitochondria-mediated cellular injury and death, he is relatively new to the field of epilepsy research. During a post-doctoral fellowship, the PI initiated an innovative collaboration with an established epilepsy researcher at the University of California at Irvine. The co-PI is an expert on mechanisms underlying the anticonvulsant actions of the ketogenic diet (KD), an effective non-pharmacological treatment for medically refractory epilepsy. The KD is a high-fat, low-carbohydrate/low-protein diet designed to reproduce the early biochemical changes seen upon fasting. Despite decades of successful clinical experience with the KD, the mechanisms underlying its anticonvulsant actions remain poorly understood. It is well known that fasting increases peripheral mitochondrial uncoupling protein (UCP) activity. However, there are no data addressing the effects of a KD on brain mitochondrial uncoupling. In preliminary studies, we have found that a KD enhances mitochondrial uncoupling and decreases reactive oxygen species (ROS) production in normal mouse cortex. The fundamental goal of the proposed studies is to determine whether a KD decreases mitochondrial oxidative damage in the hippocampus of developing epileptic mice (i.e., the Kcnal-null mutant), and following acute excitotoxic insult in normal mice. Specifically, we hypothesize that a KD increases UCP-mediated mitochondrial uncoupling and reduces subsequent ROS formation in epileptic hippocampus. Additionally, we hypothesize that the KD reduces mitochondrial dysfunction, lipid peroxidation and protein oxidation following kainic acid-induced seizures. Our preliminary data in the kainic acid model strongly suggest a direct neuroprotective effect of the KD, not related to seizure severity between groups of control diet- vs. KD-treated animals. The results of these studies will shed light on whether a KD reduces oxidative stress in a genetic model of developmental epilepsy, as well as in a well-established excitotoxic model. The clinical importance of such findings is that this therapy may ameliorate the epileptic condition itself, and not merely halt spontaneous recurrent seizure activity.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a devastating healthcare problem in the United States, however, there are currently no pharmacological treatments approved for the clinical treatment of this condition. Compelling experimental data demonstrates that mitochondrial dysfunction is a pivotal link in the neuropathological sequalae of brain injury. This proposal focuses on mild mitochondrial uncoupling as a novel therapeutic intervention following traumatic brain injury. The premise being that TBI-induced increases in mitochondrial Ca2+ cycling/overload ultimately lead to mitochondrial dysfunction. Mitochondrial uncouplers are compounds that facilitate the movement of protons from the mitochondrial inner-membrane space into the mitochondrial matrix. Uncoupling can also be mediated via the activation of endogenous mitochondrial uncoupling proteins (UCP) that can be modulated by fasting. While long-term, complete uncoupling of mitochondria would be detrimental, a transient or "mild uncoupling", could confer neuroprotection. Mild uncoupling during the acute phases of TBI would be expected to reduce mitochondrial Ca2+ uptake (cycling) and ROS production. The proposed experiments are designed to test the novel hypothesis that mild mitochondrial uncoupling is neuroprotective following traumatic brain injury. Specifically we will determine 1) if mitochondrial uncouplers increase tissue sparing and improve behavioral outcome following TBI 2) if mitochondrial uncouplers maintain mitochondrial integrity and bioenergetics following TBI and 3) examine the mechanism(s) underlying the neuroprotection afforded by fasting following traumatic brain injury. The experiments will determine the optimal dose and time post-injury to administer uncouplers to afford optimal neuroprotection and reduce cognitive defects following a mild or severe TBI in rats. Next we will examine mitochondrial function following mild or severe TBI in rats to determine if mitochondrial uncouplers maintain mitochondrial integrity. Finally, using a reductionist approach, we will employ several strategies including the use of UCP-2 transgenic mice, insulin-induced hypoglycemia and ketone administration to determine specific mechanisms involved in fasting-mediated neuroprotection following TBI. The proposed experiments may pinpoint important mitochondrial events that could be potential novel targets for the treatment of TBI and perhaps, other acute neuronal injuries.

bioimaging /biomedical imaging; brain injury

[unreadable] DESCRIPTION (provided by applicant): The Kentucky Spinal Cord & Head Injury Trust (KSCHIRT) Symposium is an annual meeting of neuroscientists, clinicians, research fellows and graduate and medical students who are involved in research and treatment of patients suffering from the effects of central nervous system (CNS) trauma. The KSCHIRT Symposium is fast becoming a premier annual meeting of the Neurotrauma community. This Symposium provides basic scientists and clinicians a rare opportunity to meet and discuss questions related to the neuropathophysiology of brain and spinal cord injury, the latest in neuroprotective strategies as well as mechanisms of recovery. This symposium is unique not only the subject matter covered but the diversity of the attendees. The KSCHIRT Symposium is organized on a biyearly basis by the University of Kentucky Spinal Cord & Brain Injury Research Center (SCoBIRC) or by our sister organization, the Kentucky Spinal Cord Injury Research Center at the University of Louisville. The goal of the University of Kentucky SCoBIRC is promote research on injuries to the spinal cord and brain that result in paralysis or other loss of neurological function. The 2008 14th annual KSCHIRT Symposium will focus specifically on the most cutting edge areas of central nervous system injury and repair including the latest breakthroughs in neuroprostheses. The Specific Aims of the 14th annual Kentucky Spinal Cord & Head Injury Trust Symposium are: 1. To provide a forum for the presentation, discussion and feedback regarding the most recent findings in Neurotrauma research and to encourage extensive interaction between those new to the field and those with extensive experience of neurotrauma. 2. To have relevant and thought-provoking presentations by researchers not directly involved in Neurotrauma research. The goal is to foster new ideas and ways of thinking into mainstream Neurotrauma as well as to encourage scientist from other disciplines to engage in Neurotrauma research. 3. To encourage participation and education of students that are entering or studying the field of Neurotrauma. The Symposium fosters student participation through poster sessions, meet and greets with the speakers, and subsidized student registration fees. 4. To encourage the involvement of researchers from women, minority groups, and persons with disabilities in neurotrauma research. The program for the 2008 symposium will feature 16 lectures and 2 dedicated posters. Their also dinner that is served the first night of the conference with seating that places students and fellows at tables with the speakers. Lunch is also arranged in this fashion. PUBLIC HEALTH RELEVANCE: The direct relevance of this project to public health rests in the exchange of new ideas and the debate generated regarding advancing laboratory findings into clinical practice. Students, basic scientists, clinician-scientists, and practicing clinicians are brought together to present and discuss their latest discoveries and advancements in the field in the field of Neurotrauma. Particular efforts are made to actively engage students to participate and interact with leading basic scientists and clinicians in the Neurotrauma field of research. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this research is to minimize the secondary cell death and resultant morbidity following traumatic brain injury (TBI). Clinical interventions to improve outcome following TBI are extremely limited. Mitochondrial dysfunction is a pivotal link in the neuropathological sequalae of traumatic brain injury (TBI). TBI-induced increases in mitochondrial Ca2+ cycling/overload ultimately lead to opening of the mitochondrial permeability transition pore (mPTP). This pore, located on the inner mitochondrial membrane, opens in response to elevated Ca2+ and oxidative stress, and is gated by the mitochondrial protein cyclophilin D (CypD). When prolonged, mPTP opening is catastrophic as a result of the loss of mitochondrial membrane potential, and the release of calcium and death-related proteins from mitochondria. The goal of this research is to minimize the secondary cell death and resultant morbidity following TBI by limiting mPTP opening. The immunosuppressant Cyclosporin A (CsA) inhibits mPTP opening by binding to CypD. We and others previously demonstrated that CsA reduces the extent of tissue damage when administered following experimental TBI. Unfortunately, CsA is toxic at high concentrations, resulting from its inhibition of calcineurin. Recently we have demonstrated that CypD levels in primary neurons are approximately double the levels found in astrocytes and that CypD levels are also significantly higher in synaptic mitochondria (neuronal origin) compared to non-synaptic mitochondria (predominately non-neuronal origin). As a result of their high CypD content, we hypothesize that neuronal mitochondria are more vulnerable to mPTP opening and also require greater CsA levels to inhibit mPTP opening. To test this hypothesis we propose to use genetic and newer pharmacologic approaches to inhibit CypD following neuronal injury. Specifically, we will use CypD knockout mice and a CsA derivative, NIM811, which does not bind to or inhibit calcineurin. The specific aims are: 1: To evaluate the hypothesis that the high CypD content of neuronal mitochondria enhances vulnerability to mPTP opening following insults that result in elevated intracellular Ca2+ and oxidative stress. 2: To evaluate the hypothesis that the levels of the CypD inhibitor NIM811 required to protect neurons from excitotoxic insult is proportional to their CypD content. 3: To examine the hypothesis that mitochondrial CypD levels modulate the neuropathologic and functional outcome following TBI in mice. 4: To examine the hypothesis that CypD inhibitor NIM811 reduces tissue damage and improves functional outcome following TBI. Based on the results of these studies, we anticipate that NIM811 will exhibit strong potential as novel therapy for TBI. PUBLIC HEALTH RELEVANCE: Traumatic brain injury (TBI) is a devastating healthcare problem in the United States, with no pharmacological treatments currently approved for clinical intervention following injury. Improved TBI treatment options are urgently needed. This proposal examines the potential of NIM811, a derivative of CsA, to limit the brain damage and dysfunction resulting from TBI.

The Microscopy, Image Analysis and Stereology Core, located on the BBSRB fourth floor, provides advanced instrumentation required for neurotrauma and other neuroscience research, including two Olympus Provis AX70 Fluorescence Microscopes (Image Pro interfaced via MagnaFire Digital Camera), an Olympus BX 41 with Video Camera, an Olympus BX 50 Epifluorescent Microscope (Bioquant Stereology package + 3D topographer interfaced with MagnaFire Digital Camera and Optronics Video Camera), a Zeiss Axiovert 200 M microscope, and a Nikon E400 teaching microscope with DAGE Video Camera. In addition to this existing equipment, SCoBIRC recently invested in three new specialized microscopes with funding from the NIH. The first, an Olympus FluoViewTM 300 point-scanning, point-detection, confocal laser scanning microscope designed for biology research applications, has excellent resolution, efficiency of excitation, and an intuitive, user-friendly interface including preset parameters for individual users. The FluoViewTM 300 is also capable of 3D rendering and time-lapse observation, and TTL I/O signals can be generated to coordinate experiments timed with external instrumentation. Additionally, two high-sensitivity photomultiplier tubes (PMTs) are located directly within the FV300 confocal fluorescence emission light path for high sensitivity detection of the fluorescence signal. The second stereology microscope is an inverted Olympus IX 81 outfitted with motorized stage encoders and the BioQuant Nova Prime software, particulariy well suited for quantification using modern stereological tools. The core is staffed by a Core Director, Patrick Sullivan, Ph.D., SCoBIRC Associate Director and Associate Professor of Anatomy & Neurobiology and an Assistant Director, Alexander Rabchevsky, Ph.D., Associate Professor of Physiology both of whom are well versed in the use of multiple image analysis endpoint measures and confocal imaging as endpoints in theirresearch. Both have expertise in developing protocols used to assess tissue sparing and cell loss in neurotrauma research.

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is a long-term health care problem in the United States, and with the exception of the modestly effective methylprednisolone, there is currently no neuroprotective intervention clinically available for treatment of acute SCI. Our published data and preliminary results demonstrate that oxidative damage to key mitochondrial enzymes and subsequent mitochondrial dysfunction is key to the neuropathological sequalae following SCI. This proposal focuses on directly targeting mitochondrial dysfunction as a novel therapeutic intervention for contusion SCI, the fundamental concept being that SCI-induced excitotoxicity increases mitochondrial Ca2+ cycling/overload and the production of reactive oxygen species (ROS), ultimately leading to mitochondrial dysfunction and glutathione (GSH) depletion. Our approach is two-pronged, aimed at reducing mitochondrial ROS production utilizing a novel, cell-permeant antioxidant and GSH precursor, NACA (the amide form of N-acetylcysteine), as well as an alternative biofuel substrate for energy production, acetyl-l-carnitine (ALC), following SCI. Our published and preliminary data signify that both NACA and ALC improve mitochondrial bioenergetics following contusion SCI in rats, and that prolonged NACA or ALC treatment increases tissue sparing following injury. The planned experiments are designed to test the novel hypothesis that reducing oxidative damage to key mitochondrial proteins will maintain mitochondrial bioenergetics, thus leading to increased neuroprotection and improved functional recovery following contusion SCI. Specifically we will: 1) Characterize oxidative damage to specific mitochondrial proteins involved in bioenergetics and test the hypothesis that NACA treatment ameliorates mitochondrial oxidative damage following SCI, 2) Test the hypothesis that a combinatorial treatment with NACA and ALC will act synergistically to preserve mitochondrial homeostasis following SCI, and 3) Test the hypothesis that a combinatorial treatment with NACA and ALC will increase tissue sparing and promote long-term functional recovery following SCI. Critically, this application is built around the utilization of several novel techniques we have developed for isolating synaptic (neuronal) and non-synaptic (soma and glia) mitochondria from the injured spinal cord, as well as an L1/L2 contusion SCI paradigm that demonstrates a significant correlation between neuroprotection and remarkable improvements in recovery of hind limb function. Collectively, the proposed experiments will pinpoint key mitochondrial events that could be potential novel targets for pharmacological interventions to more effectively treat SCI and, perhaps, other CNS injuries.

Traumatic brain injury (TBI) results in cognitive impairment, which can be long-lasting after moderate to severe TBI. Currently, there are no FDA-approved therapeutics to treat the devastating consequences of TBI and improve recovery. A wealth of experimental evidence shows that mitochondrial dysfunction is poised to be a pivotal link in the neuropathology of brain injury. We previously have targeted bioenergetic impairment with mitochondria-directed therapeutics, including mild mitochondrial uncouplers, which have shown to be neuroprotective. These uncouplers facilitate the movement of protons from the mitochondrial inner-membrane space into the mitochondrial matrix, thereby reducing the mitochondrial membrane potential (??). While complete uncoupling of mitochondria would be detrimental, we have published data showing that transient or ?mild uncoupling? confers neuroprotection in preclinical models of TBI. Recently, we demonstrated that a prodrug of 2,4-dinitrophenol (DNP), MP201, a mitochondrial uncoupler with better pharmacodynamic properties including higher tolerability and extended elimination time, rescues acute mitochondrial bioenergetics, reduces oxidative damage, increases brain-derived neurotropic factor (BDNF) and is neuroprotective. We hypothesize that the optimal dosage and timing of therapeutic intervention of MP201 is neuroprotective across species following focal contusion brain injury. Our proposed studies will explore how MP201 administration can improve acute, longitudinal, and chronic outcomes, paired with critical biomarkers, including platelet physiology and neurochemical profiles. To achieve this, we will use innovative techniques across multiple institutions to assess synaptic and non-synaptic mitochondria in both porcine and murine models of TBI. Additionally, we will extend our findings to examine therapeutic efficacy, measuring longitudinal cortical morphology (T2/DTI scanning), neurometabolite profiles (MRS scanning), platelet signature (as a novel biomarker and biosensor), and cognitive behavior. Finally, we will explore the underlying mechanism behind the long-term neuroprotection imparted by MP201 after TBI, examining BDNF levels and mitochondrial restoration. With strong preliminary data and utilizing many innovative and clinically-relevant techniques, we anticipate this proposal will generate ground-breaking data. Overall, this proposal will highlight highly translatable therapy by MP201 to alleviate negative outcomes of TBI.