Kathryn E. Saatman - US grants

[unreadable] DESCRIPTION (provided by applicant): Traumatic brain injury afflicts 2 million people each year in the United States, many of whom suffer persistent neurological disability as a result of diffuse axonal injury. Shearing or stretching of axons during traumatic brain injury initiates progressive axonal damage, ultimately leading to axotomy and neuronal death. Because traumatic injury most often causes delayed rather than immediate axotomy, the opportunity exists to intervene therapeutically prior to axotomy. To elucidate the cellular mechanisms underlying traumatic axonal injury and identify therapeutic targets, we will perform dynamic optic nerve stretch injury in mice, accurately replicating the biomechanics of injury experienced by axons in the human brain during traumatic injury. We propose to use this established model of central nervous system axonal injury to test our working hypothesis that activation of calpains after traumatic axonal injury causes microtubule damage and impairment of axonal transport which, unless reversed, will lead to axotomy and neuronal apoptosis. In Aim 1, we will evaluate anterograde and retrograde fast axonal transport as a function of injury severity and establish the time course of transport impairment relative to axotomy. In Aim 2, we will establish a mechanistic link between calpain activation and interruption of axonal transport via degradation of microtubule-related proteins. In Aim 3, we will test our hypothesis that prolonged disruption of axonal transport in optic nerve axons leads to apoptosis of retinal ganglion cells (RGCs). In Aim 4, we will use IGF-1 overexpressing mice and exogenous IGF-1 treatment to determine whether elevation of IGF-1 levels can reverse transport impairment after axonal injury by delaying RGC apoptosis and upregulating cytoskeletal protein synthesis. Because regeneration of central nervous system axons remains an elusive goal, it is vital to intervene in the pathologic cascade of traumatic axonal injury before axotomy occurs. The optic nerve stretch injury model allows correlations between axonal pathology and either mechanical injury parameters or the cell body response that are not currently possible in whole-brain axonal injury models. By exploiting these unique advantages, we hope to identify key mediators in axonal pathology and novel therapeutic strategies to effectively sustain the vulnerable neuron and repair axonal damage prior to axotomy.

Although multiple lines of experimental evidence implicate calpains in the process of secondary neurodegeneration following traumatic brain injury (TBI), little is understood about the downstream actions of calpains in the injured brain, and how these pathways might differ in focal and diffuse TBI. In addition to elucidating novel targets and cellular pathways for calpains in the injured brain, the Program will evaluate several novel strategies to inhibit posttraumatic calpain activation, including enhancing activity of calpastatin, knocking out the calpain I isoform, and administering small molecule inhibitors. The overall goal of the Animal Core (Core B) is to facilitate the Program's investigations of calpain-mediated neuropathology and evaluations of calpain inhibitory therapeutic strategies in mouse models of focal and diffuse TBI. To thisend, the Core will perform three specific functions in support of Program objectives: 1) produce consistent, predictable focal or diffuse traumatic brain injuries in mice to be further evaluated in Projects 1, 2, and 3 and in Core C, 2) provide reliable, unbiased assessments of posttraumatic cognitive and motor function in brain- injured and control mice for evaluation of strategies to reduce posttraumatic calpain activation pursued in Projects 1, 2 and 3, and 3) maintain and expand, as necessary, colonies of human calpastatin overexpressing mice, calpastatin deficient mice, calpain I knockout mice and cyclophilin D deficient mice for use in Projects 1 and 3 and Core C. Through these three major functions, the Animal Core will increase the efficiency of the Program by reducing redundancy in training, personnel and resources, and will enhance the ability of Projects and Cores to directly compare data by minimizing variability. In addition, the Core will provide behavioral assessments under blinded conditions and will provide centralized management of transgenic and knockout mouse resources vital to the Program's scientific interests. The activities of the Core are essential to the successful completion of the scientific goals of the Program.

Traumatic brain injury (TBI) results in the prolonged activation of calpains, which contributes to cytoskeletal damage, neuronal death and behavioral dysfunction. However, surprisingly few in vivo cellular substrates of calpains have been identified in the traumatically injured brain and, consequently, little is understood about the pathways through which calpains mediate posttraumatic morbidity. Calpastatin, the endogenous inhibitor of calpains, is the only known protein that exclusively inhibits calpains. As such, calpastatin represents an ideal molecular tool with which to isolate the actions of calpains within the injured brain. Efforts to translate exciting preclinical data demonstrating functional improvement in brain-injured rodents treated with exogenous calpain inhibitors have been slowed by challenges with solubility, specificity and bioavailability of small molecule inhibitors. Enhancing endogenous calpastatin activity may represent a novel and potent therapeutic approach. The overall goals of Project 1, then, are to evaluate the role of calpastatin in regulating posttraumatic calpain-mediated proteolysis and to assess the neuroprotective and behavioral efficacy of increasing calpastatin activity in the setting of TBI. Using genetically altered mice that either overexpress human calpastatin or are calpastatin deficient, Project 1 will: 1) evaluate the role of calpastatin in modulating behavioral outcome following focal or diffuse brain injury, 2) quantify the effects of altered calpastatin expression on neuronal survival and axonal injury after focal or diffuse brain injury, 3) determine the in vivo role of calpastatin in limiting trauma-induced proteolysis of neuronal cytoskeletal proteins, and 4) evaluate the role of posttraumatic calpain activation in modifying membrane proteins involved in calcium influx and in modulating mitochondria-related cell death events. Our central hypothesis is that calpastatin overexpression will prevent calpain-mediated cleavage of neuronal substrates critical for cell survival, thereby attenuating posttraumatic neuronal death and dysfunction. The proposed experiments will provide the first evidence for a functional role for calpastatin in posttraumatic pathology and elucidate differential roles for the calpain/calpastatin system in focal and diffuse TBI. In addition, this Project will provide the groundwork for novel therapeutic approaches, based on manipulation of the calpastatin system, aimed at attenuating brain damage and dysfunction due to TBI as well as other CMS injury and disease states.

The Behavioral Testing Core is housed within two rooms in the basement of BBSRB. B028 is a dedicated room for behavioral assessment of rats, in which the Core has the capability of performing a) cognitive testing of learning and memory using a Morris water maze system, b) sensory testing using a whisker nuisance task, c) nociceptive testing using a Hargreaves plantar device, and d) locomotor function testing using the Basso, Beattie, Bresnahan (BBB) scale and the foot misplacement apparatus (horizontal ladder). B030 is a dedicated room for behavioral assessment of mice, in which the Core has the capability of performing cognitive testing of learning and memory using a white Morris water maze (for black mice) or a black maze (for white mice) in conjunction with an updated digital tracking system. Mice can also be evaluated for sensorimotor function using a corner turn test, vestibulomotor ability using the rotarod apparatus, and simple and complex motor function using the composite neuroscore, neurological severity score. Basso mouse scale (BMS), grip strength meter, and string test. Specific instruments purchased with P30 funds include 2 AccuScan Instruments EzVideoDV Automated Tracking System Digital Systems, a Columbus Instruments Rotamex-5 and Foot Misplacement Apparatus, 2 analgesia meters and a wading pool for open field locomotor assessment. Kathryn Saatman, Ph.D. directs the Behavioral Testing Core along with Assistant Core Director, Alexander Rabchevsky. Stephen Onifer, Ph.D provide day-to-day management of core operations and overseeing training, data storage and dissemination, and core usage. Two senior technicians who are trained in behavioral testing in rodent TBI models (Ms. Jennifer Pleasant, 25% effort) and SCI models (Mr. Travis Lyttle, 25% effort) assist and instruct new core users with the behavioral assessments.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) afflicts an estimated 1.5 million people each year in the US. Survivors often suffer persistent neurological dysfunction, compromising their quality of life and productivity. Despite the expanding base of knowledge regarding the complex pathophysiology of TBI, no clinically proven therapeutic interventions have been identified. TBI produces acute neurovascular damage and cell death, resulting in early functional deficits. Recovery of function may be limited by the inability of the injured brain to adequately harness endogenous repair mechanisms. The central hypothesis of this proposal is that treatment with insulin-like growth factor-1 (IGF-1) will improve neurobehavioral function through the attenuation of neurovascular damage and the augmentation of posttraumatic neurogenesis and angiogenesis. We previously showed that administration of recombinant human IGF-1 (rhIGF-1) improves motor and cognitive function in brain-injured rats. However, no studies have examined the neuroprotective or neurorestorative capabilities of IGF-1 in the traumatized brain. We propose to conduct the first comprehensive investigation of the multiple, potentially synergistic mechanisms underlying the behavioral efficacy of IGF-1. Importantly, we will evaluate the efficacy of rhIGF-1 treatment initiated within a clinically relevant therapeutic window of 8 hr and demonstrate sustained protection up to 5 weeks postinjury. In the setting of TBI, IGF-1 is a promising therapeutic candidate, as it has neuroprotective properties and helps to sustain or enhance myelination. Therefore, after demonstrating in Aim 1 that a 7 day systemic infusion of rhIGF-1, initiated at 15 min, 3 hr or 8 hr postinjury, improves motor and cognitive function in mice subjected to cortical impact brain injury, in Aim 2 we will test the hypothesis that IGF-1 protects against neuronal loss and axonal degeneration. IGF-1 is also known to stimulate proliferation of progenitor cells, promote neuronal, oligodendroglial and endothelial differentiation, and increase angiogenesis in the brain. Therefore, in Aim 3 we will test the hypothesis that infusion of rhIGF-1 enhances neurogenesis in the subgranular and subventricular zones. In Aim 4, we will employ novel vascular perfusion techniques to quantify densities of total and angiogenic vessels in brain- injured mice treated with rhIGF-1 or vehicle to test the hypothesis that IGF-1 effectively enhances posttraumatic angiogenesis. Finally, in Aim 5 we will test the hypothesis that rhIGF-1 administration delayed 48 hr can selectively enhance neurogenesis and angiogenesis resulting in cognitive recovery in the absence of acute neuroprotection. These data will provide unique and valuable insights into the neurovascular targets for IGF-1 in the injured brain. By establishing long term (up to 5 wks postinjury) histological benefits and correlating these with behavioral responses, the proposed studies have the potential for high clinical impact. Ultimately our goal is to provide a catalyst to accelerate the translation of IGF-1 therapy into clinical application in order to improve the health and well-being of individuals burdened with TBI.

PROJECT SUMMARY/ABSTRACT This is a renewal application requesting continued funding for a Neurobiology of CNS Injury and Repair T32 Training Program to support 4 predoctoral fellows working toward their Ph.D. degrees. During the first 4 years of the original funding period, 11 trainees have been appointed to the 2 year program, of which 5 have completed training and their Ph.D.s (3 in Physiology and 2 in Anatomy & Neurobiology). Of those 5, 2 are currently postdoctoral fellows, 2 have been hired into university faculty positions and 1 has moved on to medical school. Of the other 6 trainees, 2 are expected to finish their Ph.D.s during year 5 of the initial funding period. The remaining 4 are anticipated to complete their doctorates within the next two to three years. The training of the predoctoral fellows will be mainly carried out by training faculty whose primary appointments are either within the University of Kentucky (UK) Spinal Cord & Brain Injury Research Center (SCoBIRC) or are the affiliated with it as SCoBIRC Faculty Associates. Three of the SCoBIRC faculty associates have their primary appointments within the UK Sanders-Brown Center on Aging (SBCoA). In addition to their primary appointments, the SCoBIRC and/or SBCoA training faculty have their academic appointments in one of three UK College of Medicine basic science departments: Anatomy & Neurobiology, Physiology, or Molecular & Cellular Biochemistry. One additional training faculty is from the UK College of Pharmacy Department of Pharmaceutical Sciences. The overall goal of the proposed program will continue to be providing broad-based training in modern research concepts regarding the acute, subacute and chronic pathophysiology of SCI, TBI and stroke, and the identification of potential disease-modifying molecular targets that can drive the discovery of pharmacological or gene therapeutic strategies by which the devastating effects of these injuries can be ameliorated. These strategies will include both ?neuroprotective? and ?neurorestorative? approaches. Although it is anticipated that most of the trainees will pursue careers in laboratory-based therapeutic discovery research, they will also receive training in clinical aspects of the targeted neurological disorders and the practical issues involved in the design and conduct of neurological clinical trials. To accomplish this, the predoctoral fellows will spend one day/week for a semester shadowing one of 6 Clinical Tutors from the Departments Neurosurgery and Physical Medicine & Rehabilitation, and will attend weekly grand rounds to gain an understanding of the clinical nature of TBI and SCI upon which their research is focused. This will enhance their ability as independent investigators to not only make therapeutic discoveries in experimental neurotrauma and stroke injury models, but also provide them with knowledge concerning how to design their basic research in a manner that will more readily enable the translation of promising therapeutic approaches into clinical studies and therapeutic trials.

Project Summary More than one million people are treated medically each year in the United States after sustaining a brain injury and traumatic brain injury (TBI) is often accompanied by the delayed development of posttraumatic epilepsy (PTE), for which there are few effective therapies. Although clinical association between TBI and epilepsy is well documented, treatments designed to prevent PTE have been largely unsuccessful. Among the most promising antiepileptogenic treatments reported to date center on inhibition of the mammalian (mechanistic) target of rapamycin (mTOR) pathway. mTOR is activated after TBI and seizures, and it's activity regulates a variety of cellular activities, including growth and proliferation, especially in developing neurons. Inhibiting mTOR activity has shown promise for altering the progression of epileptogenesis in rodent models of epilepsy, including PTE, but several caveats have also been acknowledged, specifically: Suppression of mTOR post-TBI has been proposed to prevent epileptogenesis, whereas mTOR activation has been proposed as a means of improving cognitive recovery after TBI in patients. The mechanisms by which mTOR modulation exerts its anti-epileptogenic effects are not known, and the contribution of newborn neurons and synaptic reorganization in the dentate gyrus to epileptogenesis and cognition are controversial. Preventing PTE is hampered by these fundamental knowledge gaps. This proposal will use the controlled cortical impact (CCI) model of TBI, which results in cell loss, increased neurogenesis and synaptic reorganization in the dentate gyrus, and delayed development of spontaneous seizures (i.e., epileptogenesis) to study the impact of newborn neurons on synaptic excitability changes in the dentate gyrus. The effects of both negative and positive regulation of mTOR on epileptogenesis and cognitive recovery will also be determined in the context of neurogenesis after brain injury. The overarching hypotheses are that adult born neurons contribute to synaptic reorganization after TBI and that mTOR activity-dependent regulation of neurogenesis alters epileptogenesis and post-TBI cognitive recovery. A combination of electrophysiological, histological, and behavioral techniques utilizing optogenetic and chemogenetic modification of adult born neurons will be used to address three aims: 1) Determine the functional synaptic organization of adult born DGCs after TBI; 2) Determine effects of mTOR modulation on neurogenesis and synaptic connectivity in the dentate gyrus after TBI; and 3) Determine how adult born DGCs contribute to functional recovery and seizures after TBI. A mechanistic understanding of how adult born neurons contribute to DGC circuitry and how mTOR modulation alters the circuitry of these neurons after CCI will be developed in the context of both cognitive recovery after TBI and development of PTE. A better understanding of the contribution of adult born neurons to recovery and epileptogenesis after TBI will facilitate the development of treatments to prevent PTE.

Project Summary Traumatic brain injuries (TBIs) are a major societal and public health concern with over 2.8 million TBIs reported each year in the US. Mild TBIs (mTBIs), accounting for over 80% of TBIs, can be difficult to diagnose. Because no FDA-approved therapeutic intervention for mTBI exists, a period of rest to allow symptom resolution is the primary treatment approach. It is imperative that while symptomatic, a person recovering from mTBI avoid sustaining a second mTBI, as multiple mTBIs greatly increases the risk for prolonged disability. The period of brain vulnerability after mTBI, however, extends beyond the resolution of clinical symptoms, underscoring the vital need for accurate assessment of neurophysiological recovery in order to mitigate the risks associated with repeated head injury. Unlike moderate and severe TBI, which are typically associated with neuron death and vascular disruption, mTBI results in more subtle physiological and cellular changes, such as metabolic distress and alterations in cerebral blood flow (CBF). We hypothesize that mTBI induces acute, transient changes in CBF that, coupled with metabolic dysregulation, form the basis of the window of vulnerability to repeated mTBI. We predict, therefore, that a second injury induced during the period of acute CBF alteration will result in worsened outcome as reflected by greater perturbations in CBF and metabolism. Our group has developed a novel multiple-wavelength speckle contrast diffuse correlation tomography (MW- scDCT) technique that yields non-invasive, longitudinal, regional mapping of CBF and oxygenation in mice. We have validated our technique against established methods and demonstrated its utility in detecting CBF changes in rodents. In Aim 1, we will first use MW-scDCT to measure cortical and hippocampal CBF and oxygenation after single closed head injury (CHI) to monitor temporal changes and determine the time to recovery. We will then determine whether normalization of CBF is required to prevent synergistic effects of a second CHI on cerebral hemodynamics. Neurovascular coupling and cerebrovascular reactivity will be assessed at selected time points to inform potential mechanisms underlying CBF changes. Finally, quantitative analysis of cerebrovascular structure and communication will be performed to identify anatomical plasticity or damage. In Aim 2, a targeted metabolomics approach will be used to identify metabolite profiles in cortical tissue and plasma which are unique to mice with single or repeated mTBI. We will further test whether restoration of the metabolome coincides with normalization of CBF after mTBI. Such a finding would support a dual-pronged approach for assessing concussion recovery through noninvasive CBF monitoring and assessment of plasma metabolite biomarkers. These studies will pair metabolomics with our innovative MW- scDCT technique for monitoring cerebral hemodynamics to provide new insights into the neurophysiological determinants of the brain?s vulnerability to repeated mTBI and support the development of diagnostic and prognostic biomarkers for mTBI.