Ramesh Raghupathi - US grants

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is the leading cause of death among individuals under the age of 45 in the United States and survivors are faced with chronic brain damage leading to debilitating behavioral dysfunction. Brain damage and behavioral dysfunction may be, in part, due to neuronal death following TBI. The hypothesis to be tested in this proposal is that post-traumatic neuronal cell death is a result of activation of the pro-apoptotic caspase family of cysteine proteases. The objectives of this proposal are to elucidate (1) the association between neuronal death in experimental TBI and in postmortem tissue from head-injured patients and the activation of the "executor" caspase-3, (2) whether caspase-3 activation occurs directly as a result of activation of the "initiator" caspase-8, and/or indirectly as a result of mitochondrial pathway which requires Bax translocation, cytochrome c release and caspase-9 activation, (3) whether caspase-8 activation occurs as a result of activation of the tumor necrosis factor family of death receptors, (4) the role of Bax in mediating trauma-induced caspase-9 activation and subsequent caspase-3 activation and cell death, and (5) whether post-traumatic inhibition of caspases-3, -8 and -9 will reduce the extent of injury-induced cell death. Immunoblot and immunohistochemical analyses using specific and selective antibodies will be utilized to temporal and regional patterns of activation of caspases-3, -8 and -9, resdistribution of Bax and cytochrome c, and, apoptotic neuronal damage, as indicated by the presence of cellular DNA fragmentation and morphologic analyses. Mice deficient in TNF will be used to determine the role of TNF in mediating activation of caspases-8 and -3, and eventual apoptotic cell death by using immunoblot, immunohistochemical and histological analyses. The role of Bax in trauma-induced activation of caspase-9 and -3, cytochrome c redistribution, apoptotic neuronal death and behavioral dysfunction will be examined using immunoblot and immunohistochemical techniques, and by testing cognitive and motor function in brain-injured, Bax-deficient mice, followed by histological analysis of cell death. The effect of post-traumatic treatment with peptide inhibitors selective for caspases-3 (DEVD), -8 (IETD) and -9 (LEHD), on the extent of regional cell death and behavioral dysfunction will provide the mechanistic link between caspase activation and TBI-induced pathology.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality in infants and children under the age of 4. As in the case of older children and adults, the spectrum of injury severity spans the gamut from mild to severe, with mild-moderate injured patients being the predominant population. In addition, increased efficacy of supportive neurointensive care has significantly reduced the mortality. Collectively, these phenomena result in an increasing number of survivors of TBI, who are faced with suffering life-long cognitive, emotional and social deficits. Although the pathologic alterations (cell death, axonal injury, reactive gliosis and inflammation) following closed head injury appears to be similar in both the mature and immature brains, clinical and animal studies are beginning to demonstrate that the pathogenic mechanisms in the acute and chronic post-traumatic periods are fairly dissimilar between the two ages. A second problem is that acute neuroprotective strategies, the mainstay of clinical trials and pre-clinical studies, are focused on a single "magic bullet" approach despite the multitude of pathogenic mechanisms, setting the stage for failure in clinical trials. This proposal therefore seeks to fill these two gaps in the TBI literature by using an age-appropriate, clinically-relevant model of pediatric TBI and testing whether two strategies, each aimed at limiting distinctly separate pathologic pathways, when combined, will improve functional outcome. The 17-day-old rat which is neurologically equivalent to 3-4-year-old toddler is the animal of choice. The choice of the two strategies in the current proposal arises from preliminary observations that the calcineurin inhibitor and immunophilin ligand, FK506, attenuates traumatic axonal injury following closed head injury in immature rats. In separate experiments, it was observed that the anti-inflammatory and anti-apoptotic tripeptide, Glypromate - derived endogenously from the N-terminus of insulin-like growth factor - reduced microglial activation, tissue calpain activation and attendant neurodegeneration. Using a combination of biochemical, immunohistochemical, electrophysiological and behavioral analyses, the hypothesis to be tested is that FK506, by inhibiting calcineurin-mediated neurofilament compaction and decreasing axonal injury, in combination with Glypromate which will inhibit microglial activation, decrease cytokine synthesis and reduce neurodegeneration, will together reduce acute and chronic learning and memory deficits in the brain-injured immature rat. PUBLIC HEALTH RELEVANCE: Brain trauma to the youngest section of the population is a serious health problem. Survivors are faced with life-long disabilities ranging from social and emotional problems to learning and memory deficits. Currently no treatment exists that can alleviate these deficits. This proposal seeks to evaluate approaches that will limit the multiple degenerative changes in order to improve behavioral function following closed head injury in an immature animal.

DESCRIPTION (provided by applicant): The goal of the proposed work is to determine the mechanisms of the early structural consequences of neural trauma and to develop strategies for acute intervention in traumatic brain injury (TBI) that address the primary mechanisms of axonal injury. The underlying hypothesis for this research is that initial mechanical trauma to the cell membranes leads to cytoskeletal disruptions and alterations of axonal transport and that acute intervention to restore membrane integrity and preserve axonal cytoskeleton and transport processes can dramatically reduce secondary degeneration and cell death. Specific Hypotheses to be tested are 1) Axonal cytoskeletal disruption and impaired axonal transport are causally related to membrane disruption, and acute repair of the axolemma by poloxamer P188 can prevent these effects;2) JNK-3 activation is causally related to membrane damage, and axonal transport is, in part, impaired by the actions of JNK-3;and 3) Mild injury will be manifest in axonal pathology, the time course of which is modulated by injury severity. We have developed and in vitro model of focal axonal injury in primary chick forebrain (CFB) neurons that mimics many features observed in vivo. In particular, focal swelling, or axonal beads, appeared within one hour following the mechanical insult. Co-localized with the beads were focal disruptions of microtubules and the accumulation of membrane bound organelles indicating a disruption of axonal transport. We characterized the membrane damage as a result of the mechanical trauma and showed that treatment with Poloxamer 188 (P188), a water soluble, non-ionic surfactant restored membrane integrity and significantly inhibited axonal beading in CFB neurons. We also have an in vivo model that produces axonal injury in the deep white matter. In this model, we have demonstrated membrane damage, focal accumulation of amyloid precursor protein (APP), and focal activation of JNK-3. Significantly, injured JNK-3 deficient mice do not exhibit the severe cognitive deficits seen in age-matched WT littermates. The following Specific Aims were developed to test these hypotheses: Aim 1: To determine the causal relationship between mechanically-induced membrane damage and subsequent alterations of cytoskeletal structure and axonal transport and to test whether acute treatment with an agent that promotes membrane repair can preserve axonal structure and function and thereby prevent secondary degeneration. Aim 2: To determine the mechanism of focal activation of the MAP kinase, JNK-3, in injured axons and its role in axonal pathology and to test the effect of acute membrane repair on JNK activation. Aim 3: To determine the window of opportunity for therapeutic intervention for both membrane repair and inhibition of JNK-3. PUBLIC HEALTH RELEVANCE: Currently, acute care for trauma victims deals mainly with preserving or restoring basic life support systems, e.g., cardiac and respiratory function, and managing mass lesions in the brain to prevent death and/or further brain damage. This research, if successful, will provide the basis for a new approach to the treatment of traumatic brain injury in which early intervention to preserve the structural integrity of neurons will stave off the secondary degenerative processes that result in persistent neurological deficits. Successful completion of the proposed research will hopefully emphasize the importance of the early treatment of neuronal injury as an important therapeutic consideration in addition to the current focus on delayed treatments aimed at halting secondary degeneration.

The Historically Black Colleges and Universities Undergraduate Program (HBCU-UP) through Targeted Infusion Projects supports the development, implementation, and study of evidence-based innovative models and approaches for improving the preparation and success of HBCU undergraduate students so that they may pursue STEM graduate programs and/or careers. The proposed Bridging Alliances to Infuse Neuroscience at The Lincoln University (BrainLU) Targeted Infusion project aims to establish a neuroscience program in the Department of Biology at Lincoln University. This project seeks to diversify the pipeline of neuroscientists for the 21st century. BrainLU will train undergraduate students to think like scientists and immerse them in innovative research so that they can contribute to the advancement of neuroscience. The project is transformative as it brings an interdisciplinary approach to teaching undergraduate neuroscience and transitions undergraduate students to graduate education with high-impact learning experiences.

To attain the goal of establishing a neuroscience program, the following objectives will be carried out over the 3-year funding period: 1) train and engage underrepresented undergraduate students in innovative neuroscience research in the research laboratory and classroom; 2) establish an interdisciplinary major in neuroscience at The Lincoln University that fosters creativity and original thought and builds critical thinking, analytical, quantitative, and research skills; and 3) develop a dynamic cooperative 4+1 BS/MS program in neuroscience between Lincoln University and Drexel University College of Medicine that can lead to a PhD in neuroscience. The proposed activities are geared to excite students about cutting-edge neuroscience research, bridge collaborating partners, and leverage the existing NSF HBCU-UP LEAPS Forward March program.

Almost 2.5 million people visit the emergency room each year in the United States as a consequence of sustaining a traumatic brain injury (TBI). Of these almost 75% are diagnosed with a mild TBI or a concussion, and almost a third of these patients go on to develop long-term behavioral problems. Executive function deficits, emotional disturbances such as depression and anxiety, affective disorders and substance use disorders are some of more common complaints of mild TBI patients. Adolescent boys and girls (high school and college-age) are more severely affected by concussions compared to older (adult) patients. Emerging data also suggest that girls and women are twice as likely to sustain a mild TBI, have different and more severe symptoms and take longer to recover from than their male counterparts. We have developed a model of mild TBI in the adolescent (5-week-old) rat and have demonstrated that despite similar extents of axonal injury in the early post-traumatic period only the female rats exhibit cognitive deficits. Importantly, as these animals age into adulthood, they begin to develop depression-like behavior, which manifests only in the estrus phase of the estrous cycle. This phenomenon of transient helplessness and anhedonia are hallmarks of premenstrual dysphoric disorder that some women experience. Our preliminary data demonstrate that activating the mesocorticolimbic dopamine circuit using chemogenetic approaches or agonist of the D2 receptor reversed the depressive-like behavior observed in injured animals. The activity of the D2 receptor varies with phases of the estrous cycle with the lowest activity occurring immediately after the hormone surge that occurs during proestrus. Blocking this surge using estrogen and progesterone receptor antagonists was able to reduce depressive-like behavior. The working hypothesis of this proposal is that mild TBI in the adolescent female rat results in the development of a hypodopaminergic state characterized by a decrease in the activity of the dopamine neurons in the ventral tegmental area (VTA) and the expression of D2 receptors in the medial prefrontal cortex (PFC). The specific aims are designed to test whether the estrogen receptor ? isotype is the key mediator of the decrease in D2 receptor expression (Aim 1), whether the activation of D2 receptors within the medial PFC facilitate the reversal of the depressive-like behavior (Aim 2), and whether the activation of the DA neurons in the VTA projecting to the medial PFC will reverse depressive-like behavior. Hormone receptor antagonists will be systemically administered, D2 receptor agonist and retroAAV2 will be infused directly into the medial PFC, and Gq-designer receptors exclusively activated by designer drugs will be expressed in the VTA using an AAV vector. The data from these experiments will provide the mechanistic basis for sex-specific behavioral deficits following mild TBI sustained in adolescence.