Sandra Hewett, Ph.D. - US grants

DESCRIPTION (Adapted from Applicant's Abstract): The pathophysiology of delayed neuronal death as it occurs hours or even days following cerebral ischemia is poorly understood. Recent study in animal models indicate that cytokine-inducible nitric oxide synthase (iNOS) is strongly induced in astrocytes surrounding areas of extensive neuronal degeneration 1 to 3 days following cerebral ischemic insult. Previous work by Dr Hewett demonstrated that NO derived from cytokine induction of astrocyte iNOS while not toxic alone dramatically increased the magnitude of N-methyl-D-aspartate and oxygen glucose deprivation induced neuronal injury in vitro suggesting that in vivo activation of iNOS could have the dangerous consequence of enhancing excitotoxic neuronal injury. Further it was shown that the potentiation was associated with an increase in extracellular glutamate levels and was dependent on reactive oxygen species as well as NO. Thus, the goal of this project is to elucidate specific cellular and molecular events by which astrocytic NO and reactive oxygen species contribute to the cytokine-mediated enhancement of excitotoxic neuronal injury. Experiments will be performed in vitro in primary cortical cell cultures. Astrocytic iNOS will be induced by exogenous addition of pro-inflammatory cytokines to cultures. Combined oxygen glucose deprivation as well as excitatory amino acid administration will be used in in vitro models of cerebral ischemia. Studies will be designed to answer the following questions: 1. How do NO and reactive oxygen species interact to augment excitotoxicity and what is the cellular source and enzymatic source of reactive oxygen species? 2. How does astrocytic iNOS induction lead to enhancement of extracellular glutamate? Specifically, the ability of cytokine stimulation to alter glutamate e-flux and/or re-uptake will be assessed. The long-term objectives of this study is to better understand the pathways and mechanisms by which inflammatory cytokines contribute to the enhancement of excitotoxic neuronal injury. Improved definition of these events could lead to the development of new therapeutic strategies designed to attenuate the progression of neuronal destruction following stroke.

DESCRIPTION (provided by applicant): Injury to the brain caused by cerebral ischemia (i.e. stroke) is a major public health concern. Studies have determined that the brain damage associated with cerebral ischemia is mediated by over-stimulation of excitatory amino acid receptors, oxidative stress, as well as inflammatory factors. Preliminary results obtained during the last/current grant period as well as supporting data from the literature demonstrate a link between leukocyte-type 12/15 lipoxygenase (L-12/15 LO) activation in cerebral ischemic neuronal injury in vivo and oxidative stress-induced neuronal injury in vitro. Thus, the objectives of this following 5 yr research plan of study are as follows: 1) To determine the extent to which L-12/15 LO is involved in cerebral ischemic injury. Animals genetically null for L-12/15 LO and those genetically engineered to over-express L-12/15 LO will be used to determine its contribution to cerebral ischemic damage over a detailed time course. 2) To determine whether the decreased susceptibility of L-12/15 LO null mutants to cerebral ischemic damage results from a change in cerebrovasculature function and/or a reduction in glutamate receptor-mediated damage. To assess the former, relative and absolute regional cerebral blood flow changes occurring during and at end-stage cerebral ischemia will be measured to determine whether L-12/15 LO animals (null mutants and over-expressers) sustain a similar intra-ischemic insult as compared to wild-type mice. To assess the latter, a comparison of the damage induced by direct microinjection of NMDA into brain parenchyma between L- 12/15 LO animals and wild-type controls will be made. 3) To identify an orally active, pharmacological inhibitor of the L-12/15-LO pathway for the treatment cerebral ischemic damage. Using structurally distinct, proprietary lipoxygenase inhibitors developed by Onconova Therapeutics, studies will be undertaken to identify compounds that effectively prevent injury induced by middle cerebral artery occlusion (MCAo) and/or direct hippocampal injection of NMDA as well as to elucidate their therapeutic time window. Results from this study will elucidate the contribution of L-12/15 LO to cerebral ischemic damage and could aid in the development of a novel stroke therapeutic. PUBLIC HEALTH RELEVANCE: Morbidity associated with stroke remains a huge emotional and economic burden due in large part to a void in treatment options to protect against secondary injury. It is our contention that successful completion of this proposal will advance and refine our knowledge about an important new therapeutic target (L-12/15 LO) that complements other ongoing efforts to reduce injury following cerebral ischemic insult.

DESCRIPTION (provided by applicant): Injury to the brain caused by cerebral ischemia (i.e. stroke) is a major public health concern. As much as 50% of the brain damage incurred by stroke occurs outside of the primary focus of damage with the process of tissue destruction continuing for hours to days. It is now apparent that inflammatory factors contribute to this delayed pathophysiology. Specifically, studies demonstrate that the cytokine, interleukin 1[unreadable] (IL-1[unreadable]), is upregulated following experimental and clinical stroke while additional studies implicate it in the progression of injury. However, the cellular and molecular pathway(s) by which IL-1[unreadable] contributes to neuronal cell death have yet to be identified. This is largely due to the lack of suitable in vitro models in which to assess these mechanisms. Therefore, we developed a reliable and reproducible in vitro model system utilizing mixed neuronal/astrocyte cortical cell cultures. In this model, endogenous production of IL-1[unreadable] is simulated by exogenous addition of IL IL-1[unreadable] and neuronal injury induced by depriving cells of oxygen. We found that pre-treatment? but not concurrent or post-treatment? with this cytokine dramatically potentiated neuronal cell death induced by depriving mixed murine cortical cell cultures of oxygen. The effect of IL-1[unreadable] was concentration-dependent and could be completely inhibited by the recombinant IL-1 receptor antagonist, indicating that signaling through the IL-1 receptor type I (IL1R1) was involved. Further, we found this IL-1[unreadable] -mediated enhancement of hypoxic-neuronal injury can be completely prevented by pharmacological antagonism of metabotropic glutamate receptor 1 (but not mGluRS). This is in stark contrast to a pure hypoxic neuronal injury which is unaffected by mGluRI receptor antagonism. Finally, we found that the enhancement of injury induced by IL-1[unreadable] was dependent on astrocytic expression of IL1R1 whereas loss of signaling in neurons had no effect. Thus, the objectives of this five year research plan are to 1) determine the molecular mechanism(s) by which IL1p signaling functionally synergizes with mGluRI signaling to enhance hypoxic neuronal injury;2) to determine the astrocytic factor or factors responsible for mediating the IL-1[unreadable] enhancing effect;and 3) to assess whether removal of IL-1[unreadable] signaling can effectively prevent/ameliorate hippocampal injury in vivo induced by direct hippocampal injection of NMDA and/or middle cerebral artery occlusion. Improved definition of these events could lead to the development of new therapeutic strategies designed to attenuate the progression of neuronal destruction following stroke.

DESCRIPTION (provided by applicant): Injury to the brain caused by cerebral ischemia is a major public health concern. Studies have determined that the brain damage associated with cerebral ischemia is mediated by over-stimulation of excitatory amino acid receptors, oxidative stress, as well as inflammatory factors. Our laboratory demonstrated - using an in vitro model of the ischemic penumbra - that astrocyte-mediated alterations in system xc- (cystine/glutamate antiporter) contribute to the development and progression of inflammatory (IL-12-mediated) hypoxic neuronal injury. Thus, we believe that system xc- has the potential to be a novel therapeutic target for stroke. However, to validate this hypothesis, our results must be confirmed in vivo. Mice harboring a natural loss of function mutation in the Slc7a11 (sut) gene - which encodes for xCT, the light chain dictating substrate specificity in system xc- - and xCT null mutant mice have been described in the literature. However, these are global knockouts. To date no tissue-specific knockout for this allele exists. Hence, the overall goal of this project is to develop (Aim 1) and characterize (Aim 2) an astrocyte-specific conditional knockout mouse of the Slc7a11 gene for the ultimate use in in vivo.

DESCRIPTION (provided by applicant): Injury to the brain caused by cerebral ischemia is a major public health concern. Studies have determined that the brain damage associated with cerebral ischemia is mediated by over-stimulation of excitatory amino acid receptors, oxidative stress, as well as inflammatory factors. During the last grant period our laboratory demonstrated that astrocyte-mediated alterations in system xc- (cystine/glutamate antiporter) activity contributes to the development and progression of inflammatory (IL-1beta-enhanced) hypoxic neuronal injury -a model of the ischemic penumbra. Despite this, new preliminary data demonstrate that IL-1beta-mediated upregulation of the same molecule, system xc-, can confer protection against oxidative insults. We speculate that IL-1beta upregulation of astrocyte system xc- may have evolved as a protective mechanism to counteract oxidative stress in injured tissue. However, this increase becomes maladaptive in the setting of compromised glutamate uptake, which occurs in the setting of our hypoxia model in vitro and stroke in vivo. The concept that IL-1beta and system xc- are at the crossroads of injury and protection is particularly intriguing. Studies to systematically and empirically address these ideas, as well as, to elucidate the regulation of the transporter by IL-1beta at the molecular level are solely needed. Thus, the objectives of this following 5 yr research plan of study are as follows: 1) To determine the mechanism by which IL-1beta regulates astrocyte system xc- expression. State of the art molecular biological approaches will be utilized to assess whether IL-1beta regulates xCT mRNA at the transcriptional and/or post-transcriptional level and to identify the cis and trans-acting factors responsible for the induction and/or stabilization of xCT message. 2) To determine the functional consequence of enhanced system xc- activity. The goal of this aim is to determine whether the IL-1beta-mediated enhancement of system xc- activity, a priori, increases GSH content and confers a selective resistance to oxidative injury under conditions where glutamate uptake is competent both in vitro and in vivo. 3) Using genetic approaches, studies will be undertaken to determine the extent to which IL-1beta signaling regulates system xc- expression following cerebral ischemic injury with the direct question as to whether loss of system xc- function either globally, or in astrocytes specifically, can alter the susceptibility of mouse brai to cerebral ischemic damage. Understanding the regulation of system xc- by IL-1beta is necessary so that we may use this information to devise strategies to harness the beneficial effects (i.e. to increase GSH levels to reduce oxidative injury), and when appropriate, to employ strategies to reduce its activity to decrease the probability of excitotoxic neuronal injury (i.e. under conditios where glutamate uptake is impaired).

?DESCRIPTION (provided by applicant): Injury to the brain caused by cerebral ischemia is a major public health concern. System xc? (Sxc?) is a cystine- glutamate antiporter, which functions to import cystine while simultaneously exporting glutamate. Using in vitro models, we previously demonstrated that alterations in Sxc? function contribute to the development and - progression of hypoxia- and hypoglycemia-induced neuronal injury. This led us to hypothesize that Sxc? could play a role in acute stroke injury. In keeping with idea, we have determined that mice deficient in Sxc? are less susceptible to transient cerebral ischemic injury than control mice in vivo. However, the well-characterized ability of system xc to import cystine to support synthesis of the antioxidant molecule, glutathione (GSH), has - also led us to speculate that system xc activation could also be potentially important for stroke recovery. Indeed, the same enhancement of Sxc? in astrocytes, sans energy deprivation, protects astrocytes alone and both neurons and astrocytes in co-culture against oxidative injury in a GSH-dependent manner. Whether this translates into a benefit in stroke recovery in vivo remains unknown. Hence, studies in this proposal, are designed to analyze the biphasic role of Sxc? with respect to its potential contribution to acute cerebral ischemic damage (Aim 1) as well as to recovery/ repair following cerebral ischemia in vivo (Aim 2). Utilization of newly developed pharmacological drugs as well as state-of-the-art tools to test the underlying hypothesis will allow us to obtain both preclinical information as well as to enhance our mechanistic understanding of the injury process, respectively.

PROJECT SUMMARY In several brain disorders including epilepsy, stroke and traumatic brain injury, an imbalance between the excitatory and inhibitory (E/I) neurotransmitter systems exists. Understanding fully the cellular and molecular processes that underlie normal, physiological transmission is the first step in determining how aberrations of such might be countered to provide such individuals with E/I imbalance symptomatic relief. Recent evidence from our lab demonstrates a role for the cystine/glutamate antiporter System xc- (Sxc-) ? which exports glutamate and imports cystine, the latter of which is the rate-limiting substrate for the synthesis of the thiol antioxidant glutathione ? in maintenance of E/I balance. Specifically, we find that sut/sut mice, which harbor a natural mutation in SLC7a11 (SLC7a11sut/sut) and are therefore devoid of Sxc-, are considerably more hyperexcitable than their wild-type littermates upon acute challenge with kainic acid or pentylenetetrazole. Paradoxically, after repeated sub-acute/sub-chronic administration of the same chemoconvulsants, SLC7a11sut/sut mice exhibit signs of hypoexcitability, a response polar opposite to that which occurs in wild-type littermate controls. The idea that these paradoxical findings may result from the same underlying mechanism ? namely synaptic scaling ? will be explored in this proposal. State-of-the-art in vivo sensor technology, as well as, cellular, molecular and pharmacological approaches will be used to test the hypothesis that chronic loss of Sxc- leads to a scaling up of glutamate receptors under basal conditions, whereas scaling down occurs under conditions of enhanced neuronal activity ? both in efforts to stabilize neuronal firing. Whether these finding are mediated by changes in glutamate and/or glutathione will also be explored. Studies to determine the cellular specificity of response, with specific focus on the role of the astrocyte, are also planned. Overall, these studies are designed to increase our mechanistic understanding of the contribution of astrocyte Sxc- to glutamate, glutathione and activity homeostasis in in vivo brain. More broadly, these efforts complement other ongoing efforts to identify targets to treat the E/I imbalance that exists in many neurological disorders.

This proposal requests partial support for the 4th biennial conference, Brain in Flux: Genetic, Physiologic, and Therapeutic Perspectives on Transporters in the Nervous System, a satellite to the joint International Society of Neurochemistry and the American Society for Neurochemistry biennial conference to be held in Montreal Canada. Brain in Flux will be convened at Le Buchelon Eco Resort located in Saint-Paulin (Quebec), Canada from Aug 9-12, 2019. This meeting is designed to create a stimulating event for leading researchers, junior investigators, and trainees who study brain membrane transport proteins by fostering the rich exchange of cutting-edge scientific and technical knowledge as well as supporting the professional development of and networking opportunities for junior scholars and trainees. Our goal is to convene a small, diverse group (~75-100) of geographically diverse researchers with meaningful representation at all academic ranks. Of note, one-half of the integrated topic session speakers will be chosen from junior faculty, post-doctoral and pre-doctoral abstract pools. The following themes, which complement the speakers already invited and committed, are envisioned: 1) Vesicular transport: from cells to circuits; 2) From biophysical structure to function; 3) Amino acid transporters; 4) Sexual dimorphism in transporter expression and function; 5) Transporters in information processing; 6) Contribution of transporters to astrocyte-neuron interactions and 7) Transporters in neurological disease and as therapeutic targets. Ample discussion time, two evening poster sessions and afternoon free time provide opportunity for formal and informal discussion, networking and for the formation of new collaborations. Historically Brain in Flux has demonstrated a strong record of accomplishment with respect to gender equity among its participants; however, conference organizers are keenly aware of the barriers to participation at conferences and in science faced by persons from underrepresented populations, or with disabilities. Thus, we will be making concerted efforts to promote diversity and inclusion by using a combination of existing and new recruiting and support initiatives. Representation goals include equity in woman's numbers; 50% or less tenured or equivalent, 25% early stage untenured or equivalent, and 20% groups racial and ethnically underrepresented or otherwise disadvantaged in pursuing the sciences, including persons with disabilities. Active engagement of the program committee as well as targeted outreach efforts will help us to hit these targets. Funds requested will support the participation and professional development of trainees, postdoctoral scholars and early stage investigators, including those from groups under-represented in science, allowing them the opportunity to present, discuss, and network with other scientists studying brain transporters from diverse disciplinary and technical perspectives. Career development activities offered include formal presentations on rigor and reproducibility in science as well as implicit bias and informal lunch-time discussions on topics such as work-life balance, strategies to advance one's career, or creating peer mentor supports.