Mary Stenzel-Poore - US grants

The response to stress leads to mental and physical changes that improve the ability of the organism to achieve homeostasis and increases its chance for survival. It has been postulated that dysregulation in the stress system leads to various disease states such as depression, anorexia nervosa and autoimmune disease. Corticotropin-releasing factor (CRF) is the central mediator of the stress response. Patients with clinical depression display sustained activation of the hypothalamic- pituitary-adrenal (HPA) axis that may be due to hyper-CRF secretion and such patients are considered to be subject to chronic stress. CRF has recently been shown to participate in a stress-responsive circuit between the brain and immune system. Altered regulation of central CRF and changes in the HPA axis occur in chronic inflammatory disease and infection. Cytokines from cells of the immune system cause HPA activation and have been shown to transmit information from the immune system to the nervous system. I will test the hypothesis that chronic overproduction of CRF in the central nervous system causes impaired immune function. I have developed a transgenic mouse model of chronic hyper-CRF expression 91-4). This model will be used to examine immune and nervous system interactions. This mutant mouse strain overproduces CRF in the brain and has altered HPA and behavioral parameters that parallel the stress response. Thus this animal provides a unique and powerful model for investigating the effects of prolonged CRF exposure and HPA activation. The aims of this grant are: 1) To evaluate the influence of chronic central CRF overproduction on immune function through the analysis of central CRF- overexpressing transgenic mice and 2) To determine whether expression of the metallothionein-CRF chimeric transgene is regulated by mediators known to modulate CRF expression and whether control of the endogenous CRF gene is altered in this mouse model and 3) To test whether CRF is essential for immunomodulation by the nervous system and to understand the consequences of a neuroimmune system lacking CRF, an animal model will be generated without CRF via targeted disruption of the endogenous CRF gene. These studies will test whether long term exposure to increased levels of CRF lead to sustained immunosuppression and conversely, in the absence of CRF, whether stress-induced immunosuppression fails to occur. The studies proposed here include examination of CRF expression by in situ hybridization and functional analysis of the immune system using antibody responses to antigen, NK cell cytotoxicity and T cell proliferative responses. Targeted gene disruption of the CRF gene will be accomplished using homologous recombination in embryonic stem cells.

DESCRIPTION (Adapted from the applicant's abstract): The finding of CRH-R2 in cardiac tissue leads to the hypothesis tested in this proposal: cardiac-specific responses to CRH occur via CRH receptors localized in the heart. The primary prediction is that certain specific cardiac effects of CRH occur through CRH-R2. The source of CRH that acts on the cardiac CRH-R2 may be via local synthesis in the heart and /or from production in the central nervous system. The aims are: Aim 1: Determine which cells in the mouse heart express CRH receptors. Aim 2: To examine the interaction of two peptide ligands, CRH and urocortin, with the CRH-R2 receptor and to determine the second messenger systems to which the receptor is coupled. Aim 3: Determine the mechanism(s) involved in cardiac CRH-R2 regulation by the stress-related stimulus, bacterial endotoxin.

Interactions between the immune, nervous and endocrine systems during the response to stress are key to understanding the immunopathology of stress-associated disease. Such understanding requires identification of the principle targets in the stress response as well as the molecular details Of how information is transferred between these mutually regulating systems. The neuroendocrine stress peptide, corticotropin-releasing hormone (CRH) is the primary mediator of the neuroendocrine stress response and has recently been shown to play a central role in the associated immunomodulation. The focus of this proposal is to understand the role of CRH, the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. We shall employ a transgenic animal model of CRH overproduction and chronic hypothalamicpituitary-adrenal activation, which provides the first genetic model of stress-associated immunomodulation. We have shown these transgenic mice to display the behavioral features associated with stress as well as Cushing's syndrome and immune dysfunction. Using this genetic animal model and a second provided by our collaborator Dr. R. Allen, which bears an inducible ACTH "knockout", I propose to examine the relative contributions of CRH and the direct downstream neuroendocrine products of CRH stimulation, namely, ACTH and glucocorticoids in modulating B lymphocyte development and function in chronic stress. We also propose to examine the role of peripheral catecholamines in this system. In this proposal, we shall test the hypothesis that CRH overproduction leads to loss of B lymphocytes and decreased immune responsiveness. The specific aims are: 1) To determine the mechanism of B lymphocyte loss in CRH-overproducing mice. 2) To determine the mechanism of increased susceptibility to infectious disease and altered B lymphocyte function in CRH-overproducing mice.

The long term goals of this project are to identify the cellular and molecular mechanisms that lead to inflammation in the cerebral cortex following ischemia and to determine how this inflammatory response exacerbates neuronal damage. In peripheral tissues, the inflammatory response is marked by the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6). Corticotropin-releasing hormone (CRH), a neuropeptide first identified as a hypothalamic releasing hormone that mediates the stress response, is also produced during inflammation and has been shown to induce IL-1 and IL-6. We and others have demonstrated an important potential link between CRH and ischemic damage in that infusion of a CRH antagonist produces significant attenuation of neural damage induced by cerebral ischemia. Furthermore, there is recent evidence that expression levels of CRH are rapidly upregulated in the cortex following ischemia. We have observed that in addition to neurons, a number of cells in the CNS contain functional CRH receptors including astrocytes, endothelial cells from brain microvasculature, and microglia. Importantly, CRH immunoreactivity is observed in nerve terminals throughout cerebral cortex and over varicosities located on cerebral microvessels. Our observations suggest that release of CRH induced by ischemia participates in the inflammatory reaction. We hypothesize that CRH modulates ischemia-induced inflammation via actions on non-neural cells in the CNS, leading th increased activation of resident inflammatory cells and increased infiltration by peripheral leukocytes. We propose to test these possible mechanisms in vitro and in an animal model of stroke using CRH receptor gene knockout mice to examine the contribution of CRH to the inflammatory response following MCAO. Specific Aim 1: To characterize the inflammatory response following ischemia in vivo using flow cytometry to quantify microglial activation and leukocyte infiltration in the brain and to correlate these changes with CRH and CRH receptor expression, cytokine expression, and induction of endothelial cell adhesion molecules. Specific Aim 2. To characterize the effect of CRH on purified non-neuronal cells from the CNS in culture. Specific Aim 3. To test the role of CRH in the inflammatory response following ischemia in vivo.

[unreadable] DESCRIPTION (provided by applicant): The goals of this R01 application are focused on the recovery phase of the stress response and the concept that inappropriate adaptations to stress lead to stress-associated pathology. We propose that corticotropin-releasing hormone (CRH) and its receptors are critical in achieving proper initiation and termination responses to stress. We posit that the two known CRH receptors, CRH-R1 and CRH-R2, play distinct but complementary roles in mediating the response to stress and adaptations to stress. CRH-R1 has a clear role in the initiation of the response to stress. We propose that CRH-R2 is critical in the recovery phase of the response to stress. We will use CRH receptor knockout mice lacking one or the other of these receptors to assign their positions in neuroendocrine and behavioral responses to stress. These studies will be complemented by assessment of gene expression in central CRH pathways in CRH-R2 KO and wild-type mice exposed to acute and chronic stress. In addition, we shall use CRH-transgenic (CRH-tg) mice as a model of excess production of CRH which leads to pathologic neuroadaptations manifest by increased sensitivity to stress and altered recovery. The specific aims are: Aim 1. Determine whether CRH-R2 mediates neuroendocrine recovery responses to acute and chronic stress. Aim 2. Test the role of CRH-R2 in recovery from stress using behavioral measures. Aim 3. Determine the roles of CRH and glucocorticoids in delayed recovery from chronic stress in CRH-tg mice. Aim 4. Distinguish the relative roles of CRH receptors, CRH-R1 and CRH-R2, in mediating the effect of chronic stress and recovery in CRH-tg mice. These studies are paramount to determine the functions of CRH-R1 and CRH-R2 in orchestrating the stress response. Our long-term objective is to elucidate the role of CRH pathways in governing the recovery phase of the response to stress [unreadable] [unreadable]

Neuroprotection against stroke injury can be induced by a small dose of lipopolysaccharide (LPS) given systemically prior to a stroke-a process known as LPS preconditioning or tolerance. As such, the study of LPS-induced preconditioning offers promise in identifying new mediators that can be administered systemically to confer neuroprotection centrally. The primary goal of this application is to investigate LPS preconditioning in stroke and define the specific molecular pathways that sub serve this neuroprotective process. Preliminary data suggest that LPS preconditioning leads to decreased cellular infiltration into the injured ischemic brain and suppressed cellular activation, which suggests that LPS preconditioning alters cellular responsiveness to subsequent injurious stimuli. Low dose LPS treatment of macrophages renders them resistant to the damage of subsequent high dose LPS challenge via a shift in the balance of proinflammatory/anti-inflammatory mediators referred to as genomic 'reprogramming'. Experiments in this proposal shall examine whether LPS preconditioning confers protection to subsequent ischemic injury by reprogramming the response to injury away from cell death and in favor of cell survival. TNF-alpha is an essential mediator in LPS preconditioning and may prime the events that lead to protection following stroke. Our preliminary data following stroke indicate that additional, unique pathways are induced in the brains of animals given prior LPS treatment compared to those not so treated. Interferon-associated genes are a dominant feature among the unique genes upregulated in animals given LPS treatment prior to a stroke. We hypothesize that TNF-alpha and Type I IFNs play essential, non-overlapping roles that lead to neuroprotection in LPS preconditioning. We postulate that LPS-induced TNF-alpha primes the emergence of tolerance by reprogramming the cellular response to subsequent ischemia from one of injury and cell death to that of survival. Subsequently, pathways regulated by Type 1 IFNs are activated which confer neuroprotection. Using in vivo and in vitro models of ischemic tolerance we propose to: 1) Elucidate the role of TNF-alpha signaling in priming the neuroprotective events following LPS preconditioning; 2) Determine whether Type I IFNs play a critical role in establishing a neuroprotective state following ischemic injury in LPS preconditioned mice; and 3) Test whether LPS preconditioning leads to genomic reprogramming of the response to ischemic injury. These studies should help clarify the endogenous mediators of neuroprotection induced by LPS and may ultimately lead to new therapeutic strategies for stroke.

DESCRIPTION (provided by applicant): Endogenous mechanisms of ischemic preconditioning-tolerance have reviled the brain's ability to reprogram (precondition) its response to acute ischemia from that of induced cell injury signaling cascades to induction of neuroprotective pathways (tolerance). Such endogenous neuroprotection occurs through Toll Like Receptor (TLR) signaling which reprograms an inflammatory (injurious) response to stroke into an anti- inflammatory (neuroprotective) response. We offer the preferred agonists (CpG ODNs and imiquimod - IMQ) of TLR 9 and 7 respectively as lead compounds for prophylactic neuroprotection against stroke. Although robust rodent data have been produced, past and recent translational failures require additional preclinical evaluation. Accordingly, we have developed a new primate stroke model for assessment of putative pharmacotherapeutics and propose to perform rigorous trials of our recently discovered neuroprotectants to establish essential efficacy and pharmacokinetic data. Thus our preliminary studies support new robust neuroprotective strategies for high-risk stroke patients to be further tested in the non-human primate via: Aim 1. Determine the optimal dose to achieve neuroprotective efficacy for TLR9 (K- and D-mix CpG ODNs) and TLR7 (IMQ) candidate drugs as prophylactic therapy in a NHP model of cortical stroke. Aim 2. Determine the time window of neuroprotective efficacy for K- and D-mix CpG ODNs and IMQ as prophylactic therapy in a NHP model of cortical stroke. Aim 3. Determine neuroprotective efficacy CpG ODN (K and D mix) and IMQ as prophylactic therapy in a model of cortical stroke in the aged NHP. Aim 4. Determine the neuroprotective efficacy of repeated administration of CpG ODN (K- and D-mix) and IMQ as prophylactic therapy in a NHP model of cortical stroke. Aim 5. Determine the neuroprotective efficacy of the optimal CpG ODN (K- and D-mix) and IMQ as prophylactic therapy in a model of cortical stroke in female NHPs. Aim 6. Determine pharmacokinetic and toxicity profiles of CpG ODN (K- and D-mix) and IMQ as potential stroke therapeutics. Aim 7. Submit an IND application for the optimal TLR candidate based on efficacy, pharmacokinetics and toxicity profiles. RELEVANCE: Many drug treatments to protect the brain from stroke have been tried and failed. This proposal offers a new approach based on the brain's own endogenous neuroprotective program. We will investigate 3 new drugs that have been shown to be safe in humans and test them as prophylaxis against ischemic brain injury. We will first test these drugs in a relevant primate model of stroke before moving to a clinical trial to treat humans that are at very high risk for future stroke.

(precondition) its response to acute ischemia from that of induced cell injury signaling cascades to induction of neuroprotective pathways (tolerance). Such endogenous neuroprotection occurs through Toll Like Receptor (TLR) signaling which reprograms an inflammatory (injurious) response to stroke into an antiinflammatory (neuroprotective) response. We offer the preferred agonists (CpG ODNs and imiquimod - IMQ) of TLR 9 and 7 respectively as lead compounds for prophylactic neuroprotection against stroke. Although robust rodent data have been produced, past and recent translational failures require additional preclinical evaluation. Accordingly, we have developed a new primate stroke model for assessment of putative pharmacotherapeutics and propose to perform rigorous trials of our recently discovered neuroprotectants to establish essential effacy and pharmacokinetic data. Thus our preliminary studies support new robust neuroprotective strategies for high-risk stroke patients to be further tested in the non-human primate via: Aim 1. Determine the optimal dose to achieve neuroprotective efficacy for TLR9 (K- and D-mix CpG ODNs) and TLR7 (IMQ) candidate drugs as prophylactic therapy in a NHP model of cortical stroke. Aim 2. Determine the time window of neuroprotective efficacy for K- and D-mix CpG ODNs and IMQ as prophylactic therapy in a NHP model of cortical stroke. Aim 3. Determine neuroprotective efficacy CpG ODN (K and D mix) and IMQ as prophylactic therapy in a model of cortical stroke in the aged NHP. Aim 4. Determine the neuroprotective efficacy of repeated administration of CpG ODN (K- and D-mix) and IMQ as prophylactic therapy in a NHP model of cortical stroke. Aim 5. Determine the neuroprotective efficacy of the optimal CpG ODN (K- and D-mix) and IMQ as prophylactic therapy in a model of cortical stroke in female NHPs. Aim 6. Determine pharmacokinetic and toxicity profiles of CpG ODN (K- and D-mix) and IMQ as potential stroke therapeutics. Aim 7. Submit an IND application for the optimal TLR candidate based on efficacy, pharmacokinetics and toxicity profiles.

DESCRIPTION (provided by applicant): Inflammation is a major component in the pathogenesis of brain injury during stroke. Cell signaling pathways prominently involved in the inflammatory cascade are initiated through Toll-like receptors (TLRs). Thus TLRs may be novel targets for stroke therapeutics. TLR4 is responsible for ischemic tolerance induced by systemic administration of lipopolysaccharide (LPS). Potential deleterious side effects preclude translation. We have found that additional TLRs (TLR7 & TLR9) are also potent targets to induce preconditioning against stroke. Pretreatment with the TLR9 agonist, CpG ODNs, reprograms cell signaling during subsequent stroke and the resultant inflammatory cell signaling is changed to potent neuroprotection. CpG ODNs are well tolerated in humans offering rapid translation as pre-stroke treatment for patients at high risk (e.g. new TIA, pending CABG surgery). Here we propose to characterize this novel prophylactic stroke therapy and demonstrate the efficacy and immune cell activation in the setting of stroke. We will address the potential mechanisms that underlie neuroprotection and whether these mechanisms act systemically and/or are located in the CNS as human treatments may optimally be directed systemically or centrally. Aim 1. Characterization of neuroprotection induced by the TLR9 agonist, CpG ODNs. Aim 3. Determine whether the primary inducers and effectors of LPS preconditioning are shared by imiquimod and CpG preconditioning pathways. Aim 2. Determine the relative contribution of CNS resident cells and systemic hematopoietic cells to TLR9 induced neuroprotection. Aim 3. Determine whether TNFa and IFNb are critical effectors of ischemic tolerance elicited by TLR9 (CpG) preconditioning. Aim 4. Determine whether CpG preconditioning reprograms the response to stroke through modulation of TLR signaling pathways. PUBLIC HEALTH RELEVANCE: Protecting the brain against future stroke. Here we describe a treatment by which we can modify chemical events in brain so that protection will result if a stroke were to occur. With this treatment, the damaging domino effect in brain, caused by the stroke that leads to profound brain injury, can be redirected towards a neuroprotective cascade. Studies here will test whether such treatment can be developed into a potential treatment for patients at high risk of stroke.

PROJECT SUMMARY  Brain  ischemia  is  a  leading  cause  of  morbidity  and  mortality  in  the  United  States.  We  seek  to  develop  therapeutics  to  reduce  the  extent  of  damage  and  functional  impairment  resulting  from  ischemic  injury  to  the  brain,  an  area  of  significant  unmet  medical  need.  In  a  mouse  model  of  stroke  injury  we  have  demonstrated  that the synthetic dsRNA, polyinosinic-­polycytidylic acid stabilized by poly-­L-­lysine and carboxymethylcellulose  (PIC,  Hiltonol®),  is  a  robust  prophylactic  neuroprotectant  against  ischemic  injury.  PIC  given  to  mice  one  day  prior  to  transient  middle  cerebral  artery  occlusion  reduced  the  area  of  damage  in  the  brain  by  ~95%.  We  recently published that interferon (IFN) receptor signaling is required for PIC-­induced neuroprotection against  mouse stroke, providing a potential mechanistic biomarker for predicting neuroprotective doses. Based on our  studies  in  mice,  we  hypothesize  that  preconditioning  is  mediated  through  systemic  IFN  secretion  and  consequent induction of interferon regulated genes in the brain and that these markers can be used to identify  efficacious doses for translation to non-­human primates (NHP) studies and ultimately human clinical trials.   R21 Specific Aims:  Aim 1. Validate biomarkers in rhesus macaques that best reflect PIC bioactivity induced by efficacious  doses  in  the  mouse.  Milestone  Define  a  dose  of  PIC  that  results  in  the  induction  of  the  mechanistic  biomarker IFN? and at least 3 of the 4 other cytokine biomarkers in the NHP to levels within the interquartile  range (25th-­75th percentile) of fold changes seen in the mouse at efficacious doses. If we fail to identify a dose  that meets both conditions we will rely solely on induction of the mechanistic biomarker IFN?. R21 Criteria for  Success.  The  primary  goal  is  to  establish  a  neuroprotective  dose  across  species  based  on  the  induction  of  systemic biomarkers associated with efficacious doses. A no-­go decision will be reached if we fail to identify a  dose of PIC that result in an increase of the mechanistic biomarker, IFN?.     R33 Specific Aim:  Aim 1. Evaluate the potential of PIC to protect against cerebral ischemic injury in NHPs. Milestone 1)  Establish  a  dose  that  demonstrates  a  >25%  reduction  in  neurological  score  in  a  majority  of  animals  preconditioned with PIC and/or >25% reduction in infarct volume (T2 magnetic resonance images;? day 2). We  view this milestone as essential for progression of PIC to clinical preconditioning studies. 2) Establish a clinical  monitoring  strategy  of  biomarkers  that  correlate  with  improved  outcomes.  A  biomarker  strategy  would  greatly  reduce the risk of failure in clinical studies by providing a means to better establish target doses in populations  with significant co-­morbidities.