Jonathan Godbout, Phd - US grants

[unreadable] DESCRIPTION (provided by applicant): Excessive or prolonged exposure to peripheral infections may be permissive to cognitive and behavioral complications in the elderly. We have recently reported that peripheral stimulation of the innate immune system with lipopolysaccharide (LPS) causes an exaggerated brain inflammatory cytokine response and prolonged sickness behavior in aged Balb/c mice. While transient exposure to cytokines is beneficial in the host's response to a pathogen, excessive or prolonged exposure is associated with a myriad of neurobehavioral complications including mood, cognitive, and depressive disorders. Here, we provide novel evidence that this LPS-exacerbated inflammatory cytokine response in the brain of aged mice promotes depressive-like behaviors that are evident even after the acute effects of LPS have been resolved. Furthermore, our preliminary findings suggest that these age-related depressive-like symptoms following LPS challenge are associated with increased cytokine-mediated indoleamine 2, 3-dioxgenase (IDO) activity and altered serotonin (5-HT) metabolism in the brain. In this application, we will test the hypothesis that activation of peripheral innate immune system in the aged promotes an exaggerated inflammatory response in the brain that disrupts the normal metabolism of the neurotransmitter serotonin causing pronounced and prolonged depressive-like symptoms. To address this issue, we propose two specific aims using an aged mouse model. In the first aim we will characterize depressive-like behavior in mice challenged with LPS using two distinct behavioral tests: forced swimming and sucrose preference, and determine if neuroinflammatory pathways are associated with these age-related alterations in behavior. In the second aim we will delineate if prolonged depressive-like symptoms in aged mice following LPS challenge are a consequence of impaired tryptophan (TRP) and 5-HT metabolism in the brain. Successful completion of this project will yield a better understanding of the relationship between cytokines and depression in the aged. It is known that the elderly have a higher incidence of mood and depressive disorders concomitant with illness or infection; however, the mechanisms involved are not well understood. To address this issue we have designed behavioral and biochemical experiments using a BALB/c mouse model of aging to determine if age-associated changes in the brain are permissive to the onset of depression following activation of the peripheral innate immune system. We hypothesize that a heightened inflammatory cytokine response in the brain disrupts normal serotonin metabolism and results in long- lasting depressive symptoms. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Recent evidence indicates that microglia, innate immune cells of the brain, become more reactive with age. A potential consequence of a significant reactive microglia population is an amplified neuroinflammatory response to immune activation. This premise is supported by our recent study demonstrating that peripheral stimulation of the innate immune system with lipopolysaccharide (LPS) caused an exaggerated neuroinflammatory cytokine response and prolonged sickness behavior in aged BALB/c mice. Importantly, excessive or chronic exposure to inflammatory cytokines may be permissive to cognitive and behavioral complications in the elderly. In this application, we show that an LPS-exacerbated inflammatory cytokine response in the aged brain causes protracted depressive-like behavior and is associated with impaired brain metabolism of the monoamine neurotransmitter serotonin (5-HT), a critical regulator of mood and behavior. Our findings also indicate that this impaired brain 5-HT metabolism is a result of the heightened activity of indoleamine 2, 3 dioxygenase (IDO), an enzyme that catabolizes tryptophan (TRP). TRP is the rate limiting amino acid in 5-HT synthesis, so elevated TRP catabolism could reduce 5-HT-mediated neurotransmission leading to depressive behavior. Thus, a heightened neuroinflammatory response may underlie the depressive- related complications that frequently occur in the elderly. The objective of this project is to test the hypothesis that activation of the peripheral innate immune system in the aged promotes a prolonged inflammatory response in the hippocampus that disrupts 5-HT metabolism causing pronounced and long-lasting depressive symptoms. To address this issue, we propose two specific aims using an aged BALB/c mouse model. In the first aim we will delineate if attenuation of microglial activity prevents LPS-induced neuroinflammation, impaired 5-HT metabolism, and depressive-like behavior in aged mice. In the second aim we will determine if abrogation of IDO activity reverses these same biochemical and behavioral deficits in aged mice following LPS challenge. The goal of this proposal is to understand the effects of aging on depressive disorders associated with illness to develop strategies for therapeutic intervention to improve the likelihood of successful aging. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In the elderly, peripheral infection is associated with higher incidence of depressive complications, but the mechanisms underlying these complications are unknown. In this proposal, we present novel data that indicates that microglia of aged mice become hyperactive following peripheral lipopolysaccharide (LPS) challenge, exhibiting exaggerated induction of interleukin (IL)-12 and indoleamine 2,3 dioxygenase (IDO). Moreover, this amplified microglial response in aged mice is paralleled by prolonged sickness and depressive- like behaviors. Excessive microglia-specific IDO induction may be a pivotal event as recent studies indicate that IDO is central to inflammatory-mediated depression. The IDO pathway metabolizes tryptophan into several reactive intermediates and glutamate receptor agonists that can impact behavior as well as influence neuronal restructuring. In support of this premise, we show preliminary evidence of decreased apical dendrite length in hippocampus of aged mice at a time corresponding with depressive-like behavior. Taken together, these findings underscore the importance of understanding how microglia activation is dysregulated in the aged brain. One significant mechanism for microglia regulation is the fractalkine (FKN) system. Complementary expression of FKN on neurons and FKN receptor (FKNR) on microglia establishes a unique system whereby neuron-derived FKN restrains/modulates microglia activation. In this proposal, we provide evidence that impaired microglia regulation by FKN may be the basis for this LPS-induced microglial hyperactivation in the aged brain. For example, FKN mRNA levels are decreased in the brain of aged mice. In addition, peripheral LPS injection causes a greater microglia-specific reduction in FKNR mRNA in aged mice than adults. The objective of this 5 year project is to test one major hypothesis: When FKN regulation of microglia activation is impaired, peripheral innate immune challenge elicits microglial hyperactivity (excessive cytokine production and IDO activation) that causes prolonged depressive-like behavior and hippocampal dendritic atrophy. To address these issues, we propose three specific aims using a BALB/c mouse model of aging and a FNKR-/- transgenic mouse model of impaired microglia regulation. In the first aim we will determine the degree to extent to which impaired fractalkine (FKN/FKNR) interactions are associated with microglial hyperactivity in aged mice. In the second aim we will ascertain the degree to which peripheral immune challenge causes microglial hyperactivity and depressive-like behavior in mice with or without functional FKNR. In the final aim, we will determine the extent to which attenuation of microglia hyperactivation blocks prolonged depressive-like behavior and hippocampal dendritic atrophy in aged mice after peripheral LPS challenge. Addressing these aims is relevant to understanding age-associated changes in microglia regulation and is potentially important in developing interventions to protect against prolonged neuroinflammatory responses that contribute to long- lasting neurobehavioral complications. PUBLIC HEALTH RELEVANCE: In the elderly, peripheral infection is associated with higher incidence of cognitive and behavioral complications but the mechanisms involved are not well understood. We propose that these inflammatory-related complications are caused by impaired regulation of microglia activation in the aged brain. In this application, we will use two mouse models, one of aging and one of impaired fractalkine (FKN)-dependent microglial regulation, to test one major hypothesis: When FKN regulation of microglia activation is impaired, peripheral innate immune challenge elicits microglial hyperactivity (excessive cytokine production and IDO activation) that causes prolonged depressive-like behavior and hippocampal dendritic atrophy. Thus, microglial hyperactivity with prolonged IDO activation may underlie inflammatory-related depression in the elderly.

? DESCRIPTION (provided by applicant): Microglia and astrocytes are key cells that recognize, interpret, propagate and regulate immune responses. In the elderly, peripheral infections can trigger delirium and behavioral disturbances. Moreover, ongoing inflammatory processes in the elderly are associated with depressive-like complications. These complications are not a normal part of aging and negatively affect quality of life and life-span. The cause of this altered communication between the immune system and the brain with age is unclear, but our data points to an impaired regulation of microglia. We have reported that microglia from aged mice are primed and become hyperactive following a peripheral immune challenge with lipopolysaccharide (LPS). Aged microglia produce amplified levels of both inflammatory (IL-1) and anti-inflammatory (IL-10) cytokines. Although IL-10 is a potent anti-inflammatory cytokine, LPS-induced microglial activation in the aged brain is prolonged and is associated with development of depressive-like behavior. Our revised proposal addresses this critical problem in an innovative manner by identifying astrocytes as the pivotal cell in the failure to resolve microglial activation in the aged brain following immune challenge. Novel data provided here indicate that activated astrocytes in the aged brain have decreased IL-10 receptor-1 expression with corresponding reduced sensitivity to IL-10. This IL-10 insensitivity of aged astrocytes results in failure to produce TGF? and attenuate microglial activation. These findings fill a majo gap in the field about how microglia are regulated to prevent exaggerated neuroinflammation. Thus, the goal of this project is to test the hypothesis that prolonged neuroinflammation in the aged brain following a peripheral inflammatory challenge is caused by impaired astrocyte-dependent regulation of microglia. To address this hypothesis, three specific aims are proposed. In Aim-1, we will determine the degree to which reduced IL-10 sensitivity of aged astrocytes impairs their ability to regulate active microglia in vivo and ex vivo. Age-related issues in the functionality of IL-10/IL-10R1 signaling in astrocytes following immune challenge will be delineated and corresponding deficits in TGF?/TGF?R signaling in microglia will be determined. We will use several unique approaches to determine microglia and astrocyte mRNA copy number, protein expression, morphology, and ex vivo interactions in adult and aged mice after immune challenge. In Aim-2, we will ascertain the extent to which viral gene therapy enhancement of IL-10R1 specifically on astrocytes restores microglial regulation and prevents depressive-like behavior in aged mice following immune challenge. In Aim-3, we will determine if restoration of TGF? in the aged brain circumvents IL-10 insensitivity of astrocytes after immune challenge and attenuates microglia hyper-activation and prevents depressive-like behavior. Understanding how impaired IL-10 re-programming of astrocytes contributes to neuroinflammation and depressive-like behavior will lead to new interventions that target astrocytes in order to reduce neuropsychiatric complications associated with inflammatory challenge in the aged.

PROJECT SUMMARY/ABSTRACT: Traumatic brain injury (TBI) leads to secondary neuropsychiatric complications that develop and persist years after injury and negatively affect health span. Mounting evidence indicates that neuroinflammatory processes advance after the initial head injury and worsen with time. The ongoing inflammatory processes after TBI are mediated by microglia and astrocytes. In fact, these glia are the primary inflammatory responders to the injury. Therefore, it is important to identify interventions that can be provided immediately after TBI to reduce glial- mediated inflammation and facilitate recovery. It is also critical that immediate interventions prevent the development of TBI-related long-term complications in behavior, cognition, and pathology. New data are provided to show that immediate intervention with methylene blue (MB), an antioxidant and anti-inflammatory agent, reduces neuroinflammation in mice after moderate and diffuse TBI, improves functional recovery, and limits the development of primed and reactive microglia 1 month after injury. Development of a primed microglial phenotype is relevant because it represents an increased state of inflammation. These primed glia are highly reactive to subsequent immune challenges, which trigger the development of neuropsychiatric complications. Overall, we show that there are both acute and long-term benefits of immediate MB intervention after TBI. MB is used clinically in sepsis, ischemia, and vasoplegic syndrome but it has not been used clinically for TBI. MB is safe, crosses the blood brain barrier, and is administered intravenously. Therefore, our goal is to determine the degree to which MB intervention shifts the activation profile of microglia and astrocytes and protects against secondary complications following TBI including cognitive decline, glia-mediated inflammation, and glial reactivity to immune challenge. To address this, three objectives are proposed using a midline fluid percussion injury model of TBI in mice. In Aim-1 we will ascertain if MB intervention after TBI promotes a neuroprotective ?repair? profile of microglia and astrocytes. We will use unique approaches to determine the effects of TBI and MB intervention specifically on microglia and astrocyte mRNA expression, morphological profiles, and ex vivo interactions with neurons. In Aim-2 we will determine if MB intervention (immediate or delayed) prevents or reverses glia-mediated inflammation and cognitive deterioration months after TBI. Cognitive ability will be assessed for 6 m after TBI. Parallel to these assessments, glial inflammatory states and associated axonal injury and myelination changes will be determined 1, 3, and 6 m after TBI. In Aim-3, we will determine if immediate MB intervention prevents TBI-induced immune-reactivity of microglia and the development of neuropsychiatric complications. To address this, mice will receive an immune challenge 1 m after TBI and glial profiles and depressive-like behavior will be determined. Collectively, completion of these aims will provide new insight into TBI-induced glial priming and immune-reactivity and will address the efficacy and long-term benefit of methylene blue intervention.

Program Director/Principal Investigator (Sheridan, JF & Godbout JP): PROJECT SUMMARY/ABSTRACT: Dynamic inflammatory signaling in the brain may have a causative role in anxiety. We report that repeated social defeat (RSD) in mice induces sympathetic-mediated release of primed monocytes from the bone marrow that are actively recruited to the brain. Collectively, we show that RSD promotes prolonged anxiety-like behavior that depends on activation of microglia and recruitment of inflammatory monocytes to threat appraisal regions of the brain. This application demonstrates that microglial recruitment of IL-1? producing monocytes to neurovascular endothelium is necessary for the potentiation of anxiety-like behavior in response to social stress. Despite this knowledge, three key mechanistic questions remain to be answered, which will allow for the development of novel interventions for treatment of neuropsychiatric complications associated with stress: 1) How does RSD activate microglia in threat appraisal regions? 2) How are inflammatory monocytes selectivity recruited to neurovascular endothelium in threat appraisal regions? 3) What are the key factors produced by neurovascular endothelia cells that potentiate neuroinflammation and anxiety? We show that microglial activation in response to RSD depends on neuronal activation within threat appraisal regions. Moreover, preliminary evidence suggests that this is mediated by activation of the purinergic receptor, P2RX7, on microglia. Novel data presented here show that monocyte recruitment and the development of anxiety-like behavior after RSD requires microglial activation. Furthermore, cell-specific transcriptional profiling indicates that microglia recruit monocytes to the brain via chemokine ligand (CCL2) secretion. These recruited monocytes produce IL-1??and promote anxiety-like behavior by endothelial IL-1 Receptor-1 activation. Notably, this IL-1R1 activation is associated with increased neurovascular expression of COX2, the enzyme that synthesizes neuroactive prostaglandins. Thus, the goal of this project is to test the hypothesis that recruitment and subsequent interaction of inflammatory monocytes with neurovascular endothelial cells is critical for the augmentation of neuroinflammation and potentiation of anxiety-like behavior following RSD. To address this hypothesis, three specific aims are proposed. In Aim-1, we will ascertain the extent to which RSD-induced microglial activation is dependent on stimulation of the P2RX7 purinergic receptor. In Aim-2, we will determine the degree to which microglial production of CCL2 is required for monocyte recruitment to threat appraisal regions and the induction of anxiety-like behavior after RSD. In Aim-3, we will assess the role of endothelial COX2 following RSD and determine other key factors produced by IL-1R1-stimulated endothelial cells that facilitate anxiety-like behavior after RSD. Understanding how microglial recruitment of IL-1? producing monocytes to threat appraisal regions potentiates neuroinflammation and anxiety-like behavior will lead to new interventions for neuropsychiatric complications associated with stress. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

PROJECT SUMMARY/ABSTRACT: Neuroinflammation propagated by peripherally-derived monocytes/macrophages may have a causative role in chronic anxiety disorders. We report that repeated social defeat (RSD), a preclinical mouse model, induces a sympathetic-mediated release of myeloid cells from the bone marrow that traffic to regions of the brain associated with fear and anxiety. These peripheral myeloid cells promote a robust interleukin (IL)-1 inflammatory signal in the brain that induces prolonged anxiety-like behavior. Recent clinical data support a link between chronic stress, inflammatory monocytes, and anxiety. Critical to this proposal, RSD causes ?stress- sensitization? which is associated with long-term changes in the myeloid cells in the spleen and brain. This is relevant because clinical studies also detect inflammatory monocytes in circulation with stress and observed increased monocyte production in the spleen. Moreover, an important consequence of stress-sensitization with RSD is recurring anxiety after re-exposure to an acute stressor. This grant proposal will focus on stress- sensitization of the myeloid population that establishes in the spleen. The recurrence of anxiety is dependent on the release of inflammatory Ly6Chi monocytes from the spleen, which traffic to the brain and augment neuroinflammation. We report that splenectomy and blockade of norepinephrine (NE) both prevent increased monocyte trafficking to the brain and both block the recurrence of anxiety in stress-sensitized mice. Furthermore, novel data show that RSD causes substantial engraftment of hematopoietic stem progenitor cells (HSPCs) in the spleen. We highlight novel data that HSPCs in the spleen serve as a generator of ?stress- sensitized? myeloid cells that are readily released following acute stress-induced activation of the sympathetic nervous system (SNS). Thus, we hypothesize that RSD promotes the establishment of a unique splenic myeloid population that becomes a critical cellular inflammatory mediator of recurring anxiety-like behavior. To address this hypothesis, three aims are proposed here using RSD in mice. (Pts.3, 4&12) In Aim-1, we will determine the kinetics and transcriptional profiles of splenic monocytes and HSPCs in stress-sensitized mice. (Pts.5, 11&12) In Aim-2, we will determine the sympathetic-dependent stromal cues that facilitate the development of splenic myelopoiesis with stress-sensitization. Several pharmacological and genetic interventions will be used to address the specific contribution of ?-adrenergic receptor activation in the release, establishment, and maintenance of this unique splenic HSPC population. In Aim-3, selective ablation of splenic monocytes will be used to prevent stress-sensitization and block the recurrence of anxiety. Several advanced strategies will be used to do this. Collectively, completion of these aims will provide new insight into long-term sensitization of myeloid cells by RSD and their critical contribution to recurring anxiety.

Neuroimmunology is the interdisciplinary study of interactions between the immune system and the central nervous system in health and disease. The Ohio State Wexner Medical Center is a unique training environment in this context because the faculty mentors of the proposed training program have expertise that covers all major aspects of basic and clinical Neuroimmunology. The goal of this training grant is to provide comprehensive training across key areas of relevance within the field of neuroimmunology including neurotrauma, autoimmunity, stress, neural development, aging, and biological rhythms. Students will enter the program either soon before or immediately after successful completion of their candidacy exam. These students will have an opportunity to work in a highly collaborative research environment with established mentors and clinical partnerships. Thus, we are unique nationally in terms of crossing the boundaries of traditional neuroscience and immunology training, and therefore are positioned to offer students an exceptional training experience in Neuroimmunology that will extend beyond the conventional training experiences that currently exist within ?umbrella? graduate training programs. Trainees will engage in unique program-specific learning and professional development experiences including course-work, clinical experiences/mentorship, and professional networking with scholars in the Neuroimmunology field. These trainees will be a benefit to the national neuroscience and immunology communities based on their advanced graduate training experience that is augmented by a translational experience with the clinical pairing. Our TPNI students will be positioned to excel as post-doctoral fellows and will be competitive for future faculty positions.