Susanna Rosi - US grants

DESCRIPTION (provided by applicant): In this study we propose to use immediate-early gene (IEG) expression imaging to assess the effects of brain irradiation on neuronal functioning, with the long term goal of identifying how the IEG Arc (activity regulated cytoskeleton-associated protein) is affected in the evolution of radiation- induced neuronal deficits. Therapeutic irradiation is commonly used to treat brain tumors but can cause significant damage to normal brain tissues. In general, overt tissue injury occurs after relatively high doses of irradiation, but after lower doses such tissue damage may not occur, however, hippocampus-dependent cognitive decline, including deficits in spatial learning and memory consolidation, can develop. The severity of such effects depends on the dose delivered to the medial temporal lobes, which contain the hippocampus, a region responsible for learning and memory. The pathogenesis of radiation-induced cognitive deficits is poorly understood, but is likely multifaceted, involving altered neurogenesis, chronic neuroinflammation, and chronic oxidative stress, all factors that can impact multiple neural processes and synaptic transmission. Our previous analyses of chronic neuroinflammation suggest that the depletion of synaptic activity-dependent proteins may play a critical role in cognitive decline. Particularly important in this context is the observation of alterations in the immediate-early gene product Arc, the expression of which has been used to dissect, cellular networks involved in encoding spatial and contextual information. Furthermore, the presence of chronically activated microglia is associated with the disruption of Arc expression and cognitive dysfunctions. Activated microglia may alter the coupling of neural activity with macromolecular synthesis implicated in learning and memory consolidation. Thus, Arc is closely associated with critical factors recently described as playing contributory if not causal roles in the development of radiation-induced cognitive impairments. We hypothesize that irradiation of the brain will adversely affect neuronal function, as assessed by the molecular distribution of Arc at the level of mRNA and protein expression. We further contend that this will be reflected in alterations in neurogenesis and behavioral performances, and that such effects will be dose dependent. Finally, we assert that these changes are influenced by the presence of activated microglia, and we will use mice deficient in chemokine receptor 2 to gain mechanistic insight into this relationship. Given the well-established temporal kinetics of Arc transcription, translocation and translation, we will be able to provide novel sight into the post-transcriptional infrastructure of gene expression underlying mechanisms associated with cognitive function. These studies will give new information about how ionizing irradiation impacts neuronal function in the brain. These types of data are currently unavailable and represent an essential first step for determining the risks of specific CNS-related effects and for the development of potential strategies to manage radiation brain injury. PUBLIC HEALTH RELEVANCE: Although cranial irradiation is commonly used for the treatment of brain tumors, there is a significant probability that this treatment also produces adverse effects severely impacting quality of life (i.e. cognitive impairments). The proposed studies will provide new information about how ionizing irradiation impacts mechanisms of neuronal function in brain region associated with learning and memory processes. These types of data are currently unavailable and are essential for determining the risks of specific central nervous system related effects and for the development of potential strategies to manage radiation-mediated brain injury.

DESCRIPTION (provided by applicant): In this study we propose to use immediate-early gene (IEG) expression imaging to assess the age-related long-term effects of traumatic brain injury (TBI) on the posttranscriptional infrastructure of gene expression involved in synaptic plasticity and memory. TBI is the leading cause of neurological disability in the world; the critical changes that affect cognition take place over a long period of time the initial injury, and age is a substantial factor in both the risk of and the incidence of acquired brain injury. While the pathogenesis of TBI- related cognitive impairment is uncertain, it is likely multifaceted involving diffuse axonal injury, altered neuronal integrity, imbalances in neurotransmitters, changes on brain metabolism, hippocampal vulnerability; these pathophysiological factors might ultimately alter neuronal plasticity and cause memory deficits. The goal of this proposal is to advance our limited knowledge of the age-related changes in the cellular mechanisms controlling hippocampal neuronal plasticity and memory after TBI. Our working hypothesis is that the long term cognitive dysfunctions resulting from TBI are mediated through altered de novo synthesis of plasticity-related IEGs and consequent disruption of hippocampal network activity. Our hypothesis is based on our recent published data demonstrating that dysregulation of the plasticity-related IEG Arc (activity-regulated cytoskeleton-associated protein) expression parallels cognitive dysfunctions observed two months after TBI. The IEG Arc is expressed in response to synaptic activity and is required for engaging durable plasticity processes that underlie memory; Arc is the only known activity-induced gene that correlates both temporally and spatially with the stimulus that induced its transcription. Given its critical role on synaptic plasticity and its well defined kinetics Arc represents the best candidate to study altered synapti plasticity and to test our hypothesis. Using imaging method called catFISH (cellular compartment analysis of temporal activity with fluorescence in situ hybridization) it is possible t detect the sub-cellular localization (nucleus and cytoplasm) of Arc mRNA in response to synaptic activity in a time-dependent manner. This technique provides excellent temporal and cellular resolution and facilitates mapping of neuronal activity; furthermore, it provides an innovative way to evaluate hippocampal networks mediating contextual and spatial information processing. Based upon our data on TBI, combined with our previous findings on hippocampal network activity we are proposing to use catFISH to: 1) identify the dynamics of the post transcriptional infrastructure of gene expression involved in synaptic plasticity and memory after TBI in behaviorally characterized young and old mice; 2) determine how age at the time of TBI affects hippocampal networks mediating contextual and spatial information processing. From these studies we will establish the role and the effect of age on the progression of TBI-related cognitive impairments from a behavioral, cellular and network prospective. These types of data are currently unavailable and are essential for the development of treatments and strategies to manage TBI- mediated neurocognitive dysfunctions.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a major risk factor for the development of multiple neurodegenerative diseases, including Alzheimer's disease (AD) and numerous recent reports document the development of dementia after TBI. Following the initial mechanical insult, TBI activates a cascade of molecular signaling events that can result in neurodegenerative sequelae, namely cognitive dysfunction. One of the most pronounced responses following TBI is the induction of multiple signaling mediators associated with neuroinflammation, consistently attributed to the activation of the innate immune system. Inflammation is a vital host response to injury, however excessive and unchecked propagation of inflammation can be deleterious to primarily unaffected tissues. Recent work in both humans and various animal models has shown that microglia, the brain's resident immune cells, can remain in an activated state for years after the initial insult. Despite consistent findings implicating the deleterious effects of chronically activated microglia in the brain, little is know about the role of the peripheral innate immune response and its interface with CNS tissues following TBI. Recent work has shown that cell surface expression of Ly6C and CCR2 is characteristic of an inflammatory subpopulation of bone marrow derived blood circulating monocytes. However, there is still a gap in the current knowledge as to the role and function of Ly6ChiCCR2+ monocytes in the propagation of TBI- induced pathology. The ultimate goal of this proposal is to elucidate the functional contribution of this cell subpopulation on TBI-induced neuroinflammation, as well as synaptic and cognitive dysfunction. Aim 1. Will examine if genetic and pharmacological deletion of CCR2 signaling ameliorates TBI-induced synaptic and cognitive dysfunction. TBI will be induced using controlled cortical impact on both wild type and CCR2RFP/RFP mice. We will examine hippocampal-dependent cognitive function as well as homeostatic synaptic function, 28 days after injury. Preliminary studies indicate that CCR2 deletion abrogates TBI-induced hippocampal cognitive dysfunction compared to WT mice. Aim 2. Will determine the temporal kinetics and inflammatory profile of TBI-induced Ly6ChiCCR2+ monocytes/macrophages into the brain parenchyma. TBI will be induced as in Aim 1 except using CX3CR1+/GFPCCR2+/RFP mice. Multiple time points following injury will be examined to include acute, subacute, and chronic phases. Preliminary data shows that 48 hours after injury, TBI-treated mice had a significant increase in macrophage infiltration and that a specific subset of those resembled resident microglia. Our studies will provide critical and novel information in regard to the contribution of peripheral macrophage accumulation in the pathogenicity of TBI-induced neuroinflammation and potentially a novel therapeutic target and optimal time point for its treatment.

? DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a major risk factor for the development of multiple neurodegenerative diseases, including Alzheimer's disease (AD) and dementia. One of the most pronounced responses following TBI is the induction of multiple signaling mediators associated with neuroinflammation, consistently attributed to the activation of the innate immune system. TBI-induced chronic activation of mononuclear phagocytes (microglia/macrophages, MPs) has been shown to persist for many years following the initial insult in humans and animal models. Activated MPs, the key effectors of inflammatory processes in the brain, can play a dual and decisive role in the pathophysiology of TBI, promoting inflammation (M1-polarized) or inducing repair (M2- polarized). Multiple studies have shown that MPs undergo metabolic reprogramming that differs drastically upon their polarization status. Similar to the Warburg effect observed in tumor cells, M1-polarized MPs increase lactate release, whereas M2-polarized MPs mainly employ oxidative metabolism. More precisely, recent evidences suggest that pyruvate metabolism is a key player in the differential activation of MPs. Our published data demonstrate a strong and permanent induction of the M1 preceded M2 response at acute, sub-acute and chronic time points after TBI. We demonstrated that modifying this response can rescue long term cognitive deficits. Therefore, non-invasive assessment of M1/M2 macrophages in vivo would be important for the development and validation of treatment strategies targeting TBI-dependent cognitive deficits. However, to date, no non-radioactive imaging technique can non-invasively assess neuroinflammation directly, even less distinguish between M1 and M2 macrophages. To solve this specific problem, the goal of this study is to validate, to our knowledge for the first time in TBI, a new technique, namely 13C Magnetic Resonance Spectroscopic Imaging of hyperpolarized (HP) [1-13C] pyruvate, to monitor M1/M2 neuroinflammation in vivo in the brain. Aim 1: Validate 13C MRSI of HP pyruvate to non-invasively measure MPs polarization status in vivo after TBI. We will validate 13C MRSI of HP [1-13C] pyruvate as a method to non-invasively determine MPs polarization status in vivo at acute, sub acute and chronic time points after mild/moderate and moderate TBI in rodents. Aim 2: Evaluate in vivo metabolic imaging to monitor response to an M1/M2 polarization modifying therapy: We will use our metabolic imaging approach to monitor response to a clinically relevant therapy that affect M1/M2 polarization and rescue long term cognitive outcome. This project will validate a new robust and clinically translatable metabolic imaging approach allowing for non-invasive assessment of MPs activation and response to therapy in TBI. Additionally, upon clinical translation, the method developed in this proposal could improve diagnosis and prognosis for TBI progression, help refine therapeutic regimens and, ultimately, lead to better clinical outcome and patient quality of life.

Project Summary Traumatic brain injury (TBI) is extremely debilitating for the aging community with both increased incidence and outcome severity within this population. Furthermore, TBI is a strong environmental risk factor for development of Alzheimer's disease and other dementia related illnesses. The importance of age as a prognostic factor after TBI has long been recognized but limited studies have been devoted to understand mechanisms that regulate secondary events that occur after the initial trauma. Even less research has been aimed at studying the mechanisms of cognitive loss in the elderly. The critical changes that affect cognition take place over a long period of time following the initial insult and the innate immune system activation is a key secondary injury mechanism that contributes to chronic neurodegeneration and loss of neurological function. In this proposal we will investigate the respective contribution of infiltrating macrophages and activated resident microglia in production of a neurotoxic and inflammatory milieu as well as direct interactions with neuronal synapses following TBI in an aging animal. Preliminary data for this proposal has found that TBI causes an exacerbated and prolonged CCR2+ macrophage infiltration in the aging brain. The increased recruitment of peripherally derived monocytes significantly augments TBI-induced neuroinflammatory sequelae and is paralleled by an increased expression of the superoxide-generating enzyme NOX2 which may potentiate injury-induced cognitive dysfunction observed in old animals. All together these findings demonstrate that, in the aging brain, peripherally derived macrophages have a distinct contribution to the TBI-related inflammatory response. Based upon these observations we hypothesize that the robust infiltration of peripherally derived macrophages and the consequent inflammatory response is responsible for exacerbated loss of cognitive functions by decreasing dendritic spine density. In this proposal, we will identify the temporal relationships between macrophage infiltration/microglia activation and inflammatory profiles in an aging brain after injury. Furthermore, we will identify mechanistic links between macrophage infiltration and altered dendritic spine morphology. We will determine if TBI-induced cognitive deficits are a direct result of macrophage induced ROS production and/or inappropriate synaptic pruning. Finally, we will investigate if blockade of macrophage infiltration can mitigate injury-induced neurotoxicity thereby alleviating cognitive deficits. Findings from this work will advance mechanistic understanding of secondary mechanisms associated with TBI and test two pharmacological agents (already in clinical trials) for treatment of TBI-induced cognitive deficits in an aging animal.

Project Summary Therapeutic irradiation is commonly used to treat both primary and metastatic brain tumors and can cause a number of late effects including progressive cognitive dysfunction. There is no treatment currently available that can even partially reverse cognitive changes observed after radiation injury. Specifically, irradiation of the temporal lobe can profoundly affect the cellular structures mediating learning and memory. Ionizing radiation has also been consistently shown to activate several neuroinflammatory signaling cascades that can impact multiple neural processes and synaptic transmission ultimately causing disruptions in hippocampal function. Notably, resident microglia and infiltrating monocytes, the key cellular player in neuroinflammatory processes, have distinct embryological origins and also fulfill different functions. The mechanism/s by which activation of the inflammatory response affect cognitive functions after brain irradiation and the specific role of different myeloid cells remain elusive. Thus, there is a clear need to understand the mechanisms of radiation injury and inflammation to develop strategies for preventing cognitive decline following cranial irradiation. Recent work from our group during the previous funding period has shed light in these questions and revealed specific problems in the cellular and molecular mechanisms underlying radiation-induced memory deficits. Specifically our data demonstrates a direct link between CCL2/CCR2 and cognition. These results provide a mechanistic link between peripheral innate immune system and cognition after brain irradiation. In the current proposal we will evaluate the central hypothesis that therapeutic doses of cranial irradiation induce infiltration of peripheral monocytes that modifies the resident inflammatory response and promotes synaptic dysfunction and long term cognitive deficits. Aim 1: Determine the kinetics and inflammatory phenotype of radiation-induced myeloid cell alterations after single and hypofractionated therapeutic doses of irradiation. Aim 2: Evaluate the role of peripheral monocyte recruitment into the brain as a mechanistic driver of radiation-induced altered synaptic and cognitive functions. Aim 3: Determine if temporary depletion of myeloid cells prevent the loss of synaptic function and cognition after single and hypofractionated doses of radiation. Very little is known in regard to the evolution of radiation induced pathophysiology in the context of peripherally derived macrophage accumulation or inflammation, and how this relates to altered synaptic and cognitive function. Our final therapeutic goal is to modify the cognitive changes observed after radiation injury.  

PROJECT SUMMARY Traumatic Brain Injury (TBI) is one of the most significant Public Health trauma diseases of our time. Diagnoses of various TBI forms rely on patient self-reporting, established clinical rating scales, and multi- modality imaging to an extent. TBI elicits complex, heterogeneous pathological events, including mechanical injury, hemorrhage, and progressive secondary processes such as central and peripheral inflammation dynamics and excitotoxic driven necrotic and apoptotic events. A focal contusion TBI model that recapitulates various aspects of TBI observed in human is the controlled cortical contusion impact (CCI). The CCI rodent model can be used to determine longitudinal measures of TBI promoted neuropathological tissue changes. The major central nervous system (CNS) neurotransmitter L-glutamate (L-Glu) targets the excitatory amino acid transporter 2 (EAAT2) that clears 90 % of synaptic L-Glu. EAAT2 is expressed throughout the CNS and is primarily found on astroglial (astrocyte) cell membranes. Spatiotemporal changes to CNS EAAT2 protein densities are found in TBI postmortem brain tissues, including those from rodent TBI models where 20-40% reductions of EAAT2 is observed at acute times after injury. Positron emission tomography (PET) imaging is a technique that is sensitive and quantitative at the molecular level where it uniquely determines a positron-labeled tracer binding potential to its targeted CNS protein, thereby quantifying target protein density in live CNS tissues. A first-in-class fluorine-18 labeled (18F) tracer known as 18F-FAA has been discovered and possesses outstanding in vivo PET imaging qualities for quantitative determinations of CNS astrocyte EAAT2 target protein densities in live brain. Rio Pharmaceuticals, Inc., has licensed this EAAT2 PET imaging tracer technology and is further developing it for eventual clinical use. It is thought that the EAAT2 tracer is suitable to mark severity and localization of TBI in live brain. We hypothesize that CCI-promoted traumatic brain injury results in acute and latent regional cerebral tissue pathologies that can be marked in vivo by determining CNS EAAT2 protein density changes measured by quantitative dynamic PET imaging and correlated to in-vitro regional cerebral tissue EAAT2 and GFAP density alterations over time. Our long-term objective is to advance the development of a quantitative PET imaging tracer for CNS EAAT2 astrocyte protein target as an effective marker of TBI severity and localization. Ultimately, clinical EAAT2 CNS PET imaging will aid quantitative TBI diagnoses and afford a means to follow CNS tissue changes as a result of novel TBI therapies. The goal of this Phase 1 investigation is to establish an initial proof-of-concept (POC) demonstrating that dynamic PET imaging of the astrocyte EAAT2 target protein in live CCI TBI rat brain is a quantitative marker of TBI severity and localization. Establishing the Phase 1 POC will permit subsequent Phase 2 tracer development investigation in both males and female rodents, with experiments to further interrogate EAAT2 in model TBI brain (e.g., CCI and closed-head concussive injury) and studies to satisfy FDA criteria for evaluation of the PET imaging technology for clinical safety and TBI use efficacy. The industrial-academic collaborative Phase 1 POC project goal will be accomplished with the following three specific aims over one year. Specific Aim 1: Evaluate cerebral PET-CT-MR imaging 1, 3, 7, 30, 60 and 90 days after controlled CCI to afford quantitative cerebral signatures of 18F-FAA tracer binding potentials to EAAT2 target. Specific Aim 2: Measure regional cerebral EAAT2 and GFAP densities and the expression profiles of related markers in postmortem rat brain tissues 1, 3, 7, 30, 60 and 90 days after CCI TBI injury. Specific Aim 3: Establish the Phase 1 POC by determining correlations between regional in-vivo EAAT2 PET imaging tracer binding potential values and postmortem EAAT2 and GFAP measures.

PROJECT SUMMARY: Trauma to the spinal cord and brain (neurotrauma) together impact over 2.5 million people per year in the US, with economic costs of $80 billion in healthcare and loss-of-productivity. Yet precise pathophysiological processes impacting recovery remain poorly understood. This lack of knowledge limits the reliability of therapeutic development in animal models and limits translation across species and into humans. Part of the problem is that neurotrauma is intrinsically complex, involving heterogeneous damage to the central nervous system (CNS), the most complex organ system in the body. This results in a multifarious CNS syndrome spanning across heterogeneous data sources and multiple scales of analysis. Multi-scale heterogeneity makes spinal cord injury (SCI) and traumatic brain injury (TBI) difficult to understand using traditional analytical approaches that focus on a single endpoint for testing therapeutic efficacy. Single endpoint-testing provides a narrow window into the complex system of changes that describe the holistic syndromes of SCI and TBI. In this sense, complex neurotrauma is fundamentally a problem that requires big- data analytics to evaluate reproducibility in basic discovery and cross-species translation. For the proposed TOP-VISION cooperative agreement we will: 1) integrate preclinical neurotrauma data on a large-scale; 2) develop novel applications of cutting-edge multidimensional analytics to make sense of complex neurotrauma data; and 3) validate bio-functional patterns in targeted big-data-to-bench experiments in multi-PI single center (UG3 phase), and multicenter (UH3 phase) studies. The goal of the proposed project is to develop an integrated workflow for preclinical discovery, reproducibility testing, and translational discovery both within and across neurotrauma types. Our team is well-positioned to execute this project given that with prior NIH funding we built one of the largest multicenter, multispecies repositories of neurotrauma data to-date, housing detailed multidimensional outcome data on nearly N=5000 preclinical subjects and over 20,000 curated variables. We will leverage these existing data resources and apply recent innovations from data science to render complex multidimensional endpoint data into robust syndromic patterns that can be visualized and explored by researchers and clinicians for discovery, hypothesis-generation and ultimately translational outcome testing.

Project Summary/Abstract Ionizing irradiation is commonly used to treat both primary and metastatic brain tumors and can cause a number of late effects including progressive cognitive dysfunction. These cognitive changes are particularly severe in individuals who were treated with radiation during childhood. The extent and nature of the resulting cognitive deficits may be influenced by age, treatment and gender . The neurobiological reason for this difference is unknown, and very few experimental studies have addressed this issue. Ionizing radiation in rodents has been consistently shown to activate several neuroinflammatory signaling cascades that can impact multiple neural processes and synaptic transmission, ultimately disrupting hippocampal function. Neuroinflammation, characterized by activation of brain resident microglia and recruitment of peripherally derived monocytes (collectively referred to as `myeloid cells'), has been consistently associated with the loss of cognitive function in mice after radiation. There are still no treatments for preventing or treating radiation-induced cognitive dysfunction. Despite the extensive clinical evidence linking fractionated brain irradiation with cognitive deficits, there are still unanswered gaps in the biologic basis of this observation: the mechanism/s by which activation of the inflammatory response affect cognitive function, and the effect of age and sex. Furthermore, there are no pre-clinical models that recapitulate the features of the most common clinical scenario: patients with central nervous system (CNS) tumors. Our final therapeutic goal is to prevent and treat the cognitive changes observed after fractionated whole-brain irradiation (fWBI) injury. We hypothesize that changes in the composition and function of myeloid cells following brain irradiation can both prevent and rescue cognitive deficits through durable effects on synapses. The translational objective of this proposal is to demonstrate that resetting the immune system by brief microglia depletion prevents the long-term development of memory deficits in a brain tumor model designed to mimic conventional treatment paradigms used in clinical settings. The specific aims in support of our hypothesis are: 1. Establish the effects of fWBI on memory and synaptic composition as a function of age and sex in an immunocompetent mouse glioma model. 2. Determine the role of myeloid cells in the development of fWBI-induced memory deficits. 3. Evaluate the role of myeloid cells as a mechanistic driver of the permanent memory deficits after fWBI. Very little is known in regard to the evolution of radiation induced pathophysiology in the context of peripherally derived macrophage accumulation or inflammation, and how this relates to altered synaptic and cognitive function. Our final therapeutic goal is to modify the cognitive changes observed after radiation injury.

PROJECT SUMMARY: Trauma to the spinal cord and brain (neurotrauma) together impact over 2.5 million people per year in the US, with economic costs of $80 billion in healthcare and loss-of-productivity. Yet precise pathophysiological processes impacting recovery remain poorly understood. This lack of knowledge limits the reliability of therapeutic development in animal models and limits translation across species and into humans. Part of the problem is that neurotrauma is intrinsically complex, involving heterogeneous damage to the central nervous system (CNS), the most complex organ system in the body. This results in a multifarious CNS syndrome spanning across heterogeneous data sources and multiple scales of analysis. Multi-scale heterogeneity makes spinal cord injury (SCI) and traumatic brain injury (TBI) difficult to understand using traditional analytical approaches that focus on a single endpoint for testing therapeutic efficacy. Single endpoint-testing provides a narrow window into the complex system of changes that describe the holistic syndromes of SCI and TBI. In this sense, complex neurotrauma is fundamentally a problem that requires big- data analytics to evaluate reproducibility in basic discovery and cross-species translation. For the proposed TOP-VISION cooperative agreement we will: 1) integrate preclinical neurotrauma data on a large-scale; 2) develop novel applications of cutting-edge multidimensional analytics to make sense of complex neurotrauma data; and 3) validate bio-functional patterns in targeted big-data-to-bench experiments in multi-PI single center (UG3 phase), and multicenter (UH3 phase) studies. The goal of the proposed project is to develop an integrated workflow for preclinical discovery, reproducibility testing, and translational discovery both within and across neurotrauma types. Our team is well-positioned to execute this project given that with prior NIH funding we built one of the largest multicenter, multispecies repositories of neurotrauma data to-date, housing detailed multidimensional outcome data on nearly N=5000 preclinical subjects and over 20,000 curated variables. We will leverage these existing data resources and apply recent innovations from data science to render complex multidimensional endpoint data into robust syndromic patterns that can be visualized and explored by researchers and clinicians for discovery, hypothesis-generation and ultimately translational outcome testing.