Katerina Akassoglou - US grants

Abstract not provided.

bioimaging /biomedical imaging; technology /technique development

[unreadable] DESCRIPTION (provided by applicant): Fibrin is a blood-derived protein deposited in the nervous system after disease associated with vasculature rupture; such as stroke, multiple sclerosis (MS) and brain glioblastomas. Our previous studies in mice genetically or pharmacologically deficient in fibrinogen demonstrated that fibrin inhibits peripheral nerve regeneration and exacerbates inflammatory demyelination in the central nervous system in an animal model for MS. Our long term goal is to identify the molecular and cellular interface that fibrin utilizes to induce nervous system pathology. [unreadable] [unreadable] The specific hypothesis of this proposal is that fibrin interacts with the p75 neurotrophin receptor (p75NTR) to mediate its effects in the nervous system. Our hypothesis is based on the observations that 1: Fibrinogen and its derivatives directly bind to p75NTR and thus may directly modulate receptor function; 2. p75NTR expression in vivo regulates the bioavailability of fibrin and its derivatives in the nervous system. 3. Fibrinogen and its degradation products mediate diverse survival responses via p75NTR. [unreadable] [unreadable] Based on these observations, the experimental focus of this proposal is on the biochemical, in vivo and in vitro analysis of the p75NTR interactions with fibrinogen. The specific aims are to: 1. Establish the biochemical interactions of p75NTR with fibrinogen and its derivatives, 2: Determine the biological effects induced by fibrin(ogen) in p75NTR - expressing cell types in the PNS and CNS and 3: Establish the contribution of p75NTR / fibrin interactions in nerve regeneration and inflammatory demyelination. Since p75NTR is upregulated in the adult nervous system in response to injury, identifying the molecular interactions of p75NTR with fibrin could potentially provide injury-specific targets for pharmacological intervention in a variety of diseases characterized by vascular damage, endothelial cell activation and decreased capacity for tissue repair. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Fibrinogen, a critical component of blood coagulation, extravasates across a damaged blood-brain barrier (BBB) and accumulates as fibrin deposits at specific sites of injury. The widespread deposition of fibrin within the nervous system is well documented in demyelinating plaques in Multiple Sclerosis (MS). Our published work pioneered studies of fibrinogen in CNS disease. We identified fibrinogen as a novel molecular link between BBB disruption and inflammatory demyelination. We demonstrated novel cellular functions of fibrinogen as an activator of microglia via interaction with the CD11b/CD18 integrin. Given that fibrin and its cell surface receptors play a role in both the inflammatory response in the CNS and tissue remodeling/repair, they are prime candidates to be critical determinants of inflammatory demyelination. Our ultimate goal is to design novel therapeutic approaches to specifically target the proinflammatory functions of fibrin in the CNS with potential application in MS and other neurologic diseases associated with fibrin deposition. In this grant renewal application we employ a multifaceted experimental design to determine the role of fibrinogen in T cell trafficking in the CNS using state-of-the-art in vivo two-photon microscopy (Aim 1), the mechanisms of fibrinogen/CD11b signaling in T cell immune modulation and epitope spreading using genetic models of fibrinogen depletion or specific inhibition of its interactions with its receptors in viv (Aim 2), the pathways downstream of fibrinogen/CD11b/CD18 signaling involved in the activation of the innate and adaptive immune responses in the CNS (Aim 3), and the therapeutic potential in neuroinflammatory disease of two novel inhibitors developed in our laboratory that specifically target the interaction of fibrinogen with CD11b/CD18 without adverse effects in blood coagulation (Aim 4). The proposed studies will provide a cellular and molecular definition of the role of hemostatic factors in the pathogenesis of inflammatory demyelination and will determine the therapeutic efficacy of novel inhibitors of fibrinogen function in neuroinflammation. Identifying the mechanisms of fibrin actions in the nervous system could ultimately illuminate new therapeutic strategies for the treatment of MS.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Blood-brain barrier disruption is a hallmark of nervous system diseases associated with vascular rupture, such as stroke, multiple sclerosis (MS), brain glioblastomas and spinal cord injury. However, the molecular and cellular mechanism of the contribution of blood components to CNS pathogenesis remains poorly understood. Our previous studies identified that fibrinogen, a major blood factor deposited in the nervous system after BBB disruption, inhibits peripheral nerve regeneration and exacerbates inflammatory demyelination in the central nervous system in an animal model for MS. The specific hypothesis in this proposal is that BBB disruption that leads to leakage of fibrinogen in the CNS is responsible for microglial activation. Our hypothesis is based on the observations that: 1. Using two-photon microscopy, microglia respond very rapidly by process extension and isolation of the traumatized sites to blood vessel damage in both the brain and spinal cord;2. Fibrinogen activates microglia in vitro via signaling through the CD11b/CD18 integrin receptor resulting in a dynamic rearrangement of the actin cytoskeleton resulting to increase in phagocytosis;3. Fibrinogen depletion in vivo results in decreased microglial activation in an animal model for MS. Based on these observations, the experimental focus of this proposal is on the direct demonstration of microglial activation by BBB disruption and fibrinogen leakage using in vivo imaging in the mouse brain and spinal cord. Since BBB disruption and microglial activation are hallmarks for several neurodegenerative diseases, we expect our work to provide a state-of-the-art demonstration of the molecular -crosstalk between blood factors and the brain parenchyma as it relates to glial cell activation and the development of CNS pathology.

In vivo imaging by two-photon microscopy has revolutionized our understanding of neurobiological and immunological processes and revealed unexpected information on the dynamic morphology of central nervous system structures and immune processes in health and disease. Currently, intracellular signaling cascades can only be studied in isolated cells or slices of brain tissue. The goal of this EUREKA proposal is to image signal transduction pathways in the living brain in mice, with the aim to study signaling events as they are regulated in real time by trauma, inflammation, and hypoxia. In this proposal we will (1) develop genetically modified biosensorexpressing mice to image the activation of five major signal transduction pathways-redox changes, NFB, Rho, PKA, and Ras- in microglia and macrophages in vivo;(2) generate novel genetically encoded biosensors for the PKA, Rho, and Ras pathways, optimized for in vivo imaging by two-photon microscopy;(3) develop the first pharmacologic tools (biosensor-conjugated Quantum Dots) to deliver biosensors to microglia and macrophages in the brain;and (4) demonstrate dynamic signaling events in microglia in living mouse brain in real time during hypoxia, inflammation, or traumatic injury. The tools we propose to develop will fundamentally change the methods we use to study the dynamic activation of intracellular signaling pathways. Results from this proposal will elucidate inflammatory signal transduction pathways in an unprecedented way-by correlating molecular mechanisms of microglial activation with dynamic morphologic alterations in response to extracellular stimuli in the brain of a living animal. This proposal will generate tools to make possible imaging of signal transduction in vivo. It will make available to the scientific community novel biosensors, delivery methods, and animal models to study the temporal and spatial activation of five major signal transduction pathways in the living animal. In vivo imaging of signal transduction pathways in microglia and macrophages can be applied to a wide range of diseases with an inflammatory component, including cancer, diabetes, asthma, infection, and autoimmune disorders, and nervous system disorders characterized by microglia activation, such as multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, pain, and spinal cord injury.

DESCRIPTION (provided by applicant): In central nervous system pathologies, including multiple sclerosis, stroke, spinal cord and traumatic injuries, scar formation consisting of reactive astrocytes and deposition of extracellular matrix is a major inhibitor of tissue repair. The molecular mechanisms that trigger astrocyte activation in nervous system disease remain incompletely characterized. We have shown that the neurotrophin receptor p75NTR regulates repair processes by inhibiting fibrin degradation and regulating cell differentiation. Our long-term goal is to characterize the molecular pathways that are responsible for the effects of p75NTR in nervous system pathogenesis, as a prerequisite for the development of therapeutic protocols that can specifically target p75NTR signaling and attenuate neuropathological disease processes. Our major hypothesis is that intramembrane proteolysis of p75NTR regulates TGF-¿ signaling to control astrocyte functions during development and disease. Our preliminary data demonstrate that a) the intracellular domain of p75NTR (p75ICD) is a novel component of the nuclear pore complex in astrocytes, b) p75NTR directly binds to the natively unfolded FG-domain of nucleoporin 153 (Nup153), c) TGF-¿ induces 3-secretase-dependant cleavage of p75NTR resulting in its translocation inside the nuclear pore, d) p75NTR regulates of Smad2, and e) p75NTR regulates astrocyte differentiation and TGF-¿ functions in the CNS in vivo. Our specific aims are designed to test our working model, in which intramembrane cleavage of p75NTR results in remodeling of the nuclear pore complex that allows nucleocytoplasmic shuttling of Smad2 and induces astrocyte differentiation and activation. We employ a multiphaceted experimental design that includes transgenic models of TGF¿-induced astrocyte activation, generation of new transgenic mice for cell-fate mapping of p75NTR - expressing cells, atomic force microscopy and three-dimensional electron tomography to determine the role of cleaved p75NTR in the dynamic remodeling of the nuclear pore complex in astrocytes, and biochemical experiments to define how p75NTR cleavage regulates Smad2 nucleocytoplasmic shuttling and its coupling to the TGF¿ transcriptional machinery. Identifying the molecular interplay between p75NTR and TGF¿ signaling pathways could potentially provide injury-specific targets for pharmacological intervention in a variety of diseases characterized by astrocyte scar formation and decreased capacity for tissue repair.

DESCRIPTION (provided by applicant): Funds are sought for partial support of the 2014 Plasminogen Activation and Extracellular Proteolysis Gordon Research Conference (GRC) and Gordon Research Seminar (GRS). These are paired conferences held sequentially at the same location, February 8-9, 2012 for the GRS, and February 9-14 for the GRC. The GRC has been held continuously every two years since 1990 and enjoys an outstanding international reputation. It brings together a group of highly motivated participants with diverse scientific backgrounds that engage in intensive, yet informal, discussions at the frontier of research related to the plasminogen activation system and associated extracellular proteases in an off-the-record fashion. The conference thus provides a unique opportunity for the leading researchers in the world from a broad range of disciplines to present their latest unpublished research related to plasminogen activation and extracellular proteolysis, and to discuss innovative ways to translate their research into new therapies. Previous research in our field has led to the development of tissue plasminogen activator (tPA) for the successful treatment of a wide range of thromboembolic disorders, and the discovery of important roles for the plasminogen activation system extracellular proteolysis in a number of physiologic and pathologic processes. In the 2014 meeting, emphasis will be placed on Mechanisms, Imaging, and Therapeutics. The 2014 GRC will be launched by our Keynote Speaker Roger Tsien, Nobel Laureate for the discovery of GFP, who will discuss novel protease probes. Breakthrough findings in plasminogen activation and extracellular proteolysis in vascular biology, central nervous system function and dysfunction, traumatic brain injury, Alzheimer's disease, tissue homeostasis and regeneration, hematopoiesis, stem cell biology, metabolism and obesity, tumor biology, cardiovascular function, and angiogenesis will be discussed. Novel modalities of biomedical imaging and bioengineering in the field of proteases will be presented. Since translating basic research into treatments is a major focus of the 2014 meeting, the cutting- edge in biomarkers, drug delivery, and new therapies in our field will be discussed. Attendees will be selected by invitation and from applications submitted online. They will be chosen to represent a diverse spectrum of researchers, and a strong emphasis has been made on the inclusion of women and underrepresented minorities as Discussion Leaders and Speakers, and will continue on Poster Presenters, and Attendees. The associated GRS is a novel feature that will provide a unique opportunity for young scientists to share in the GRC experience. This two-day seminar will be organized by Graduate Students and Postdoctoral Fellows with the support of leading researchers, and will allow junior researchers within the field of plasminogen activation and extracellular proteolysis to come together to discuss their current research, while building informal networks with their peers that may lead to a lifetime of collaboration and scientific achievement.

DESCRIPTION (provided by applicant): Patients with central nervous system pathologies, including multiple sclerosis, stroke, spinal cord and traumatic injuries, often present with cognitive impairment, indicative of neuronal dysfunction. Although vascular damage and blood-brain barrier (BBB) disruption, which results in leakage of blood proteins into the brain parenchyma, are hallmarks of cognitive pathologies, the molecular links between BBB disruption and neuronal dysfunction remain poorly understtod. We have shown that CNS deposition of fibrinogen, a critical component of blood coagulation, is not merely a marker of BBB disruption, but plays a causative role in the regulation of inflammation and repair in the CNS by activating integrin receptors expressed in nervous system cells. Our long-term goal is to characterize the molecular pathways that are responsible for the effects of fibrinogen in nervous system pathogenesis, as a prerequisite for the development of therapeutic protocols that can specifically target the interactions between fibrinogen and its receptors and attenuate neuropathological disease processes. Our major hypothesis is that fibrinogen activates the CNS innate immune response to induce spine alterations and cognitive deficits in nervous system pathology. Our preliminary data demonstrate that a) stereotactic injection of fibrinogen into the dentate gyrus induces microglial activation and impairs memory recall, b) injection of fibrinogen induces neuronal loss, dendrite retraction and dendritic spine density reduction in mice as shown with in vivo two-photon microscopy, and c) genetic depletion of the CD11b/CD18 microglial receptor rescues fibrinogen-induced spine elimination and dendritic retraction. Our specific aims are designed to test our working model, in which fibrinogen, deposited in the brain following BBB disruption and cerebrovascular abnormalities, activates the innate immune response and causes spine elimination and cognitive decline. We employ a cutting edge experimental design that includes in vivo two-photon imaging of neurons in transgenic mice expressing YFP under Thy1 promoter, following the dynamic interactions between microglial and spines over time in the living mouse, and pharmacologic and genetic inhibition of innate immune activation, including specific inhibition of fibrinogen interactions with CD11b that do not affects its beneficial functions in blood coagulation. Identifying the molecular interplay between fibrinogen following BBB disruption, activation of innate immunity, and neurotoxicity could potentially provide specific targets for pharmacological intervention in a variety of diseases characterized by cerebrovascular abnormalities or increased BBB permeability and cognitive impairment.

PROJECT SUMMARY/ABSTRACT The neurovascular interface fundamentally changes during CNS diseases due to increased blood-brain barrier permeability and influx of plasma proteins in the CNS parenchyma. Studying neurologic diseases through the multidisciplinary prism of vascular biology, immunology, and neuroscience could be critical for the identification of novel mechanisms of disease, discovery of imaging tools and therapeutic treatments for a wide range of neurologic diseases characterized by BBB disruption. In my laboratory we made unanticipated discoveries on the functional role of BBB disruption in CNS autoimmunity, glial cell activation, and neurodegeneration. We identified leakage of blood proteins in the brain and neurotrophin receptor signaling as novel molecular mediators at the neurovascular interface that regulate glial ? neuron cross-talk and the communication between the brain and the immune system. Furthermore, we developed novel methods for high-resolution two-photon microscopy of the neurovascular interface in vivo. Our aim is to understand the mechanisms that control the communication between the brain, immune and vascular systems with the ultimate goal to design novel therapies for neurologic diseases. In this application we propose a multipronged approach to determine the role of neurovascular dysfunction in neurodegeneration, CNS repair, and glial cell biology and discover novel genetic regulatory circuits that control vascular-driven CNS innate immune mediated neurotoxicity. We use an innovative experimental design consisting of in vivo two-photon, super-resolution and electron microscopy of the neurovascular interface, electrophysiology, cell biology and signal transduction, new genetic tools and animal models, and genomic and proteomic approaches. The proposed studies will set the foundation how neurovascular dysfunction regulates brain functions and the outcomes of this research would be applicable for the understanding of the etiology and the development of new treatments for several neurologic diseases, such as multiple sclerosis, stroke, spinal cord and brain injury.

Project Summary / Abstract Traditionally, neurological diseases have been classified into mechanistically distinct categories, such as neurodegenerative, inflammatory, and vascular. However, recent insights have led to a reassessment of the complex relationship between diseases and their mechanisms. Emerging evidence supports a role for vascular dysfunction as an early feature in AD that is an equal and independent predictor for cognitive decline compared to amyloid and correlates with worse prognosis in AD. However, the cellular and molecular mechanisms at the neurovascular interface that promote cognitive decline are poorly characterized. Furthermore, whether and how vascular alterations contribute to neuronal network and synaptic dysfunction, one of the earliest manifestations of AD, is unknown. A fundamental change at the neurovascular interface in AD is the deposition of the blood coagulation factor fibrinogen, which is deposited as insoluble fibrin in the AD brain. Our ultimate goal is to determine the dynamic interactions between innate immunity, vascular, and blood-derived signals and their causal relationships in regulating impaired synaptic activity as a prerequisite for devising novel therapies to improve synaptic and cognitive functions after vascular impairment. Studies from our laboratory and others have shown that genetic or pharmacologic depletion of the blood coagulation factor fibrinogen protects from neuroinflammation in several models of neurological disease. Our preliminary data demonstrate that dendritic spine elimination occurs around fibrinogen deposits in AD mice and fibrinogen-CD11b signaling promotes dendritic spine loss and cognitive impairment in AD mice. The four specific aims will are designed to determine the role of fibrinogen/CD11b signaling in microglial-synapse interactions and neuronal network abnormality, determine the mechanisms underlying fibrin-induced innate-immune driven neuronal dysfunction, and the therapeutic implications of targeting fibrin-microglia interactions in protecting from spine elimination and neuronal dysfunction. Our experimental design is based on a cutting-edge multi-pronged experimental approach consisting of in vivo two-photon imaging of neuronal activity and microglial dynamics, EM co-registration, iDISCO, unbiased transcriptomics and proteomics, and combined two-photon imaging with in vivo EEG recordings. The proposed studies will set the foundation how neurovascular dysfunction regulates synapse elimination and neuronal activity and the outcomes of this research would be applicable for the understanding of the etiology and the development of new treatments for vascular cognitive impairment including in AD and related conditions.