Barbara L. Hempstead - US grants

Nerve growth factor (NGF) is an important growth regulatory polypeptide which promotes the survival and differentiation of immature neuroblasts in the developing nervous system. Unlike most growth hormones which stimulate cell replication, NGF inhibits cell proliferation and induces the maturation of cells bearing specific NGF receptors. Thus, the NGF-NGF receptor interaction serves as an important model system to use in investigating the normal mechanisms which regulate cell growth and differentiation. Furthermore, since the effects of NGF extend even to neoplastic cells bearing functional receptors, NGF is of potential clinical significance as a growth inhibitor of neoplasias of neural crest origin. Although NGF was the first growth factor to be identified, its mechanism of action is not well understood, except that its actions are initiated by binding to a high affinity cell surface receptor. This proposal is directed towards defining the role of the NGF receptor in initiating cellular responses to NGF, using the recently cloned gene that encodes the human NGF receptor. A combined molecular biological and biochemical approach will be used to determine the structure of the functionally active high affinity receptor. Specifically we plan to: (a) transfect the human NGF receptor clone into cells derived from the neural crest to determine the cellular characteristics which are responsible for responsiveness to NGF; (b) define the molecular basis of the high and low affinity forms of the receptor by membrane fusion experiments using PEG-mediated fusion; (c) define the domains of the receptor responsible for high affinity ligand binding, internalization and interactions with cell membrane proteins; (d) biochemically characterize the high affinity receptor complex using affinity chromatography and (e) use gene transfer to identify other gene products in the cell membrane which are required for functionally active high affinity receptor.

DESCRIPTION (provided by applicant): Neurotrophins, a family of highly conserved growth factors, are best characterized for critical roles in sculpting the developing peripheral and central nervous system through contrasting actions that regulate neuronal differentiation, survival and cell death. More recent studies reveal important roles in the adult in modulating behavior, synaptic plasticity, and in injury protection. Neurotrophins mediate these effects through two receptors; the Trk receptor tyrosine kinases that mediate survival, plasticity and behavioral effects, and p75, a TNF receptor family member that can induce survival and apoptotic actions. Neurotrophins are initially synthesized as precursors (proneurotrophins, proNTs) that are cleaved to mature forms, long thought to be the biologically active moiety. However proNTs can be released by cells, and our studies indicate that they selectively bind p75 but not Trk receptors to promote neuronal apoptosis. We have uncovered a third receptor, sortilin, which binds to the pro-domains of neurotrophins, and forms a co-receptor complex with p75 to convey apoptosis. In vivo models suggest that proNGF is an inducible cytokine which is upregulated following injury, and strategies that impair proNGF function in the injured CNS are neuroprotective. In contrast, proBDNF is an endogenous ligand that is released from central neurons, and must be locally processed at the synapse to generate mature BDNF to induce L-LTP. The long-term goal of our work is to understand the biochemical and molecular basis of neurotrophin function. We propose three interrelated approaches to identify the mechanisms by which proNTs mediate distinct actions. Specifically we propose to: (a) define the signaling pathways that are unique to proNTs to initiate p75-mediated cell death. Using cells which co-express p75, TNF receptor and Fas, we will identify the "death adaptor complex" that is selectively recruited to ligand-activated p75 to determine the specificity of apoptotic signaling, (b) Analyze the biological consequences of ectodomain shedding of the sortilin receptor, which is induced upon proNT binding. We will identify the proteases that mediate shedding, and examine if sortilin ectodomain acts as a decoy receptor to abrogate apoptosis, or whether shedding is required to initiate cell death, (c) Genetically dissect the actions of proNGF and proBDNF in the central and peripheral nervous system using inducible, gene replacement strategies. These will be used to evaluate the consequences of proNGF and proBDNF expression in (i) sculpting peripheral and central nervous systems by mediating developmentally regulated cell death, (ii) altering synaptic plasticity in the juvenile and adult hippocampus, and (iii) mediating apoptosis in the adult nervous system.

The intracellular mechanisms which are required to induce vascular smooth muscle cell migration and proliferation in response to injury are poorly understood. Although several receptor systems are capable of initiating smooth muscle cell migration and proliferation following the binding of ligand (bFGF, PDGF and most recently, NGF) the specific intracellular pathways which are activated by these receptor tyrosine kinases in vascular smooth muscle cells have yet to be characterized. Recent studies from our laboratory have demonstrated the expression of a novel growth factor/receptor system, the neurotrophins and their receptors, trk receptor tyrosine kinases and p75, in medial smooth muscle cells of both rat and human aorta and in cultured vascular human and rat smooth muscle cells. Moreover, their expression is exquisitely regulated during the development of vascular restenosis, as demonstrated by their expression in human atherosclerotic lesions and in the neointima which forms following balloon injury to the rat aorta. Finally, neurotrophins are potent mediators of vascular smooth muscle cell migration, in a response comparable to PDGF. The coexpression of the neurotrophins and trk receptors and their effect on smooth muscle cell migration suggests that these growth factors play an important role in regulating the response of smooth muscle cells to vascular injury. Trk is a receptor tyrosine kinase which has been shown to activate a number of intracellular signaling pathways in neuronal cells, such as the ras/MAP kinase pathway, phospholipase C (PLC-gamma) and phosphotidyl inositol 3-OH kinase (P13-kinase). The mechanisms of p75 intracellular signaling have not yet been defined, although an activation of sphingomyelinase and subsequent release of intracellular ceramide has recently been demonstrated by several laboratories. The overall aim of this grant proposal is to define the signaling pathways for smooth muscle cell migration in response to the neurotrophins. These pathways will be compared with those activated by the PDGF and bFGF receptors, to define those pathways which are required for the induction of vascular smooth muscle cell migration. Specifically, we intend to: I. Establish a vascular smooth muscle cell system to express trk receptors, as well as other receptor tyrosine kinases implicated in initiating vascular smooth muscle cell migration using gene transfer techniques. II. Identify the post-receptor pathways which mediate smooth muscle cell migration using cells which express trk, PDGF-beta and FGF-1 receptor tyrosine kinases. Mutant receptors, incapable of activating defined signaling enzymes, will be used to confirm that these specific pathways mediate cell migration. III. Examine the role of p75 receptor signal transduction in neurotrophin- mediated smooth muscle cell migration. These studies involve critical interactions with Dr. Gross, and Drs. Hajjar, Pomerantz, and Lander.

Nerve growth factor (NGF) is a polypeptide growth factor that plays critical roles in the differentiation of immature neuroblasts and the subsequent survival of a subpopulation of mature neurons in both the peripheral as well as central nervous systems. Although the precise mechanism of NGF actions remains incompletely undefined, NGF initiates neuronal diffentiation by binding to the receptor tyrosine kinase (TRK A). This induces the phosphorylation of TRK A on intracellular tyrosine residues which recruits downstream signal transducing enzymes as well as adaptor proteins that contain only the Src homology (SH) domains 2 and 3. Formation of this receptor-signal transducing complex mediates the numerous cellular responses that ultimately lead to a diffentiated neuronal phenotype. Paradoxically, activation of other receptor tyrosine kinases by their cognate ligands, such as the binding of epidermal growth factor (EGF) to its receptor, also appears to initiate a similar spectrum of downstream biochemical events and yet, these growth factors induce mitogenesis rather than differentiation. This dichotomy of action suggests that downstream events are critical in determining the cellular consequences of receptor tyrosine kinase activation. The long term goal of our work is to understand the biochemical and molecular events accompanying NGF actions, and to delineate how NGF signaling promotes neuritogenesis. We have established a model system which can augment, or inhibit NGF induced neurite formation by the ectopic expression of the v-Crk adaptor molecule, or SH2 mutant v-Crk proteins, respectively, in the neural chest cell line PC12. This model system will be used to dissect the mechanisms encoding differentiative signaling . Specifically we propose to: (1.) Identify the intracellular substrates that interact with v-Crk to determine which pathways are critical for neuritogeneis. V-crk, or mutant v-Crk proteins will be expressed in PC12 cells by gene transfer to delineate the domains of v-Crk which regulate neuronal differentiation. We will also explore the potential role of v-Crk associated phosphatase/s in modulating trk receptor signaling . (2.) Determine the role of native adaptor proteins and v-Crk in linking activated receptor tyrosine kinased to the cytoskeleton. (3.) Characterize the trk A/crk/cytoskeletal interactions which result in enhanced receptor internalization.

DESCRIPTION (adapted from the applicant's abstract): The aim of this project is to define the role of neurotrophin-3 (NT-3):trk C signaling during cardiac morphogenesis. The investigators plan to: (1) define the expression of NT-3, and each trk C receptor isoform during cardiac development, using immunohistochemistry and in situ analysis. These studies will define the timing of alternative splicing of trk C from transcripts encoding kinase active proteins, to transcripts encoding proteins lacking tyrosine kinase activity; (2) compare the cardiac abnormalities resulting from the lack of expression of NT-3 and trk C using null mutant mice, to define the roles of this neurotrophin:receptor system in cardiac neural crest and cardiac myocyte development; (3) directly test the ability of trk C receptors to modulate cardiogenesis in vivo, using replication defective retroviruses to direct the expression of trk C transgenes in cardiac myocytes or cardiac neural crest of chicken embryos. Retroviruses encoding the full length or truncated trk C isoforms will be used to infect and tag cardiac myocyte or neural crest precursors, and the ability of the transgene to modify the migratory, proliferative and differentiative capacity of these cells will be determined.

The histotechnology core will function to provide extensive services to each of the four projects. The services can be grouped in three specific areas, which are utilized in the analysis of in vivo models of angiogenesis. First, services are provided in the procurement of tissue, including specialized fixation and perfusion fixation needed for vessel morphometric analysis. In the second aspect, harvested tissues are processed for routine histology, and a wide range of immunohistochemical techniques. Lastly, service is provide in the imaging of such histology and immunohistochemistry, by the use of electron microscopy and confocal imaging. These imaging techniques also include statistical morphometric analysis and digital image analysis. Thus, the core facility will provide a complete range of services for projects utilizing in vivo models, with oversight by a single PI and technical support staff to standardize procedures and maintain quality control, and to facilitate interactions with the extensive imaging facilities at Weill Medical College. Additionally, this will result in significant savings in terms of immunohistochemical supplies, and in tissue preparation and sectioning, as well as reduced rates for the imaging facility.

Migration of medial smooth muscle cells (SMC) into the intima in response to vascular injury is a major component of the remodeling which occurs in the development of atherosclerotic and restonotic lesions. Growth factors such as PDGF and the neurotrophins, which activate the PDGF and trk receptor tyrosine kinases, respectively, are potent chemotactic agents for vascular SMC. Cellular migration is highly regulated, requiring first, the detachment of cells from the extracellular matrix, followed by the reorganization of the cytoskeleton, and finally, the release of matrix, metalloproteinases, to degrade the basement membrane and permit SMC to egress from the media into the intima. The signaling mechanisms which coordinate the complex processes of cellular detachment, cytoskeletal reorganization and MMP release are poorly defined. The overall aim of this proposal is to dissect the downstream pathways regulating SMC migration in response to the neurotrophins and PDGF, and thus, determine if cellular or complementary signaling mechanisms are utilized by these two different receptor tyrosine kinases to induce directed cell migration. Specifically, we plan to: I. Identify signaling pathways activated by growth factors and adhesion molecules which initiate cellular detachment, cytoskeletal reorganization and turnover t focal adhesions in response to migratory stimuli. II. Identify the contributions of metalloproteinases in growth factor-initiate SMC migration. III. Directly evaluate the role of the neurotrophin, BDNF, on lesion development in well defined models of vascular injury. Lesion development in Apo E (-/-) mice deficient in either BDNF or the BDNF receptor, trk B, will be studied chronically in mice maintained on a high fat diet and acutely using the flow dependent-carotid artery ligation model off vascular injury. These studies will allow us to test whether impaired BDNF:trk B signaling reduces neointimal formation. These studies involve critical interactions with Dr. Roy Silverstein (Project III), Dr. Kathy Hajjar (Project II) and Dr. David Hajjar (Project VI).

DESCRIPTION (provided by applicant): Neurotrophins are a family of highly conserved polypeptide growth factors that play critical roles in the differentiation of neuroblasts and the survival of mature neurons. More recent studies have revealed additional actions in mediating axonal guidance, synaptic plasticity and injury protection. Neurotrophins are synthesized as preproproteins which are subsequently cleaved to smaller, mature forms which dimerize. At the molecular level, neurotrophins exert their effects by interacting with two structurally unrelated receptors: p75, a member of the TNF receptor superfamily, and the Trk receptor tyrosine kinases. Neuronal signaling and gene regulation mainly reflect Trk activation, while p75 can modulate ligand binding when both receptors are expressed. In addition, ligand activation of p75 can initiate apoptosis when p75 is expressed independently of Trk. Tyrosine phosphorylation of activated Trk recruits downstream signaling enzymes and adaptor proteins that contain protein interacting domains. Although the formation of receptor-adaptor-enzyme complexes is believed to mediate the numerous biological responses ascribed to the neurotrophins, only a limited number of signaling modules, such as the Ras-MAP kinase cascade and the PI3-kinase-Akt pathway, have been identified to date. Another confounding issue is that many of the downstream effectors of Trk are not unique to the neurotrophins, but are activated of other receptor tyrosine kinases to yield different biological endpoints. The diversity and specificity of neurotrophin actions on neuronal populations therefore suggest that additional mechanisms exist which determine the cellular consequences of p75 and/or Trk receptor activation. The long-term goal of our work is to understand the biochemical and molecular basis of neurotrophin function. Using the nerve growth factor (NGF) responsive cell line PC12 and the brain derived neurotrophic factor (BDNF) responsive primary cortical neurons, we have identified novel signaling paradigms at the levels of ligand:receptor interaction, post-receptor signaling and transcriptional activation. The combination of in vitro and in vivo approaches outlined below are designed to test three inter-related hypotheses. Specifically we propose to: 1. Define the biological activities of the pro-forms of NGF and BDNF in the selective activation of Trk or p75 and to determine their biological relevance in neuronal signal transduction. 2. Characterize the unique mechanism by which activated TrkA and TrkB receptor tyrosine kinases is linked to the cellular adaptor molecule CrkL to generate neuronal specific and transcriptional responses. 3. Define the mechanism and functional consequences of TrkA- and TrkB-mediated activation of the transcription factor STAT5.

DESCRIPTION (provided by applicant): Angiogenesis is a physiological process in which new blood vessels are formed to meet the oxygenation demands of local tissues. Thus, the identification of growth factors which specifically promote angiogenesis would provide a means to ameliorate the ischemic consequences of atherosclerosis occluding the arterial system. Although factors such as VEGF and bFGF appeared promising in pre-clinical trials, studies in humans have been mixed, prompting a search for additional factors with angiogenic activity. Here we propose to study the role of one of the neurotrophins, BDNF, in promoting collateral vessel formation in the ischemic heart and skeletal muscle. Best known as a differentiative and survival factor for neurons, we have recently identified BDNF as a critical regulator of cardiac vessel stabilization and survival through analysis of BDNF null mutant mice. In addition, BDNF inhibits angiogenic actions in nonischemic animal models. Although our long term goals are to determine whether BDNF can promote vessel formation in ischemic human myocardium, we propose three interrelated aims to identify the mechanisms by which BDNF promotes vessel growth and stability in animal models of vascular insufficiency. First, we will identify the actions of BDNF on purified microvascular endothelial cells, assessing chemotaxis, proliferation and survival, and will characterize the signaling pathways which regulate these events. Second, we will study the induction of BDNF in response to tissue ischemia, and identify the cell types which upregulate BDNF. Lastly, we will characterize the angiogenic response to BDNF, alone or in combination with VEGF in the well characterized models of hindlimb ischemia and coronary constriction to assess effects on capillary formation, ensheathment of vessels by vascular smooth muscle cells and vascular remodeling. These studies will allow us to determine whether local production of BDNF by skeletal or cardiac myocytes promotes vessel growth in ischemic tissue. In addition, these studies will test whether BDNF acts coordinately with VEGF to promote the stability of newly formed vessels.

DESCRIPTION (provided by applicant): This application is for partial support of the 6th and 7th Neurotrophic Factors Gordon Research Conference to be held at Salve Regina College, June 8-13, 2003 and in June, 2005. This international conference, which has been held every two years since 1993, will provide an overview of the most exciting recent developments in the biology of neurotrophic factors. Both basic molecular and structural advances together with in vivo actions of neurotrophic factors will be discussed. Emphasis will be placed on the emerging roles of neurotrophic factors in regulating the function of adult neurons, and in promoting recovery in the injured nervous system. Eight sessions are planned and will cover; (1) Stem cell regulation by neurotrophic factors; (2) Neurotrophic factor signaling and transport; (3) Neurotrophic factors and ion channels; (4) Regulation of dendritic and axonal growth; (5) Modulation of synaptic transmission by neurotrophic factors; (6) Neurotrophic actions upon connections and pathfinding; (7) Regulation of glial biology by neurotrophic factors; (8) Neurotrophic factions in models of central nervous system diseases. This conference will be a major vehicle for the integration of new knowledge in the field of diverse biological processes regulated by neurotophic factors. It should be noted that this is the only national meeting at which the most recent and exciting work on neurotrophic factors is presented biannually. This conference has an excellent past record in including women, minorities, young investigators and foreign scientists as session leaders, speakers and participants, and these goals are reflected in the preliminary program for 2003. It is the serious intentions of the Co-chairs of the Gordon Conference ( B.Hempstead and Y.-A. Barde) to profile new and young investigators as speakers in this Conference and for them to have a leadership role at this meeting.

The development of atheroma is regulated in part by the localized expression of growth factors that promote the recruitment of hematopoietic cells as well as vascular smooth muscle cells into the neointima. We previously demonstrated that the neurotrophins and their receptors, the trk family of receptor tyrosine kinases and the p75 neurotrophin receptor (p75NTR), are highly expressed in atherosclerotic lesions. Three specific roles for the neurotrophins in regulating vessel development and in modulating the vascular response to injury have been identified: (1) neurotrophin-mediates survival of Trk B-expressing cardiac endothelial cells (2) neurotrophin-induced recruitment of Trk A and Trk B-expressing vascular smooth muscle cells to the developing neointima following injury; (3) neurotrophin-induced activation of p75NTR-expressing smooth muscle cells in the neointima induces apoptotic cell death. The paradox of neurotrophin actions in the vasculature, mediating both pro-survival and pro-death outcomes, has recently been clarified by our identification that the pro-forms of the neurotrophins selectively bind to the proapoptotic p75NTR, whereas the mature ligand selectively activates the chemotactic and survival promoting Trk receptors. Our preliminary studies indicate that both the pro- and mature forms of the neurotrophins NGF and BDNF are expressed in human atherosclerotic lesions and atheroma from murine models, and that selective MMPs and plasmin can cleave pro-forms to mature forms. The long term goals of this project are to understand how the differential expression of pro- and mature forms of neurotrophins regulate the dynamics of lesion formation and vascular remodeling. Studies in Specific Aim 1 will define the spatial and temporal expression of the pro- and mature forms of the neurotrophins in human lesions and in murine models of atheromata formation and correlate their expression with the co-ordinate expression of p75NTR and Trk receptors, as well as MMPs and components of the plasminogen protease system. Studies in Specific Aim 2 will identify the biological actions of the pro-neurotrophins on vascular smooth muscle cells, monocytes/macrophages and endothelial cells using in vitro analysis of chemotaxis, survival and apoptosis and compare them to the actions of the mature neurotrophins. Finally, in Specific Aim 3, we will genetically dissect the actions of the pro-neurotrophins from mature neurotrophins in lesion formation in vivo by replacing the native BDNF coding exon with a cleavage resistant mutant to generate only pro-BDNF. The effects of pro-BDNF overexpression in murine models of vascular injury will be assessed. The results of these studies may identify unique targets to regulate the microenvironment of the developing atheroma.

BDNF induces structural and functional changes in central neurons to modulate synaptic efficacy; our goal is to identify molecular mechanisms that regulate BDNF targeting and release at synapses to modulate neurotransmission. BDNF is synthesized as a precursor, proBDNF, sorted to a regulated secretory pathway, and released in an activity-dependent manner. At the synapse, proBDNF can bind selectively to p75 to induce LTD, and potentially reduce spine density and dendritic complexity. If proBDNF is converted to mature BDNF in the secretory vesicle or synaptic cleft, TrkB is selectively activated to enhance synaptic transmission and promote axonal branching and dendritic growth. Thus, mechanisms that regulate conversion of proBDNF to mature BDNF, and regulate trafficking to dendrites or axons critically modulate structural and functional neuronal plasticity. We have generated knock-in mice expressing HA-tagged BDNF to markedly enhance detection of endogenous BDNF. We have also identified intracellular chaperones, including sortilin, and other sortilin family members that bind proBDNF. With these tools, three interrelated aims are proposed: (1) Using neurons from the BDNF-HA mouse, identify if conversion of proBDNF to mature BDNF occurs during sorting to secretory vesicles, or following vesicle fusion and release. We postulate that the location of BDNF conversion may differ among neuronal subtypes. (2) We will identify the sortilin family members that chaperone proBDNF to the constitutive or regulated secretory pathways, and to dendrites or axons. We posit that different sortilin members direct intracellular trafficking to different subcellular compartments, delivery to the synapse, and regulate cleavage to mature BNDF. Using BDNF-HA mouse, and acute silencing of different chaperones, we will assess the developmentally regulated changes in the ratio of proBDNF/mature BDNF release, and in retrograde and anterograde traffiking of BDNF isoforms. (3) We will generate knock-in mice to conditionally delete relevant sortilin family members. These animals will permit us to dissect the roles of select BDNF chaperones in regulating BDNF levels, targeting to axons or dendrites, and effects on neuronal morphology and connectively in the intact, postnatal brain. RELEVANCE (See instructions): This project identifies mechanisms that regulate BDNF release, and modulates morphology and connectivity of hippocampal and cortical neurons; disregulation of these processes contributes to neurodevelopmental disease. A human SNP that reduces BDNF release results in anxiety and depressive symptoms in mice, and correlates with these human diseases. Abnormal frontolimbic connectivity underiies conditions of anxiety anri autism Ths mfjchanlRms irifintifiert hfirR will vieiri npw tarnRt.¿; fnr fixaminatinn in thRSfi riiseases

DESCRIPTION (provided by applicant): Neurotrophins induce structural and functional changes in neurons to modulate synaptic efficacy; our long term goal is to identify molecular mechanisms that regulate BDNF targeting and release at synapses to modulate neuronal structure and neurotransmission. BDNF is initially synthesized as a precursor form (proBDNF) that is sorted to a regulated secretory pathway, and released in an activity-dependent manner. When proBDNF is released at the synapse, it can bind to p75 receptors to induce LTD, and potentially reduce spine density and dendritic complexity. However, if proBDNF is converted to mature BDNF in the secretory vesicle or synaptic cleft, TrkB is selectively activated to enhance synaptic transmission and promote axonal branching and dendritic growth. TrkB receptors are present both pre- and post-synaptically in the Schaffer collateral pathway, and mature BDNF can activate both pre- and post-synaptic TrkB receptors to facilitate neurotransmission. Thus, the molecular mechanisms that regulate conversion of proBDNF to mature BDNF, and that regulate intracellular trafficking to dendrites or axons are critical to modulate structural and functional neuronal plasticity. We have developed new genetic tools to facilitate detection of endogenous BDNF, and identified new sorting receptors that direct BDNF intracellular trafficking. Specifically, we have generated knock-in mice that express HA tagged BDNF to markedly enhance detection of endogenous BDNF. We have also identified intracellular chaperones, including sortilin, and other sortilin family members that bind to proBDNF. With these tools, three aims are proposed to dissect BDNF trafficking, cleavage, and depolarization dependent release: (1) Using neurons from the BDNF-HA tagged mouse, identify if conversion of proBDNF to mature BDNF occurs during synthesis and sorting to secretory vesicles, or whether conversion occurs following vesicle release. We predict that the location of BDNF conversion may differ among neuronal subtypes and across early postnatal time points when synaptic connections are being refined and synaptogenesis is robust. (2) We will investigate how sortilin family members alter intracellular cleavage of proBDNF and modulate pro- vs. mature BDNF release in neuronal cultures. (3) We will identify the sortilin family members that chaperone proBDNF to the constitutive or regulated secretory pathway, and to dendrites or axons. We posit that different sortilin family members direct intracellular trafficking to different subcellular compartments and regulate cleavage to mature BDNF and its release. These studies will rely on the BDNF- HA tagged mouse, and overexpression or shRNA knockdown of different chaperones. These studies will identify molecular mechanisms that regulate BDNF processing and trafficking, to induce structural and functional changes in the developing postnatal central nervous system.

? DESCRIPTION (provided by applicant): It is now well-accepted that structural and biochemical alterations in brain circuitry during childhood and adolescence can affect learning, memory, and functional circuitry later in life. A common human single nucleotide polymorphism (SNP) in the BDNF prodomain that leads to valise-to-methionine substitution at codon 66 has provided insights into the role of BDNF in altered learning and memory, especially in the realm of fear-related processes. In vitro studies to date have suggested that this SNP acts as a loss of function mutation that impairs BDNF secretion. Thus, the abnormalities found in humans and knock-in mice with this SNP have been attributed to a loss of function model based on decreased bioavailability of mature BDNF. The potential function(s) of the isolated prodomain generated after proteolysis of proBDNF remain cryptic. Here, we provide evidence that the Met prodomain is secreted in an activity-dependent manner, acts as an independent ligand, and elicits antagonistic biological actions to mature BDNF, by activating an alternate set of receptors, p75NTR and SorCS2. Recently, we have made two key findings using the BDNF Met knock-in mouse that suggest that the Met prodomain affects the maturation of a specific brain circuit, leading to functional impairments in fear- based learning that is not evident with BDNF deficiency (BDNF+/- mice). We will directly test the hypothesis that the human Met prodomain of BDNF is a biologically active ligand that induces morphological neuronal remodeling, and explains the significant impact of this SNP on fear circuitry and function. We will identify the mechanisms by which Met prodomain signals through a p75/SorCS2 co-receptor complex to alter neuronal morphology in cultured neurons and affect the developing fear circuitry between the hippocampus and prefrontal cortex. Finally, we will determine the impact of the Met prodomain in fear extinction-related behaviors during a sensitive period for fear regulation during the transition into adolescence. Collectively, these studies are designed to investigate an additional potential mechanism by which the BDNF SNP may impact brain function. The hypotheses tested represent a significant reconceptualization of the biological actions of a key brain growth factor.