Rejji Kuruvilla - US grants

[unreadable] DESCRIPTION (provided by applicant): The proper functioning of the nervous system relies on the establishment of precise neuronal circuits. Neurotrophins are extracellular instructive cues secreted by neuronal targets that influence wiring of the nervous system by regulating survival, growth and synaptic plasticity. Despite considerable progress in understanding functions of target-derived neurotrophins, a fundamental question still remains: How do a limited repertoire of neurotrophins and their receptors orchestrate the diverse events required to establish and maintain precise neuronal circuits? We hypothesize that one mechanism by which target-derived neurotrophins influence neuronal function is by regulating neuronal expression and signaling of other secreted growth factors. Such a mechanism of crosstalk between neurotrophins and other growth factor families could potentially broaden the repertoire of neurotrophin actions in the nervous system. We have recently identified an interaction between the neurotrophin and Wnt signaling pathways during development. Wnts are a large family of secreted cysteine-rich proteins, initially characterized as morphogens during embryonic patterning. However, recent studies identify surprisingly versatile roles for Wnts in neuronal development. The overall goal of this proposal is to test the hypothesis that interactions between Wnts expressed in neurons and neurotrophins secreted by their target tissues regulate neuronal development and establishment of precise neuronal circuits. Thus, our specific aims are; (1) To test the hypothesis that neuronal expression of Wnts is regulated by target-derived neurotrophins: We found that several Wnts and their receptors are expressed in sympathetic neurons, a tractable model system for studying neuron-target interactions. Of these, Wnt-5a is robustly expressed at a developmental time when sympathetic axons are reaching final targets. The goal of this aim is to demonstrate that neuronal expression of Wnt-5a is regulated by interactions with target tissues and target-derived neurotrophins. (2) To test the hypothesis that neuronal Wnts are mediators of neurotrophin function in sympathetic neurons: We found that Wnt-5a is sufficient to recapitulate the growth-promoting effects of the neurotrophin, Nerve Growth Factor (NGF), in cultured sympathetic neurons. The goal of this aim is to demonstrate that Wnt-5a functions downstream of NGF, to mediate axonal growth required for innervation of target tissues. (3) To identify mechanisms of Wnt signaling in sympathetic neurons: We will characterize the signal transduction machinery within sympathetic neurons that couples an extracellular Wnt-5a signal to changes in the cytoskeletal machinery necessary for axonal growth and branching. Our study will provide new insight into molecular mechanisms underlying neuronal development and allow for new strategies for promoting growth and regeneration of neurons following injury or disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A fundamental question in developmental neurobiology is how a limited number of growth factors coordinate the establishment of precise neuronal circuits during nervous system development. Neurotrophins provide one of the best examples of target-derived developmental cues regulating neuronal survival, axonal and dendritic growth and synaptogenesis. An intriguing question in neurotrophin research is how a very large number of biological events are triggered by such a limited set of neurotrophins and their receptors. We previously reported that two different neurotrophins, Nerve Growth Factor (NGF) and Neurotrophin-3 (NT-3), employ the same TrkA receptor to promote sequential stages of axonal growth during sympathetic nervous system development. NT-3, secreted from the vasculature along the trajectory of projecting sympathetic axons, promotes early axon outgrowth. However, NGF derived from peripheral targets is required for final innervation of end- organs. How does a common TrkA receptor respond to two different neurotrophins to facilitate distinct phases of axon growth? We identified a calcineurin-dependent TrkA endocytic pathway that is critical for NGF-, but not NT-3-dependent trophic functions. Calcineurin is a calcium-responsive phosphatase that influences diverse aspects of neuronal development by translating small changes in intracellular calcium levels to morphological and transcriptional changes. Thus, the overall goal of this proposal is to test the hypothesis that the two neurotrophins, NGF and NT-3, differentially regulate TrkA signaling and trafficking to promote distinct stages of sympathetic axon growth. To this end, we will employ biochemical assays to identify TrkA signaling pathways that allow target-derived NGF and NT-3 to differentially activate calcineurin in sympathetic neurons. Using mutant mice lacking calcineurin or its downstream target, the endocytic GTPase, dynamin1, we will test the hypothesis that endocytosis of TrkA receptors is specifically required for NGF, but not NT-3-mediated sympathetic axonal growth in vivo. We will also define the mechanisms by which TrkA endocytosis promotes axonal growth. Together, these studies will provide novel insights into how two target-derived neurotrophins signal via a common TrkA receptor to cooperatively regulate axonal growth during neuronal development, as well as provide the foundation for addressing the role of these axonal growth programs in mediating regenerative growth of adult neurons following injury or disease. PUBLIC HEALTH RELEVANCE: The initiation, extension and targeting of axons during development underlies the formation of precise neuronal circuits, necessary for physiological and cognitive functions. Axon degeneration is a hallmark of spinal cord injuries and almost all neurodegenerative disorders. Thus, it is essential to gain a better understanding of molecular mechanisms that promote axonal growth during nervous system development, with the goal to apply this information to axonal repair strategies during neurological disorders or injury. This proposal aims to identify a critical receptor trafficking mechanism by which neurotrophic growth factors promote axon growth during development. The proposed research has the potential to impact future neurotrophin-mediated therapeutic strategies to enhance regenerative growth following injury and disease.

DESCRIPTION (provided by applicant): Neurotrophins are peptide growth factors best studied for their roles in promoting neuronal survival and axonal growth in the developing nervous system. However, neurotrophins have also been implicated in development and function of several non-neuronal tissues including the cardiovascular, immune and endocrine systems. In the pancreas, in vitro studies have shown that the neurotrophin, Nerve Growth Factor, (NGF) regulates survival and function of insulin-producing [unreadable]-cells. NGF also prolongs survival of transplanted mouse islets in vitro and in vivo. These studies implicate NGF signaling in pancreatic islet development and regeneration. However, to date, there have been no attempts to use genetically modified mice lacking NGF or its receptor tyrosine kinase, TrkA, to address the in vivo role of neurotrophin signaling in the pancreas, and specifically islet development. Employing mice deficient for NGF, we observed that developing pancreatic islets are disorganized and that the normal spatial arrangement of endocrine cell types within islets is disrupted. In adult heterozygous NGF+/- mice, pancreatic islets are reduced in size and fragmented. Since NGF and its receptor, TrkA, are expressed in islet cells, these deficits could arise due to a direct requirement for NGF signaling in the pancreas or indirectly due to the death of NGF-dependent neurons innervating the pancreas. Based on our preliminary results, the goal of this proposal is two-fold;(1) Use genetically engineered mouse models to examine the cell-autonomous requirement for NGF-TrkA signaling in islet development, and (2) determine the contribution of sympathetic neurons to pancreas development by employing a combination of in vitro neuron-pancreas co-culture assays and in vivo analyses of pancreas development in mutant mice lacking sympathetic innervation. By focusing on the in vivo requirement for neurotrophin signaling and nerve-derived signals in pancreas development, our studies will provide unique insight into extrinsic growth factors that regulate pancreas development. Knowledge about extrinsic signals regulating islet development will facilitate the design of better therapeutic strategies to promote islet survival during type I diabetes and injury, as well as enhance islet survival following transplantation. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to identify novel extrinsic growth factors that influence development of pancreatic islets. Gaining insight into extrinsic signals regulating islet development has important implications for treatment of type I diabetes, promoting pancreatic regeneration following injury or disease, and prolonging islet survival after transplantation. In this study, we focus on the neurotrophin, Nerve Growth Factor (NGF), a soluble growth factor best studied for its role in promoting neuronal survival and connectivity in the developing nervous system. NGF and its receptor, TrkA, are also expressed in the pancreas and NGF signaling has been shown to promote survival of pancreatic islet cells, in vitro. However, to date, there have been no attempts to use genetically modified mice to address the in vivo role of NGF signaling in the pancreas and specifically islet development. The goal of this study is to use innovative tools including mouse genetics and neuron-pancreas co-cultures to address whether NGF signaling influences pancreas development directly by signaling within the pancreas, or indirectly via promoting innervation of NGF-responsive sympathetic neurons. Together, these studies will provide significant insight into a previously uncharacterized question of the influence of neurotrophins and nerve-derived signals in islet development.

DESCRIPTION (provided by applicant): The autonomic nervous system is known to regulate glucose homeostasis by modulating hormone release in the adult pancreas. Pancreatic islets of Langerhans are richly innervated by sympathetic nerves of the autonomic nervous system and the onset of innervation is coincident with stages of islet growth and maturation in the developing pancreas. Yet, whether, sympathetic innervation contributes to pancreas organogenesis has not been defined so far. We recently reported that ablation of sympathetic nerves results in profound defects in the shape and cyto-architecture of islets during development in mice (Borden et. al, 2013). Sympathectomized mice exhibit reduced insulin secretion and glucose intolerance later in life. Thus, the overall goal of this proposal is to elucidate the molecular mechanisms by which sympathetic neurons promote islet formation and the acquisition of functional maturity. Based on preliminary findings, we hypothesize that the nerve-derived signal is the neurotransmitter, norepinephrine, that acts via pancreatic ?-adrenergic receptors to promote ?-cell migration and islet organization. Thus, we will determine if norepinephrine is necessary and sufficient for islet architecture by assessing islet formation i vivo in mutant mice that lack noradrenergic neurotransmission, as well as by examining the effects of norepinephrine on ?-cell migration and aggregation in vitro (Aim 1). In Aim 2, we will identify the molecular mechanisms by which norepinephrine signaling influences islet architecture. By deep sequencing-based profiling of sympathectomized islets, we observed a dramatic down-regulation of PTTG1 (pituitary tumor-transforming gene 1), that encodes for a protein reported to have cytoskeleton-regulatory functions. Thus, we will determine if PTTG1 is a transcriptional target of norepinephrine signaling. Additionally, we will assess whether PTTG1 is an essential regulator of ?-cell migration, employing available PTTG1 knockout mice. Finally, we will elucidate the mechanisms by which nerve-derived signaling influences islet maturation by examining the effects of norepinephrine on the glucose-sensing machinery and insulin granule trafficking in ?-cells (Aim 3). The significance of our studies is that it is the first to address how the nervous system controls islet development and will also initiate a new line of research in current translational efforts to treat pancreatic dysfunction.

? DESCRIPTION (provided by applicant): Decreased function of insulin-producing ?-cells in the pancreas is a key contributor to the etiology of type 2 diabetes. Blunted ?-cell secretion is one f the earliest features of the disease, that precedes the onset of overt hyperglycemia. Thus, it is critical to identify factors that regulate normal ?-cell secretory responses and identify their mechanisms of action, so that therapeutic strategies can be targeted toward early intervention during ?-cell failure. Neurotrophins are soluble peptide growth factors that have been best-studied as essential regulators of neuronal development and synaptic transmission in the vertebrate nervous system. Despite the wide-spread expression of neurotrophins and their Trk receptor tyrosine kinases in non-neuronal tissues, including the pancreas, little has been done to investigate their in vivo functions outside of the nervous system. Employing genetic mouse models, we uncovered an endogenous role for the classical neurotrophin, Nerve Growth Factor (NGF) in glucose-stimulated insulin secretion. Thus, the overall goal of this proposal is to define this new role for neurotrophin signaling in ?-cell function, and to elucidate the underlying molecular mechanisms. Based on preliminary findings, we hypothesize that NGF is a vascular-derived signal that acts on ?-cell-localized TrkA receptors to acutely augment glucose-stimulated insulin secretion. The goal of Aim 1 is to define the essential roles of NGF and TrkA in adult ?-cell function, by assessing glucose homeostasis and insulin secretion in mice in which NGF and TrkA receptors have been inducibly deleted from vascular and ?-cells, respectively. In Aim 2, we will identify the molecular mechanisms by which TrkA signaling in ?-cells influences insulin secretion. Preliminary results suggest that TrkA-mediated vesicular trafficking and actin cytoskeletal mechanisms are responsible for mobilizing insulin granules to the ?-cell plasma membrane. Thus, we will elucidate the role of Trk receptor endocytosis and endosomal signaling in glucose-stimulated changes in actin and insulin granule exocytosis, employing biochemical, live-imaging and ultra- structural assays. The significance of our study is that it is the first to address the essential functions of neurotrophins, classically studied as neuronal factors, in the endocrine system. Our findings have the potential to inform a new line of research in current translational efforts to treat metabolic disorders.

Abstract This proposal is to provide support for junior investigators, post-doctoral fellows, and students to attend the Gordon Research Conference (GRC) on Neurotrophic Mechanisms in Health and Disease. The meeting will be held in Salve Regina University, Newport RI from June 2-7, 2019. Neurotrophic factors, broadly defined as factors that have critical roles in circuit connectivity, synaptic plasticity, and behavior, are essential for the development, maintenance, and functions of the nervous system. Importantly, several treatments that exploit knowledge of neurotrophin biology are now in human clinical trials for chronic pain, Alzheimer?s disease, and cancer. Thus, diverse communities of basic researchers and clinicians address problems rooted in neurotrophin biology. The 2019 Neurotrophic Mechanisms in Health and Disease GRC is unique in bringing together a multi- disciplinary group of researchers in areas spanning fundamental problems in molecular and cellular neuroscience to translating neurotrophin biology into therapies. This meeting will provide a unique venue to integrate basic research on neurotrophic factor biology with clinical needs and industry interest, with the ultimate goal of accelerating the development and adoption of new treatments for human neurological conditions, psychiatric diseases, and some forms of cancers. The conference will bring together speakers, many new to the meeting, who will speak to the intersection of neurotrophin biology with circuit formation, learning and memory, depression, chronic pain, cancer, Alzheimer?s disease and chronic inflammation. Additional topics and late- breaking developments will be incorporated via talks selected from abstracts, specifically encouraging participation of junior researchers. Several new features distinguish the 2019 GRC Neurotrophic Factor conference from earlier meetings. These include an emphasis on the intersection of neurotrophin biology with systems approaches and therapeutic avenues, recruitment of a cohort of junior investigators new to our meeting who will speak to emerging areas and will provide fresh perspectives, introduction of a faculty mentoring system for junior trainees, and offering family friendly policies such as on-site child-care and subsidies to offset costs of childcare/accommodation.

A fundamental question in neuronal cell biology is how membrane proteins are transported long-distance to axons after biosynthesis in cell bodies. Axon targeting of membrane proteins is critical for the formation and maintenance of neuronal connections and for a functional nervous system. Yet, how most membrane proteins are delivered to axons remains undefined. A long-held view in neurobiology is that signaling receptors are constitutively delivered to axons via secretory trafficking. In contrast, we found that TrkA neurotrophin receptors that are essential regulators of neuron survival, axon growth, and inflammatory pain are actively recruited to axons via transcytosis, an endocytosis-based mechanism where receptors embedded in soma surfaces are internalized and anterogradely transported to axons. Strikingly, anterograde TrkA transcytosis is triggered by the ligand, Nerve Growth Factor (NGF), acting on axon terminals, suggesting a positive feedback mechanism that serves to dynamically scale up receptor availability in axons during times of need. Furthermore, we identified that TrkA transcytosis is primed by the activity of PTP1B, an ER-resident protein tyrosine phosphatase, in cell bodies. The overall goal of this application is to elucidate the signaling and trafficking mechanisms underlying a poorly characterized mode of ligand-triggered targeting of receptors to axons. In Aim 1, we will define NGF-mediated mechanisms that initiate transcytosis in cell bodies, elucidate the trafficking itinerary and transport kinetics of receptor transcytosis, and investigate TrkA transcytosis in vivo. In Aim 2, we will test the hypothesis that ER- anchored PTP1B phosphatase promotes a gain of TrkA biological function by controlling the long-distance transcytosis of receptors. We will employ live imaging, biochemical, and functional analyses in compartmentalized neuron cultures in combination with in vivo analyses of genetically modified mice to accomplish these goals. These studies will address a fundamental, yet poorly studied, cell biological question of how signaling receptors are directed to axons, and will provide insight into specialized mechanisms that enhance neuronal responsiveness to spatially acting extrinsic cues.

Axons continuously respond to external signals in their environment as they grow and navigate toward their target tissues during development, and for maintenance and repair in adult life. mRNA localization and local protein synthesis is a conserved mechanism that allows for rapid and axon-autonomous responses to external trophic and guidance cues. Despite growing appreciation for the functions of axonal protein synthesis, a major gap in our knowledge is in understanding how the nascent proteins are further processed and localized in axons to generate fully functional proteins. Our preliminary results suggest that a neurotrophic factor (Nerve Growth Factor) acutely regulates local protein synthesis and lipidation (prenylation) of newly-made proteins in axons. It has been assumed that protein prenylation is a constitutive mechanism that occurs throughout the cytoplasm and is permissive for cellular functions. In striking contrast, we find that, in neurons, prenylation is under exquisite spatio-temporal control by extrinsic signals. The goal of this application is to define whether coupled axonal protein synthesis and prenylation serves as a regulatory mechanism to localize and enrich proteins in axonal compartments to ensure dynamic and spatial responses to extrinsic growth-promoting cues. We will use a combination of imaging, biochemical, and functional analyses in compartmentalized neuronal cultures, as well as in vivo analyses in mice to accomplish this goal. These studies will advance the knowledge of spatial modes of signaling in neurons that underlie axon development, maintenance, and regeneration.