Warren G. Tourtellotte, MD, PhD - US grants

DESCRIPTION (Adapted from applicant's abstract): Complex nervous system processes involving neurotransmitter release and membrane depolarization, or neurotrophin/membrane receptor interactions, exert their control by activating cellular signal transduction pathways that effect long-term phenotypic changes by altering prevailing patterns of gene expression. Two general classes of genes are considered to be coupled to neuron membrane receptor activation, namely, early and rapidly transient gene activation (immediate early genes; IEGs) and late response genes. The Egr gene family of IEGs has been consistently implicated in trans-synaptic activation and neurotrophin/membrane receptor interactions, and consists of four known transcription factors that include egr1 (also known as NGFI-A, zif/268, and Krox24), egr2 (also known as Krox20), egr3 and egr4 (also known as NGFI-C and pAT133). A variety of correlative studies have implicated their function in an astounding number of cellular processes as diverse as cell proliferation, lymphocyte activation and apoptosis, neuronal synaptic activity, long-term synaptic potentiation (LTP), neuronal plasticity, neuronal kindling, and circadian rhythm generation. Using gene targeting strategies to study mice having loss-of-function mutations for each gene family member, this research program will focus on their essential functions in the developing and adult mammalian nervous system. As the expression of these genes is extensively colocalized in neurons throughout the nervous system, by studying mice with polygenic-loss-of function mutations (i.e., mice having two or more Egr gene mutations), their potential cooperative/redundant interactions will be examined in the nervous system. Specifically, their roles in mediating neurotrophin signaling involving neuron survival and differentiation will be central areas of active investigation during this research program. Additionally, by examining mice with multiple Egr gene mutations, differential expression analysis will be used to identify down-stream target genes regulated by this family of transcription factors. The postulated central nervous system functions of these genes are potentially highly relevant to learning and memory mechanisms as well as neurodegenerative diseases and dementia. Their activation has been specifically associated with pharmacologically altered dopaminergic neurotransmission in basal ganglia, raising the possibility that they may play a role in cognitive and/or motor impairments associated with a variety of neuropsychiatric illnesses. Moreover, the roles that these genes may play in mediating some aspects of neurotrophin-mediated neuron survival and differentiation is germaine, as these processes are likely to be central to mechanisms involving cell death and aberrant plasticity responses associated with intractable epilepsy and neural kindling, as well as to neurodegenerative diseases such as Parkinson's, Huntington's and Alzheimer's diseases. Finally, these genes may play a role in long-term synaptic potentiation and structural plasticity mechanisms, and may therefore be relevant to normal learning and memory processing.

DESCRIPTION (From the Applicant's Abstract): Nerve-muscle interactions play an important role in altering gene expression required for growth and differentiation of muscles. In particular, sensory innervation of skeletal muscle plays a critical role in the genesis of muscle spindles, mechanosensory organs within vertebrate skeletal muscle that provide limb position (proprioceptive) information to the central nervous system. Muscle spindles are innervated by both sensory and motor axons but their genesis is induced specifically by sensory afferents. The tropic effects of sensory afferents are instructive for the transformation of a subpopulation of myotubes to form the complex spindle structure. The molecular mechanisms involved in this complex process are poorly understood. However, we have recently discovered that the zinc-finger transcription factor Egr3 is critically involved in spindle morphogenesis since it is expressed at high levels within forming spindles at a developmental time point that coincides with their induction and since Egr3-deficient mice lack muscle spindles. Egr3 appears to serve as an essential signal transduction molecule expressed in myotubes that have been contacted by sensory axons to form spindles. We have outlined a research program to study the function of Egr3 in mediating the signal transduction mechanisms involved in muscle spindle morphogenesis. Using a variety of in vivo and in vitro molecular techniques we will examine the role of Egr3 in orchestrating gene expression in myotubes during spindle morphogenesis. Motor and sensory innervation to muscle spindles depends upon the neurotrophic factors NT-3 and GDNF which are produced by spindles. We will investigate whether Egr3 regulates these neurotrophins and plays a role in the sensory and motor neuron defects observed in Egr3-deficient mice. Finally, using "gain-of-function" models to overexpress Egr3 both in vivo and in vitro, we will attempt to identify target genes regulated by Egr3 and begin to define the reorganization of gene expression that occurs during spindle morphogenesis. This model system for studying one aspect of nerve-muscle interaction as it relates to the genesis of muscle spindles may be applicable to other mechanosensory organs. It is well appreciated that the genesis of other mechanosensory organs such as Pacinian corpuscles (vibratory sensation), Golgi tendon organs (muscle tension) and Merkel cells (light touch) are also induced by their respective sensory afferent innervation. A more thorough understanding of the reciprocal tropic-trophic interactions between sensory neurons and mechanosensory organs may provide greater insight into the etiopathogenesis of a variety of sensory neuronopathies.

DESCRIPTION (provided by applicant): This is an Independent Scientist Award for Warren G. Tourtellotte, M.D., Ph.D. who is currently an Assistant Professor of Pathology, Neurology and Neuroscience at Northwestern University, Feinberg School of Medicine. Dr. Tourtellotte devotes greater than 75% professional effort to research focused on transcriptional regulation of genes involved in central and peripheral nervous system development. This Research Career Award will assure Dr. Tourtellotte stable salary support to allow him to commit 75% professional effort to research. The institution is supportive of a 75% research effort as evidenced by minimized clinical service commitments and recent expansion of the research space available to Dr. Tourtellotte. The career development plan will extend the focus of Egr transcription factor function in the laboratory to include studies of cellular physiology, signal transduction and genome wide regulatory network analysis. Dr. Tourtellotte will use the career development resources to strengthen collaborative networks both within the institution and externally to strengthen the research program by improving the methodology, scientific staff and expertise available to the laboratory. The Research Plan will characterize the role of Egr transcription factors in sympathetic nervous system development, neurotrophin mediated gene regulation and signal transduction. The planned career development activities will take full advantage of the rich collaborative and career development resources available at Northwestern University.

DESCRIPTION (provided by applicant): The molecular mechanisms mediating the development, innervation and stability of sensory axon- mechanoreceptor interactions are very poorly understood, yet they are likely to be important in human neuropathies associated with mechanoreceptor denervation and axon loss. Some human sensory neuropathies primarily affect large diameter sensory axons which preferentially innervate muscle and tendon mechanoreceptors (muscle spindle stretch receptors and Golgi tendon organs) to provide skeletal and muscle position sensation (proprioception) to the central nervous system. During prenatal skeletal muscle development, sensory axons regulate the expression of a specific repertoire of genes in contacted myotubes to mediate stretch receptor morphogenesis and stabilize their innervation. The specific molecular signals that predominate are unknown but identifying them is of considerable interest since proprioception deficits are a common and debilitating aspect of many sensory neuropathies. Without a better understanding of the molecular factors involved in establishing and maintaining innervation to muscle spindle stretch receptors, it will be difficult to formulate rational therapies to slow or reverse proprioceptive axon loss. The signal transduction pathways engaged in myotubes that are contacted by large diameter axons (la- afferents) and the molecular signals involved in maintaining sensory and motor innervation to them are very poorly understood;la-afferents provide instructive signals that transform a subpopulation of myotubes into spindle stretch receptors presumably by engaging gene regulatory networks that are specific for spindle morphogenesis. In previous work, we identified Egr3 as an essential transcriptional regulator of spindle development which is induced in myotubes by la-afferent innervation. This research plan is outlined in three specific aims: (i) to characterize the function of novel Egr3 regulated target genes in stretch receptor morphogenesis and innervation, (ii) to examine whether Egr3 mediated gene expression is sufficient to transform myotubes into intrafusal muscle fibers in the absence of la-afferent morphogenetic signaling and i) to determine whether Egr3 is necessary to fate specify myotubes to an intrafusal muscle fiber lineage. We anticipate that these studies will provide greater understanding of how sensory innervation controls muscle stretch receptor morphogenesis and will better define the role of Egr3 in regulating stretch receptor specific genes that may be involved in stabilizing sensory and motor innervation to them. Muscle and cutaneous thermo- and mechanoreceptors all depend upon sensory innervation for their morphogenesis suggesting that some common molecular mechanisms may be revealed by our studies focusing on nerve- muscle stretch receptor interactions.

DESCRIPTION (provided by applicant): The sympathetic nervous system (SNS) is a division of the autonomic nervous system that has a major role in tissue and organ homeostasis. It is the target of a wide variety of congenital and neurodegenerative diseases, and the source of several types of malignant pediatric and adult tumors. Millions of humans are afflicted with diseases involving the SNS, yet we understand very little about the mechanisms regulating growth and differentiation of sympathetic neurons or the mechanisms mediating the establishment and maintenance of target organ innervation. Familial Dysautonomia (FD, Riley Day Syndrome, HSAN3) is a devastating genetic autosomal recessive disease involving the sympathetic and sensory nervous systems. In greater than 99.5% of cases, it is caused by a single highly conserved point mutation of the IKBKAP gene. IKBKAP encodes a protein (IKAP) with very poorly characterized function. Here, we propose to study the function of IKBKAP in sympathetic and sensory neurons most affected by FD using in vitro and in vivo methods. This exploratory/developmental proposal is outlined with 2 specific aims: (1) to study IKAP function in sympathetic neurons in vivo by generating a mouse model to conditionally ablate (knockout) IKBKAP and (2) to examine the role of IKAP in growth, differentiation and axon outgrowth of sympathetic and sensory neurons in vitro. We anticipate that these studies will generate new insights into how IKAP functions during development of sympathetic and sensory neurons. Moreover, the conditional IKBKAP knockout mouse will provide a valuable tool for further studies aimed at elucidating prevailing gene regulatory networks controlled by IKAP that are involved in sympathetic and sensory nervous system development and maintenance in humans. PUBLIC HEALTH RELEVANCE: The sympathetic nervous system (SNS) is critical for controlling many unconscious processes in the body and it is the target of a wide variety of developmental, degenerative and cancerous diseases. Familial Dysautonomia (FD) is a devastating genetic disease involving the sympathetic and sensory nervous systems and is caused by mutation of the IKBKAP gene. Very little is known about IKBKAP and its role in developing and adult sympathetic neurons. Here, we will generate an animal model that will make it possible to study in detail how mutation of IKBKAP leads to FD.

DESCRIPTION (provided by applicant): The Candidate is a board certified Anatomic Pathologist and Neuropathologist with extensive experience in generating and studying genetically-modified mice to model disease pathogenesis and basic biological mechanisms. Dr. Tourtellotte's independent research program focuses on immediate early gene regulation in neurodevelopment and behavior, and it serves as a training program for undergraduate, predoctoral and postdoctoral students in mouse pathobiology research at Northwestern University. Specifically, Dr. Tourtellotte's research program focuses on: (1) molecular mechanisms mediating nerve-muscle interactions, (2) autonomic nervous system development and dysfunction and (3) the mechanisms of IKBKAP gene function in human familial dysautonomia. Dr. Tourtellotte also serves as a mentor to clinical pathology residents and fellows, many of whom are engaged in research projects that use murine pathobiology to model mechanisms of human disease. Dr. Tourtellotte is proposing to expand his mentoring role by helping to enhance the mouse pathobiology training and research infrastructure at Northwestern University. Through his leadership of the Transgenic and Targeted Mutagenesis Laboratory and the Mouse Phenotyping Core Laboratory at Northwestern University, he is proposing to enhance the training and service in mouse pathobiology research that is available to both junior and senior investigators who wish to use and/or develop mouse models for their own independent research programs. Dr. Tourtellotte is seeking career development support to enable him to decrease his clinical service commitment and focus his efforts on mouse pathobiology research and training. Specifically, he will continue to strengthen his independent research and training program and enhance mouse pathobiology related core laboratory resources and training programs at Northwestern University. The latter efforts will enable him to maximize his influence as a mentor to a future generation of mouse pathobiologists beyond the relatively small number of trainees he currently mentors in his independent research program. PUBLIC HEALTH RELEVANCE (provided by applicant): Mouse pathobiology has become important for modern biomedical research because mice can be genetically manipulated with relative ease. Genetically modified mice serve as experimental model systems for studying disease mechanisms and for formulating therapies. The PI seeks support to train mouse pathobiologists who will be able to generate and manipulate mouse genetic models in their research programs.

? DESCRIPTION (provided by applicant): Familial Dysautonomia (FD) is a rare genetic disease characterized by severe and progressive sympathetic and sensory neuron loss caused by a highly conserved germline point mutation of the human Elp1 (IKBKAP) gene. Elp1 is a highly conserved subunit of the hetero-hexameric transcriptional Elongator complex, but how it functions in disease vulnerable neurons is very poorly understood. We propose to study the role of Elp1 in sympathetic neuron development and innervation. The project is outlined in three specific aims to: (1) characterize Elp1 function in sympathetic neuron target tissue innervation and in maintaining adult sympathetic neuron innervation homeostasis, (2) to identify signaling pathways and interacting proteins that mediate its function in the neuron cytoplasm and (3) to characterize its role in nerve growth factor signaling which is essential for their normal survival and target tissue innervation. Millions of humans are afflicted with diseases involving sympathetic and sensory neurons, yet almost all of them are untreatable because the mechanisms regulating their growth and differentiation are very poorly understood. We anticipate that these studies focused on a rare neuropathy-associated protein will identify new signal transduction pathways that are essential for peripheral neuron survival, differentiation and innervation homeostasis. Moreover, we anticipate that these studies will elucidate essential disease-relevant signaling pathways in sympathetic neurons that may be exploited to treat developmental and degenerative peripheral neuropathies.

PROJECT SUMMARY / ABSTRACT Alzheimer?s Disease is a debilitating degenerative disease without effective treatment that is increasing in prevalence. Developing effective therapies has been impeded because the underlying biological mechanisms driving disease pathogenesis are still poorly understood. Abnormal cleavage of amyloid precursor protein (APP) that generates aggregating forms of neurotoxic amyloid-beta (A?) protein has been a focus of investigation for many years. Although very rare forms of early onset familial AD caused by mutations in APP or processing-associated proteins PSEN1 and PSEN2 substantiate a role for A? in the pathogenesis of AD, most cases (>95%) have no definitive genetic cause. Many risk-associated genes (>26) have been identified, but the role of most of them in AD pathogenesis remains very poorly understood. Microglia are resident innate immune cells that mediate persistent neuroinflammatory responses to A? protein characterized by increased inflammatory cytokine production, synapse loss and neurotoxicity. Many of the identified risk-associated genes appear to encode proteins that are expressed in microglia and involved in phagocytosis and endolysosomal trafficking and proteolytic degradation, raising the possibility that fundamental abnormalities in microglia may contribute to poor A? processing and persistent neuroinflammation that leads to neurotoxicity and neurodegeneration in AD. Angiotensin Converting Enzyme (ACE) is a very poorly studied risk-associated gene. Previous studies published by us and strong preliminary data indicate that it has a significant role in A? clearance from the brain and in enhancing A? protein phagocytosis, its endolysosomal trafficking and proteolytic degradation in microglia. The project is outlined in three specific aims to: (1) examine the role of ACE specifically in microglia in novel transgenic mice and in an animal model of AD, (2) examine the molecular mechanisms of ACE- regulated gene expression in microglia and determine their role in phagocytosis, endolysosomal trafficking and proteolytic degradation and (3) characterize the function of ACE in human induced microglia engrafted into mice brains to study their response in vivo in a model of AD. Millions of humans are afflicted with AD, yet prevention and treatment remain very poor. We anticipate that these studies focused on the AD risk-associated gene ACE and its role in A? processing in microglia will identify novel signaling pathways that enhance A? protein processing. Moreover, we anticipate that these studies may elucidate mechanisms in microglia that may be exploited to develop treatments to enhance A? proteostasis and mitigate neuroinflammation in the brain in AD.