Michael J. Galko - US grants

DESCRIPTION (provided by applicant): The capacity for tissue repair is a fundamental property of multicellular organisms that is often critical for survival. Despite its biological and clinical importance, the signals that initiate and terminate epidermal wound closure remain obscure, as do the genes required to execute this process. This is partly due to the complexity of wound repair responses in vertebrate model organisms and the fact that tissue repair has not been studied extensively in simpler model genetic organisms where rapid gene discovery and precise analysis of gene function are greatly facilitated. This research project focuses on the molecular genetic control of postembryonic wound healing. Our guiding hypothesis is that there is a conserved molecular genetic program of cell-cell signaling events that initiates, regulates, executes, and terminates the repair process. To test this hypothesis in a genetically tractable model system we have established epidermal wound healing assays using Drosophila larvae and demonstrated that wound closure requires the Drosophila Jun N-terminal kinase (JNK) and eight other genes we identified in a pilot conditional genetic screen for wound closure mutants. For this pilot screen we developed transgenic reporter larvae that allow live visualization of wound closure as well as epidermal-specific expression of exogenous RNAi transgenes that could interfere with or enhance this process. Our long term objective is to use our unique assays and tools to identify the complement of genes required for efficient wound closure and determine the function of these genes during wound closure. Our shorter term goals are enumerated in the following specific aims: 1. to test the hypothesis that wound- induced JNK signaling is activated by the Drosophila homolog of the PDGF/VEGF receptor, PVR, a receptor tyrosine kinase identified in our pilot screen. 2. To test the hypothesis that JNK signaling pathway activation initiates cell migration in wound-responsive cells. 3. To identify and characterize novel genes required for wound closure by expanding our conditional genetic screening strategy. This project represents the first systematic study of postembryonic wound closure in a model genetic organism and has great potential for uncovering the elusive signals that control wound closure. Given the conservation of genes required for most fundamental processes we expect that this project will inform our understanding of wound closure in vertebrates and in pathophysiological states, such as cancer, where the wound healing response is thought to be improperly activated or regulated. This research project employs a model genetic organism, the fruit fly, to uncover the genetic control of postembryonic wound healing, a process of immediate relevance to human health. Wound healing is critical for recovery from trauma and surgery, both of which are common occurrences. Given the conservation of genes required for most fundamental biological processes we expect that this project will inform our understanding of wound closure in vertebrates and in pathophysiological states, such as cancer and a variety of skin diseases, where the wound healing response is thought to be improperly activated or regulated.

DESCRIPTION (provided by applicant): Multicellular organisms have evolved mechanisms to protect damaged tissue while it heals. One mechanism is a form of sensory neuron plasticity called nociceptive sensitization. This sensitization can manifest as aversive withdrawal to previously non- noxious stimuli (allodynia). Despite its biological and clinical importance, the genes required for nociceptive sensitization remain obscure. This research project focuses on the molecular genetic control of nociceptive sensitization in a model genetic organism where gene discovery is greatly facilitated. Our guiding hypothesis is that there is a conserved molecular genetic machinery that initiates, regulates, executes, and terminates nociceptive sensitization. To test this hypothesis we have established a nociceptive sensitization assay using Drosophila larvae and demonstrated that development of thermal allodynia requires the Drosophila homolog of the tumor necrosis factor (TNF) receptor and seventeen other genes we identified in a genetic screen for sensitization mutants. For this screen we tested whether larvae expressing gene-specific inhibitory RNAi transgenes specifically in nociceptive sensory neurons could develop allodynia normally following UV irradiation of the barrier epidermis. Our long-term objective is to use our unique assays and genetic tools to identify the complement of genes required in nociceptive sensory neurons for development of allodynia and to determine the specific function of these genes during sensitization. Our shorter-term goals are enumerated in the following specific aims: 1. To test the hypothesis that physical tissue damage (wounding) induces allodynia through different signals and mediators than UV-induced tissue damage. 2. To test the hypotheses that TNF induction following UV irradiation is apoptosis-independent, transcriptional, and/or translational/post-translational. And, 3. To characterize and identify novel genes required for nociceptive sensitization by expanding our genetic screen. This project represents the first systematic study of nociceptive sensitization in a model genetic organism and has great potential for uncovering the genes that initiate, execute, and regulate this process. Given the conservation of genes required for most fundamental neuronal functions we expect that this project will inform our understanding of nociceptive sensitization in vertebrates and in pathophysiological states, such as cancer and chronic pain syndromes, where this form of sensory neuron plasticity is thought to be improperly activated or regulated. PUBLIC HEALTH RELEVANCE: This research project employs a model genetic organism, the fruit fly, to uncover the genetic control of nociceptive (pain) sensitization, a process of immediate relevance to human health. Nociceptive sensitization is critical for organisms to protect sites of tissue injury, such as those caused by trauma or surgery, while they heal. Given the conservation of genes required for most fundamental biological processes, including neuronal function, we expect that this project will inform our understanding of nociceptive sensitization in vertebrates and in pathophysiological states, such as cancer and a variety of chronic pain syndromes, where the sensitization response is thought to be improperly activated or regulated.

DESCRIPTION (provided by applicant): The 2011 Gordon Research Conference on Tissue Repair and Regeneration (TRR-GRC) will be held from June 5-10 at Colby Sawyer College, New London, New Hampshire. In conjunction with this meeting, a Gordon-Kenan Graduate Research Symposium (GRS) will be held June 4-5 at the same site. This latter pre- meeting is designed to provide a platform for graduate students and post docs to present work relevant to tissue repair/regeneration so that they become more fully integrated into the TRR-GRC that follows. This will be the tenth iteration of the full TRR-GRC and the first that includes a GRS pre-meeting. Both conferences address topics that sit at the crossroads of cell/developmental biology and medicine/tissue engineering- how the body's different tissues sense and respond to tissue damage. These topics are a major public health/clinical concern as inadequate wound healing following trauma or surgery and diseases that involve misregulated tissue repair responses, such as fibrosis and cancer, affect millions in the US per year. Nine scientific sessions are planned which span the basic and translational science of tissue repair and regeneration: 1) Danger Signaling, 2) Barrier repair and regeneration, 3) Tissue Engineering, 4) Organ repair and regeneration, 5) Cancer as a disease of misregulated wound healing, 6) Regenerating pattern throughout the animal kingdom, 7) Inflammation, 8) Stem Cells and Regenerative Medicine, and 9) Matrix and Fibrosis. In all sessions we have invited speakers who have recently published high profile stories of broad interest. Sessions 2-4, 6-7, and 9 are traditional for this meeting whereas the sessions on danger signaling, cancer and wound healing, and stem Cells/regenerative medicine are meant to highlight emerging new directions in the field to which attendees from any discipline would benefit from exposure. Indeed, this TRR-GRC is unique in that it has traditionally sought a very diverse group of scientists, from basic cell biologists/bioengineers to clinicians, attacking the problems of tissue repair and regeneration with a variety of experimental approaches and models. Exposing these groups to each other and fostering the discussions and collaborations that result from this exposure, is a major longstanding goal of this TRR-GRC. Our diverse speakers will emphasize unpublished results and be encouraged to make their presentations accessible to attendees from outside their immediate disciplines. Participation by junior scientists (students and postdocs) will be greatly facilitated by the GRS pre-meeting and indeed many of the TRR-GRC invited speakers and discussion leaders are junior investigators actively taking the field in exciting new directions. These will be complemented with eminent senior investigators who have a comprehensive and historical view of the field. Participation by underrepresented minorities will be encouraged and actively sought and scholarships subsidizing their involvement will be offered through existing TRR-GRC programs. Generous NIH support will substantially enhance the quality of the program and collaborative opportunities fostered by the planned program. PUBLIC HEALTH RELEVANCE: This proposal requests funds to support a scientific meeting whose topic is tissue repair and regeneration. These subjects are a major public health concern as traumatic injury is a leading cause of death for young Americans and inadequate healing/repair due to a variety of physiological factors (age, infection, fibrosis) or disease states (diabetes, vascular disease, cancer) is a major clinical preoccupation and public health burden. The philosophy behind the meeting is that a thorough understanding of the basic science of repair/regeneration can be translated into effective therapies for tissue repair and regeneration.

DESCRIPTION (provided by applicant): Pain sensitization is an adaptive response to tissue injury because it fosters behaviors that protect the site of injury while it heals. Sensitization cn occur across multiple sensory modalities such as thermal and mechanical and can manifest as responsiveness to previously non-noxious stimuli (allodynia) or exaggerated responsiveness to noxious stimuli (hyperalgesia). My laboratory has been modeling the thermal mode of nociceptive sensitization in Drosophila larvae since precise quantitative assays for this mode have been developed and refined. Clinically, however, mechanical hypersensitivity following tissue trauma, surgery, or in a variety of disease states that cause chronic pain (such as cancer and diabetes) is a more serious problem than the thermal mode. This is because patients encounter mechanical stimulation much more commonly in the course of daily life. Our goal in this R21 proposal is to develop the assays and tools and a sufficient basic genetic depth of understanding for the field to begin to dissect mechanical pain hypersensitivity in Drosophila. Our guiding hypothesis is that there is conserved molecular genetic machinery that initiates, regulates, executes, and terminates mechanical nociceptive sensitization. We have developed customized Von Frey filaments suitable for testing mechanical nociceptive responses in Drosophila larvae, have defined the precise noxious range for these animals, and have demonstrated the existence of mechanical allodynia and hyperalgesia following UV-induced tissue injury. Further, in a small-scale pilot screen we have identified a small number of genes required for mechanical nociception. Our project goals are enumerated in the following specific aims: 1. To test the hypothesis that particular types of tissue damage (physical wounding, burns, chemotherapy) are more potent at inducing mechanical nociceptive hypersensitivity. 2. To test the hypothesis that mechanical nociceptive hypersensitivity uses the same inducing signals as thermal nociceptive hypersensitivity. And, 3. To characterize novel genes required for mechanical nociceptive sensitization. This project represents the first systematic study of mechanical nociceptive sensitization in a model genetic organism and has great potential for uncovering the genes that initiate, execute, and regulate this process. It uses a template that we have already used successfully in our approach to studying thermal nociceptive sensitization. Given the conservation of genes required for most fundamental neuronal functions we expect that this project will inform our understanding of mechanical nociceptive sensitization in vertebrates and in pathophysiological states, such as cancer, diabetes, and chronic pain syndromes, where this form of sensory neuron plasticity is thought to be improperly activated or regulated.

PROJECT SUMMARY/ABSTRACT Pain and pain sensitization are devastating side effects of tissue injury, cancer treatment, and certain diseases like diabetes. Treating pain effectively is an enormous clinical obstacle for many injuries and disease states. Pain research leaders have argued that new models in which novel conserved gene targets can be efficiently identified are urgently needed. Even more essential is the translation of new conserved targets functionally identified in these new and simple models to more clinically relevant vertebrate models. Our long-term goal is to identify novel pain/pain sensitization regulators with high potential for clinical translation. Over the last ten years my laboratory has performed a number of genetic screens in the fruit fly Drosophila, by combining unique tissue damage and pain assays. These screens have identified highly conserved regulators of pain biology (in particular acute and chronic pain sensitization following tissue damage) whose roles in regulating pain were not previously appreciated from work in other models. The two most promising screen hits as defined by conservation, robustness and uniqueness of their Drosophila phenotype, and novelty to pain biology, are Smoothened, the signal transducer of the Hh signaling pathway, and the Insulin receptor. In fly larvae, both genes are required in peripheral nociceptive sensory neurons? Smoothened to regulate acute thermal pain sensitization and the Insulin receptor (InR) to regulate the cessation of acute sensitization. InR is particularly interesting as the neuron-specific role in regulating the transition from acute to chronic sensitization may provide an entirely new way of looking at the pain associated with diabetes. Our goal in this exploratory R21 proposal is necessary and urgent. For both Smoothened and the Insulin Receptor we propose to perform parallel experiment(s) in mice to those that we have already performed in flies: we will conditionally knock these genes out in pain-sensing sensory neurons and assess baseline, acute, and chronic pain phenotypes in two standard assays. This is necessary because this type of forward translation of Drosophila-based results, though standard and useful in developmental biology and innate immunity, has not been previously attempted in any systematic way in the pain field. It is urgent because successful translation (meaning the genes do actually regulate pain biology in some way in mice) could establish a veritable pipeline of new conserved pain gene targets for testing in vertebrate models. This is especially true as we have many other conserved targets that have been identified in our screens. The results could be both scientifically compelling- providing new insight into conserved regulation of pain responses. They could also be clinically useful- pointing the way to new druggable conserved targets that have been difficult to identify systematically in other complementary experimental systems. The importance of pain-sensing and sensitizing mechanisms to the animal, reflected in the high evolutionary conservation of these mechanisms, suggest to us that this approach is likely to work.

PROJECT SUMMARY/ABSTRACT Tissue repair is essential to multicellular organisms that occasionally encounter a hostile environment. The ability to rapidly repair and restore function to barrier tissues is critical to organismal survival and is highly conserved. My laboratory pioneered the use of Drosophila larvae to study postembryonic epidermal wound healing. Over the past eleven years (9 of them funded by R01GM083031; Genetic control of postembryonic wound healing in Drosophila) we have investigated the signaling mechanisms that initiate wound closure, that recruit inflammatory macrophage-like cells to the wound, that actually orchestrate wound-induced cell migration, and that mediate the curious phenomenon of wound-induced epidermal cell-cell fusion. This NIGMS R35 MIRA application is intended to provide more stable and flexible funding to my laboratory as it continues to pursue important cellular and mechanistic questions regarding organismal tissue repair responses. Our long- term goal is to identify the full suite of genes that initiate each important wound response (migration, cell shape change, dedifferentiation, fusion, inflammation) and understand how these genes function and work together to orchestrate a successful wound repair program. Our work over the next five years will focus on three essential questions that emerge naturally from our ongoing studies: 1. How does epigenetic reprogramming and a coordinated transcriptional response in wound edge cells help facilitate closure? This question emerges from our observation that some epigenetic regulators are cleared from wound edge cells and others, along with certain transcription factors, are directly required for wound closure. A major effort will be devoted to understanding the transcriptional and epigenetic changes that are necessary for healing at the wound edge. 2. We have now set up a viable platform for identifying and studying the function of genes that are necessary for the curious phenomenon of wound-induced cell-cell fusion. This effort is likely to lead to major novel insights into how the understudied process of epithelial cell-cell fusion is controlled and orchestrated. 3. How do blood cells adhere to and spread at a wound to serve their function of clearing cellular debris? We have now identified at least one signaling pathway (Vascular Endothelial Growth Factor [VEGF] signaling) that is required for blood cell spreading at wound sites. This suggests that attachment and spreading are genetically separable events and we have developed a strategy for both identifying more players in this process and studying the function of known players that act downstream of VEGF in this context. My lab's substantial history of creative high-impact publications on diverse aspects of tissue repair suggests strongly that we will continue in this vein, especially if our ongoing grant-writing burden is reduced. Our system complements others in the field and the likelihood of continued novel basic insight into how organisms cope with tissue damage at the cellular and molecular/genetic levels is high.