Leah R. Reznikov, PhD - US grants

DESCRIPTION (provided by applicant): Exaggerated airway smooth muscle (ASM) contraction and airway narrowing are hallmark traits of asthma. Contraction of ASM is predominantly regulated by the nervous system. In airway diseases such as asthma, inflammation causes neural regulation of ASM to become defective, thus promoting hypercontraction. We recently generated a novel porcine model of a chronic airway disease (cystic fibrosis, CF), and discovered that newborn CF pigs display hypercontracted ASM in the absence of airway infection and inflammation. Thus, the mechanism of ASM hypercontraction in CF remains unknown. In the current application, the candidate hypothesizes that defective neural regulation of ASM causes hypercontraction independent of airway inflammation. She hypothesizes this based upon her work indicating that there are several novel nervous system phenotypes in newborn pigs with CF. These include reduced axon density, decreased innervation of the airway, and decreased nerve function. The candidate proposes to: 1) determine whether inhibitory neural control (pro-relaxation) of ASM is defective in CF pigs; and 2) investigate whether blocking pro-contractile neural input ameliorates ASM hypercontraction in CF pigs. The candidate's long-term career goal is to become a recognized leader in neuroscience and airway disease research. She plans to advance both fields by examining neural regulation of ASM using porcine models. The selection of the porcine model is particularly relevant because the airway anatomy and physiology, as well as the nervous system, more closely resemble humans than traditional rodent models. In the current K99/R00 application, the candidate will gain intellectual, professional and technical skills that will allo her to become an independent and successful investigator specializing in neural regulation of ASM. During the mentored phase, she will learn ASM biology, lung slice culturing, Ca2+ imaging, morphometry, and whole animal pulmonary mechanics using flexiVent. She has created an exceptional mentoring team and training plan to ensure she learns these skills. In addition, the candidate will give formal presentations at Mayo Clinic, attend the American Asthma Foundation Funding Breakthrough Research Annual Meeting of Awardees, review manuscripts for American Journal of Respiratory Cell, serve as a group leader for a medical students Problem-Based Learning course, and attain skills important for managing budgets. The candidate will utilize these skills during her independent phase to investigate neural regulation of ASM in acid-induced airway injury. This topic is highly significant as acidification f the airway occurs in asthma and acidic pH potently activates axons innervating the airway. Hence, the candidate has an unprecedented opportunity to elucidate neural mechanisms involved in ASM hypercontraction using models with direct relevance to human disease. In summary, this award will train the candidate to become a leader in neuroscience and airway disease research, thereby advancing the field and enhancing the lives of people living with airway disease.

PROJECT SUMMARY Large animals are increasingly utilized in biomedical research and offer an advantage in that their anatomy and physiology closely parallel humans. Yet despite these advantages, rodent models dominate many areas of research, including neuroscience. This might be due in part to the greater number of technologies available to rodent researchers. In the current application, the investigator proposes to create a new technology to facilitate large animal researchers and bridge this gap. Specifically, she proposes to create transgenic pigs with red-shifted channelrhodopsin-citrine fusion proteins or green fluorescent protein/calmodulin protein sensors expressed in neurons. This will allow for simultaneous visualization of neurons (and their innervation), and the ability to precisely control or monitor neural activity. An important advantage of the red-shifted channelrhodopsin variant is that it can be activated by near far red light (~630 nm), thus decreasing phototoxic events and opening the door to less-invasive methods of neural activation (i.e. through the skin). Moreover, the fusion of the green fluorescent protein derivative, citrine, will allow for identification of specific neural elements expressing channelrhodpsin, as well as enable visualization of organ innervation. To create the transgenic pigs, the investigator proposes to target porcine fetal fibroblasts using piggyBac transposon technology. The piggyBac transposon system allows for ?cut and paste? integration of the targeting construct into the porcine genome. An advantage of this approach is that it promotes stable transgene expression. Transgenic pigs will be derived from the targeted fetal fibroblasts at the University of Missouri where Dr. Randall Prather and his team will perform somatic cell nuclear transfer. Embryos containing the targeted nuclear material will be transferred to a surrogate gilt and allowed to develop until term. Transgenic pigs will then be studied and characterized in the investigator's lab at the University of Florida. The proposed work is completely aligned with the priorities of SPARC and will facilitate the imaging and targeting of peripheral nerves with end organs in a large animal model. Moreover, its utility is not limited to peripheral nervous system researchers, as central nervous system neurons will also express channelrhodopsin-citrine fusion proteins or green fluorescent protein/calmodulin protein sensors. Thus, the work proposed in this application has the potential to greatly accelerate the neuroscience field and propel the bench-to-bedside process.

PROJECT SUMMARY Exacerbations (asthma attacks) account for nearly one-third of all asthma deaths. Despite psychiatric illness as a risk factor for death from asthma, and many connections between asthma exacerbations and anxiety, the brain region that initiates anxiety, the amygdala, has received limited attention as a driver of asthma exacerbations. This represents a considerable gap in the field, which, if addressed, may lead to new mechanism-based approaches to treat asthma exacerbations and reduce patient deaths. Exciting preliminary data from control mice suggest that acute optogenetic activation of the basolateral amygdala, a key input center essential for anxiety, reduces airway resistance. In asthmatic mice, which show anxiety, optogenetic activation of the basolateral amygdala fails to reduce airway resistance, suggesting amygdala dysfunction. Amygdala dysfunction is mechanistically linked to anxiety and characterized by heightened activity and spinogenesis (e.g., development of new dendritic spines). Asthmatic mice showed spinogenesis, heightened activity, and elevated expression of genes important for functional and structural remodeling in the basolateral amygdala. To investigate whether these changes were mechanistically linked to impaired regulation of airway resistance, we blocked NMDA glutamate receptors with MK-801, an anxiolytic drug that prevents anxiety-associated basolateral amygdala spinogenesis and in a class of drugs that reduce airway resistance in asthma. We found that MK-801 mitigated bronchoconstriction and diminished elevated gene expression in asthmatic mice. Broadly disrupting the cAMP- responsive element-binding protein (CREB), a transcription factor downstream of NMDA receptor signaling necessary for maintenance of amygdala neuroplasticity, also attenuated bronchoconstriction in asthmatic mice. These data guide our central hypothesis that the basolateral amygdala undergoes NMDA-CREB-dependent plasticity that disrupts airway regulation and promotes pathologic bronchoconstriction. To test this hypothesis, we propose 3 Specific Aims. In Aim 1, we will use optogenetic approaches to activate or inhibit excitatory neurons of the murine basolateral amygdala to test the hypothesis that the basolateral amygdala regulates airway resistance. In Aim 2, we use pharmacologic approaches, magnetic resonance imaging, RNAscope, and Golgi staining to test the hypothesis that experimental asthma structurally and functionally remodels the basolateral amygdala through NMDA receptor signaling. Finally, in Aim 3, we use CRE-lox technology and transgenic mice to test the hypothesis that ablation or overexpression of CREB in the basolateral amygdala alleviates or promotes, respectively, bronchoconstriction. Completion of this proposal will establish NMDA-CREB signaling in the basolateral amygdala as a key driver of asthma exacerbations and highlight NMDA receptor antagonists as a stand-alone or adjunct relief medications for asthma.