Carmen W. Dessauer - US grants

DESCRIPTION (provided by applicant): The cAMP pathway has key physiological effects on heart function and is essential for the chronotropic, inotropic and lusitropic effects during the 'fight-or-flight' response. However, chronic activation of the cAMP pathway induces hypertrophic growth and ventricular dysfunction, which ultimately leads to the development of chamber dilatation and heart failure. Despite the description of a number of alterations in the cAMP pathway during hypertrophy and heart failure, the exact mechanisms accounting for the cardiotoxicity of this pathway are not fully understood. Much research has been dedicated to understanding the roles in heart for types 5 and 6 adenylyl cyclase (AC), the enzymes that synthesize cAMP. However roles for additional ACs expressed in cardiac myocytes have been largely overlooked. We have recently shown that type 9 AC is not only expressed in adult mouse cardiomyocytes, but is the only AC isoform to associate with the Yotiao- IKs complex in both transgenic mouse models and guinea pig heart. Sympathetic responses increase cAMP signaling which increases IKs current and shortening of the action potential duration to allow sufficient diastolic intervals in the face of increased heart rate. Mutations that disrupt interactons between the IKs channel subunit (KCNQ1) and the scaffolding protein Yotiao give rise to Long-QT syndrome. We have provided significant evidence that adenylyl cyclase (AC) is an integral part of signaling scaffolds known as A-kinase anchoring proteins (AKAPs) that coordinate events both upstream and downstream of cAMP production. We hypothesize that AC9 plays a key role in cAMP regulation of cardiac function and IKs regulation via interactions with Yotiao and possibly additional AKAPs. Specific Aims will 1) examine the enzymatic regulation of AC9 and real-time production of cAMP in cardiac myocytes, 2) identify and characterize AC9 complexes in cardiac myocytes, and 3) examine the functional roles of AC9 in heart.

DESCRIPTION (provided by applicant): D2 dopamine receptors have been implicated in neuropsychiatric and neurologic disorders including schizophrenia, drug abuse, and Parkinson's disease. Acute activation of D2 dopamine receptors inhibits cyclic AMP accumulation; however, persistent activation of D2 dopamine receptors enhances subsequent drug-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylyl cyclase (AC) signaling occurs following persistent activation of several G?i/o-coupled receptors in vitro and in vivo. The overall objective of this research proposal is to elucidate the molecular mechanisms involved in heterologous sensitization of AC following persistent activation of D2-like dopamine receptors. Previous studies support a hypothesis that heterologous sensitization requires the activation of G?i/o subunits to induce sensitization through a G??-dependent mechanism. We hypothesize that G?? subunits lead to heterologous sensitization of individual AC isoforms through both direct and indirect mechanisms. The indirect mechanisms may involve protein-protein interactions as well as G?s. The general approach for these studies will be to express heterologously D2L dopamine receptors together with well characterized wild-type or mutant ACs (e.g. AC1, AC2, and AC5) for intact cell experiments in unique cellular backgrounds (i.e., G protein subunit deficient). This strategy takes advantage of recently discovered molecular and cellular tools to study G protein signaling as well as novel fluorescent technologies. The first specific aim will test the hypothesis that heterologous sensitization of select isoforms of AC involves G??-AC interactions and requires G?? subunit signaling. These studies will use a series of AC mutants, unique cellular models, small molecule inhibitors of G?? subunit signaling, and striatal neurons. The second specific aim will determine the roles and requirements for G protein subunits in modulating receptor-AC and AC-AC interactions. These experiments will use bimolecular fluorescence complementation (BiFC) to probe the specific role of G?? and G?s subunits in modulating basal and drug-induced protein-protein interactions in living cells. The third specific aim will identify and characterize the AC sensitization interactome using BiFC in a neuronal cell model. These studies will use BiFC to perform cDNA library screening to identify sensitization-induced interacting proteins of AC in living cells. Completion of the proposed studies will deliver mechanistic information regarding specific G protein subunits and new protein targets that could ultimately be used to prevent the development and expression of heterologous sensitization in vivo.

DESCRIPTION (provided by applicant): Persistent activation of G?i/o-coupled receptors leads to enhanced adenylyl cyclase (AC) signaling that has been described using many different names, including cAMP overshoot and heterologous sensitization of AC. This adaptive response has been implicated in several psychiatric and neurological conditions. Previous studies support a hypothesis that persistent activation of G?i/o-coupled receptors promotes the dissociation/rearrangement of G? and G?? subunits in a pertussis toxin-sensitive manner that induces sensitization through a yet unknown mechanism. We hypothesize that drug-induced protein interactions with AC are responsible for the enhanced AC response. Previous studies have examined closely-related proteins or established AC interacting partners preventing the discovery of truly novel mechanisms. Thus, an unprecedented approach will be used to identify the sensitization interactome of adenylyl cyclase type 5 (AC5) in a neuronal cell model. These studies will use Bimolecular Fluorescence Complementation (BiFC) to perform cDNA library screening to identify sensitization-induced interacting proteins of AC5 in living cells. Specific am 1 will construct and characterize a neuronal cellular model for drug-induced BiFC of AC5. These studies will use CAD cells stably expressing an engineered AC5 fusion construct capable of fluorescence complementation with appropriate binding partners. Specific aim 2 shall construct the retrovirus-based BiFC cDNA library of potential AC5 binding partners for FACS screening. Specific aim 3 shall execute both primary and secondary screening for drug-induced BiFC. These studies will infect the neuronal cell model with the retroviral cDNA library followed by treatment with a G?i/o receptor agonist to induce heterologous sensitization of AC activity. Cells revealing drug-induced BiFC will be identified using FACS and isolated for cDNA amplification. Specific aim 4 will initiate a series of biochemical and functional studies to characterize those interacting proteins that represent the AC5 sensitization interactome. The anticipated outcome of these aims is the identification and characterization of novel AC5 interacting proteins relevant to heterologous sensitization. The impact of these scientific outcomes is substantial and will address a long-standing scientific question, develop a novel methodological approach, and have the potential to provide drug targets for in vivo studies.

Project Summary: Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment, while the mechanisms that drive and/or maintain chronic pain remain unclear. We have shown that chronic nociceptor hyperexcitability after severe injury is maintained by cAMP signaling through multiple cAMP effectors, including PKA, EPAC and HCN channels. These pathways are enhanced by AKAP-mediated complex formation with AC and show significant cross-talk with Ras/MAPK signaling. Activation of cAMP- and Ras-mediated pathways initiate at the plasma membrane (PM) and are uniquely sensitive to clustering of lipids within the PM. We have also shown that spinal cord injury reduces AC inhibition by G?i, resulting in reduced potency of opioids in DRG neurons. This reduced sensitivity can be mimicked in DRG neurons from naïve animals by overnight exposure to neurotrophic factors or by a 5 min, modest depolarization that approaches the firing threshold of DRG neurons after severe injury. Importantly, nociceptor hyperexcitability and reductions in opioid potency, induced by either injury, neurotrophic factors or acute depolarization, can be reversed by inhibition of Ras-dependent signaling or reorganization of lipids in the plasma membrane. We hypothesize that the sustained depolarization that occurs in many injury models drives alterations in PM lipid organization, leading to increased ERK signaling and decreased opioid responses. Release of neurotrophic factors reinforce these pathways and, in conjunction with cAMP signaling, drives nociceptor hyperexcitability and a chronic pain state. To address these hypotheses, we propose three Aims. 1) Determine the mechanism for reduced MOR-G?i inhibition of AC by C-Raf, 2) Define the mechanism of Ras activation and nociceptor hyperexcitability by depolarization and SCI, and 3) Define functional consequences of interactions among depolarization and cell signaling by cAMP, C-Raf, and ERK. Importantly, our model identifies multiple FDA-approved drugs that could simultaneously enhance endogenous opioid responses and block nociceptor hyperexcitability after severe injury.

PROJECT SUMMARY / ABSTRACT Training Interdisciplinary Pharmacology Scientists (TIPS) will train predoctoral students to address critical issues in drug development, by providing them the tools and knowledge to navigate the ever- changing, interdisciplinary landscape that makes up today?s basic, translational, and clinical research. This T32 program is designed to create leaders in the broadly defined field of Pharmacological Sciences. TIPS will provide interdisciplinary training in drug development including screening, computation, medicinal chemistry, and structural biology relevant to target discovery and validation, while exploring how to maximize therapeutic benefit, minimize toxicity and implement precision medicine. Through the participation of 3 premier and neighboring biomedical research institutions and their graduate schools (University of Texas Health Science Center at Houston, Baylor College of Medicine, and Rice University), TIPS will provide interdisciplinary training, career development activities and industry shadowships that will prepare students to advance biomedical research through successful careers in academia, government and regulatory agencies, and industry. Program leadership includes the program director and co-director and a steering committee of representatives from the participating graduate schools, with assistance from an advisory committee of diverse experts. TIPS program requirements and benefits will include co-mentoring from two faculty in a collaborative research project; specific didactic and elective courses (available to TIPS trainees at all participating graduate schools); monthly trainee meetings to build a unified cohort; career development activities, and required training in the responsible conduct of research. Mentors will include 26 faculty members at 3 institutions, working in 9 different departments and two interdepartmental graduate programs, who have training and research expertise in drug/compound screening, pharmacology, computation, medicinal chemistry, structural biology, systems biology, biochemistry, genetics, and molecular and cellular biology. All participating departments recruit nationwide, especially for under-represented minority students and students with disabilities. This provides a large, diverse pool of predoctoral students from a variety of backgrounds for our 5 requested slots. Students will apply to TIPS after joining a lab near the end of their first year or during their second year of graduate school, and if selected through a competitive application process, will receive two years of support. Reappointment to a second year will be contingent on a thorough oral and written review of trainee research progress and completion of program requirements. TIPS will complement the degree requirements of a trainee?s home institution, causing no delay in degree completion. The interdisciplinary skills emphasized by this program will produce pharmacology scientists well positioned to advance biomedical research and to address the complexity of drug development.

PROJECT SUMMARY / ABSTRACT Training Interdisciplinary Pharmacology Scientists (TIPS) will train predoctoral students to address critical issues in drug development, by providing them the tools and knowledge to navigate the ever- changing, interdisciplinary landscape that makes up today?s basic, translational, and clinical research. This integrated T32 program is designed to create leaders in the broadly defined field of Pharmacological Sciences. TIPS will provide interdisciplinary training in drug development including screening, computation, medicinal chemistry, and structural biology relevant to target discovery and validation, and an understanding of regulatory issues involved in advancing candidate therapeutics to clinical application. Through the participation of 3 premier and neighboring biomedical research institutions and their graduate schools (University of Texas Health Science Center at Houston, Baylor College of Medicine, and Rice University), TIPS will provide interdisciplinary training, career development activities and industry shadowships that will prepare students to advance biomedical research through successful careers in academia, government and regulatory agencies, and industry. Program leadership includes the program director and co-director and a steering committee of representatives from the participating graduate programs, along with an external advisory committee of diverse experts. TIPS program requirements and benefits will include co-mentoring from two faculty in a collaborative research project; specific didactic and elective courses (available to TIPS trainees at all participating schools); monthly trainee meetings to build a unified cohort; a wide variety of career development activities, and required training in the responsible conduct of research and rigor and reproducibility. Mentors will include 31 faculty members at 3 institutions, working in 14 graduate programs, who have training and research expertise in drug/compound screening, pharmacology, computation, medicinal chemistry, structural biology, systems biology, biochemistry, genetics, and molecular and cellular biology. All participating departments recruit nationwide, especially for students of diverse backgrounds who are under-represented in the biomedical sciences, including individuals with disabilities, from disadvantaged backgrounds, and from under-represented racial and ethnic groups. This provides a large, diverse pool of predoctoral students from a variety of backgrounds for our 5 requested slots. Students will apply to TIPS after joining a lab near the end of their first year or during their second year of graduate school, and if selected through a holistic application process, will receive two years of support. Reappointment to a second year will be contingent on a thorough oral and written review of trainee research progress and completion of program requirements. TIPS will complement the degree requirements of a trainee?s home institution, causing no delay in degree completion. The interdisciplinary skills emphasized by this program will produce pharmacology scientists well positioned to advance biomedical research and to address the complexity of drug development.