Mark M. Voigt, Ph.D. - US grants

DESCRIPTION: (Investigator's Abstract) Evidence accumulated over the past three decades supports the role of adenosine-triphosphate (ATP) as a neurotransmitter/neuromodulator in many adrenergic, cholinergic and non-adrenergic/non-cholinergic pathways. Its role as a chemical mediator of intercellular communication has been established in tissues such as the central and peripheral nervous systems, endocrine glands, heart and smooth muscle. However, the mechanisms which mediate ATP's role as a chemical signal is poorly understood. A primary reason for this is the presence of multiple receptor subtypes in most tissues coupled with a lack of subtype-selective agonists and antagonists. Additionally, in some tissues there are high levels of cell-surface enzymes (ecto-ATPases) that can quickly degrade many of the ATP analogues used for receptor classification, resulting in further complications. In order to clearly understand the role of ATP-mediated transmission, it is essential to define the pharmacological and physiological properties of ATP receptors. Currently, ATP receptors fall into one of two classifications, P2gamma and P2x receptors. The focus of this grant is the ATP receptors which belong to the p2x category. These receptors are ligand-gated channels that are permeable not only to monovalent cations such as Na+ and K+, but also to Ca+. In this project, the investigators will circumvent the difficulties associated with investigating multiple receptor subtypes in their native settings through the use of recombinant receptor expression in mammalian cells. This approach allows for the expression of individual receptor subtypes in a controlled environment, thus permitting detailed characterization of the basic properties of a receptor subunit. The applicants have embarked on the investigation of P2x receptors at the molecular and functional level using a compare and contrast methodology, utilizing novel P2x receptor subunits that they have recently cloned from brain and heart. In addition, they will elucidate the topological conformation of the proteins within the membrane and the subunit composition of receptor assemblies. They will also search for additional proteins that participate in the formation of functional assemblies. These experiments will provide essential information regarding P2x receptor function and the role of ATP as a neurotransmitter in the central and peripheral nervous systems. The overall goal of this project is to provide a clearer understanding of the pharmacological and physiological properties associated with specific P2x receptor subtypes.

Evidence accumulated over the past three decades supports the role of adenosine-tripphosphate (ATP) as a neurotransmitter/neuromodulator in many adrenergic, cholinergic and non-adrenergic/non/cholinergic pathways. Its role as a chemical mediator of intercellular communication has been established in tissues such as the central and peripheral nervous systems, endocrine glands, heart and smooth muscle. However, the mechanisms which mediate ATP's role as a chemical signal is poorly understood. A primary reason for this is the presence of multiple receptor subtypes in most tissues coupled with a lack of subtype-selective agonists and antagonists. Additionally, in some tissues there are high levels of cell-surface enzymes (ecto-ATPases) that can quickly degrade many of the ATP analogues used for receptor classification, resulting in further complications. In order to clearly understand the role of ATP-mediated transmission, it is essential to define the pharmacological and physiological properties of ATP receptors. Currently, ATP receptors fall into one of two classifications, P2Y and P2X receptors. The focus of this grant are the ATP receptors which belong to the P2X category. These receptors are ligand-gated channels that are permeable not only to monovalent cations such as Na+ and K+, but also to Ca2+. In this project, we will circumvent the difficulties associated with investigating multiple receptor subtypes in their native settings through the use of recombinant receptor expression in mammalian cells. This approach allows for the expression of individual receptor subtypes in a controlled environment, thus permitting detailed characterization of the basic properties of a receptor subunit. We have embarked on the investigation of P2X receptors at the molecular and functional level using a compare and contrast methodology. In this study we will elucidate the stoichiometry of receptor assemblies, elucidate the domains involved in subunit interactions and identify amino acid residues that participate in agonist (binding and gating of the channel. These experiments will provide essential information regarding P2X receptor function and the role of ATP as a neurotransmitter in the central and peripheral nervous systems. The overall goal of this project is to provide a clearer understanding of the pharmacological and physiological properties associated with specific P2X receptor subtypes.

[unreadable] DESCRIPTION (provided by applicant): A zebrafish embryo goes from a single-cell to an organism with a functioning nervous system within its first 24 hours of existence. By 24 hours post-fertilization, the embryo has mechanosensory-mediated reflex motor behaviors and by 96 hr the fish is a free-swimming larva. The manifestation of these behaviors is due to the establishment of a simple but effective neural network in the spinal cord and hindbrain of the developing fish. This simple nervous system provides the scaffolding for further expansion during maturation of the organism. Elucidating the mechanisms underlying early nervous system formation in the zebrafish could have a huge impact on our understanding of how many neurological disorders arise in man. The properties of the embryonic/larval zebrafish has made it uniquely suited to study the formation of a neural network- it's fertilized externally, transparent during its development and lends itself easily to molecular techniques such as anti-sense treatment and transgenesis. Anatomical studies using dye labeling have yielded information regarding the morphologies of spinal neurons during these early time points, suggesting the presence of some 11-15 neurons per hemi-segment of the cord between 24-96 HPF. In addition, these studies have provided some insight into how the neurons connect to one another to form a network. However, almost nothing is known about how these neurons communicate amongst each other. It is the purpose of this proposal to generate transgenic fish lines that will enable the elucidation of the neurochemical anatomy of the developing spinal cord. Not only will these fish provide information as to the connectivity of the various spinal and supraspinal pathways, but they will also provide markers for use in functional studies utilizing either co-expression (e.g., with calcium indicators) or electrophysiological approaches. In addition, they can provide markers for specific neuronal types that can be used in forward genetic screens aimed at identifying genes required for either phenotype or connectivity decisions in these cells [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There are numerous clinical conditions in which altered sensory signaling from the face, head and/or viscera are manifested, e.g. peripheral neuropathies arising from diabetes, HIV/AIDS and herpes, trigeminal neuralgia, the various hereditary sensory neuropathies, neuropathies arising from trauma or surgical outcomes, and others. We have recently engineered a transgenic fish line that expresses an eGFP marker protein in neurons of the cranial sensory ganglia (CSG). The eGFP produced is soluble and fills the cell body together with its peripheral and central projections. This expression begins at the time these neurons are forming the ganglia and extending their axonal processes, and is maintained through at least three weeks of age. We propose using these transgenic fish in a ethylnitrosourea-based mutagenesis screen in an effort to identify genes involved in any of the following three processes: formation of the individual cranial ganglia, innervation of specific targets by different ganglia, and innervation of different targets by individual neurons within the same ganglia. There are two aims for the work proposed in this application: (1) to introduce ENU mutated genes into the TG(3.2::eGFPGR) line and use an F3 recessive screening paradigm to identify carrier families that produce embryos exhibiting defects in CSG development and connectivities and (2) to characterize the identified mutants. We have initiated a preliminary screen using a small number of carrier families (21). To date, we have already identified two mutants (out of 11 families screened) that have developmental abnormalities in their CSG by 96 hpf. Allele sl14 exhibits disrupted formation of the trigeminal ganglia while having normal epibranchial ganglia, whereas allele sl19 has improper targeting of the central and peripheral projections of the epibranchial ganglia while the trigeminal ganglia appear unaltered. This R21 proposal is designed to increase the size of our screen to 150-200 families (genomes) in order to generate a body of mutants that cover the wide spectrum of mechanisms responsible for CSG formation. A successful outcome of this proposal will also strengthen a nascent collaboration between the Voigt lab (SLU), which is focused on peripheral neurons and signaling issues and is adopting zebrafish as a model, and the Appel lab (Vanderbilt), an established zebrafish lab which is interested in mechanisms underlying the myelination of central and peripheral neurons. PUBLIC HEALTH RELEVANCE Millions of people worldwide suffer degraded health and quality of life issues due to peripheral sensory neuropathies. Currently, treatment of such disorders is limited by the lack of clinically useful tools. The principal goal of this project is to provide a springboard for the development of new approaches to the treatment of these diseases by identifying genes, and thus potential therapeutic targets, involved in the normal development of the sensory nervous system.

? DESCRIPTION (provided by applicant): The focus of this project is the peripheral non-myelinating glia that ensheath the majority of sensory axons. Unmyelinated axons are often damaged and/or lost in neuropathies. The non-myelinating glia are thought to play key roles in the repair, degeneration and/or regeneration of the affected nerves. We have chosen to focus on the response to degeneration of sensory axons, one feature common to many neuropathies. To gain a better understanding of the mechanisms involved in the glial response to axonal damage/degeneration, we developed an inducible model of peripheral sensory nerve degeneration in zebrafish. This model permits the conditional and selective ablation of peripheral sensory neurons and their axons. In our initial studies, we have found evidence that upon axon degeneration, peripheral glia are critical to the removal of axonal debris and recruitment of immune cells to sites of the involved nerves. The hypothesis that will be tested in this proposal is that axonal damage and degeneration induces glia to alter their gene expression, resulting in phagocytic behaviors and signaling to the immune system. In this application, we propose two aims: (1) identify genes involved in the response of peripheral glia to sensory neurodegeneration and (2) to validate the candidate genes identified in Aim 1. This will be achieved through transcriptome analysis of control and metronidazole- treated fish. Validation of candidate genes will be performed using qPCR, in situ hybridization and CRISPR/Cas9 mediated deletion analysis. Identification of involved genes will provide an important foundation for future investigations into the mechanisms by which peripheral glia react to death and degeneration of sensory circuits, and can be used as the starting points for directed studies in higher vertebrates, with the eventual goal of developing new directions in clinical efforts to treat this aspect of many debilitating sensory neuropathies.

? DESCRIPTION (provided by applicant): This application is a request for the 26+ years of continuous support for the Training Program in the Pharmacological Sciences of the Saint Louis University School of Medicine. We seek financial support for six outstanding predoctoral students who will be working for a Ph.D. degree with research emphasis on cellular communication and disease. This is a broadly based, multidisciplinary effort, which involves 21 faculty members from five departments in the Saint Louis University School of Medicine. These departments include: Pharmacological and Physiological Science; Biochemistry and Molecular Biology; Chemistry; Internal Medicine; Otolaryngology and the Center for World Health and Medicine. Students will be selected from candidates who have successfully completed the one year Core Program in Basic Biomedical Science, directly enter with advanced degrees, or M.D./Ph.D. students who have completed the first two years of medical school. We have designed a curriculum that provides in-depth training in Pharmacological Science regardless of the student's background. During the first year of study, all traditional Ph.D. students enroll in the interdisciplinary Core Graduate Program in Biomedical Sciences. This program is designed to provide students with a strong foundation in all aspects of basic biomedical science and the freedom to explore diverse research opportunities. The curriculum combines lectures, small group discussion and seminars. Students entering the Pharmacological Sciences Training Program take advanced pharmacology, journal clubs and seminars. Key to this training program is integration of quantitative pharmacological approaches, application of chemical biology tools, and drug discovery techniques into the formal training program. We utilize several key colleagues from the pharmaceutical industry to enhance this portion of the training program. Subsequent training for all Ph.D. candidates will concentrate on the development of research and teaching competence in a specific area of inquiry under the mentorship of one or more members of the Pharmacological Sciences Training Faculty. The mentors and laboratories participating in this program are well equipped to provide state-of-the-art research training. In addition, core and shared facilities for advanced technologies are available for enhancement of the research training of the participating candidates. Students supported by the T32 Pharmacological Sciences Training Grant have access to a range of unique enrichment activities. The overall objectives of this training program are to provide individuals with the opportunity to achieve a high degree of competence in the area of pharmacological sciences thus preparing them for teaching and research careers.