Timothy E. Glass, Ph.D. - US grants

DESCRIPTION (provided by applicant): Fluorescent chemical sensors for small organic compounds will be developed. Chemical sensors are synthetic probes which produce a visible signal upon interaction with a specific analyte. In the biochemical community, these probes have been used as sensitive, nondestructive methods for quantifying the concentration of a particular analyte in cells. Chemical sensors have played a pivotal role in unraveling the cellular function of a number of metal ions, most notably cell calcium. Similar fluorescent sensors for organic molecules have not reached this level of success. The specific aims of this research are to develop novel chemical sensors for neurotransmitters and neuromodulators. This is an important problem with broad ramifications. This project will produce sensors for glutamate, aspartate, lysine, and the catecholamines which function under physiologically relevant conditions of pH and salinity. Furthermore, fluorophores which are pH dependant will be produced to expand the utility of this class of sensors. Biological studies will be performed with the aid of collaborators in which cells will be stained with the fluorescent sensors and the location and concentration of neurotransmitter will be measured.

The Macromolecular, Supramolecular and Nanochemistry Program of the Chemistry Division supports Professor Timothy Glass of the University of Missouri on a project focusing on the design and preparation of fluorescent sensors and receptors for various classes of biologically important lipids. These sensors are synthesized from tube-like hydrophobic receptor molecules that selectively recognize certain lipids by shape-selective interactions. Both macrocyclic 'closed tubes' and non-macrocyclic 'open tubes' are being explored. Because most lipids are embedded in a membrane or lipid compartment, the receptors are designed to extract the lipids from the membrane. The introduction of head-group binding units is a central focus of this project. This work lays a foundation for approaching many different problems in lipid recognition.

Fundamental work on lipid recognition and sensing may eventually lead to the ability to selectively detect such lipids in humans. For example, such analyses can be used to test for oxidized phospholipids which have been found to be involved in atherosclerosis; or certain glycolipids which are know to be overproduced on the surface of cancerous cells. Moreover, students involved with this project are trained not only in the design and synthesis of novel compounds, but more broadly in methods of approaching biological problems from a chemical perspective.

? DESCRIPTION (provided by applicant): Neurotransmitters are critical to the regulation of the central and peripheral nervous systems and command a number of functions such as learning, memory, sleep, and movement. Discerning the machinery involved in vesicular fusion, the spatiotemporal mechanisms of synaptic release, and the chemical activity of neurotransmitters is vital to understanding both normal and atypical neuronal processes. The ability to effectively measure neurotransmitters in neurons is an essential tool in the study of neurophysiology and neuropsychiatric disorders. Furthermore, understanding the mechanisms of vesicle fusion and transmitter release via exocytosis is of broad medical significance because it will not only aid in the development of therapies for diseases where release of neurotransmitters is compromised, but it will also advance our understanding of FDA-approved treatments that modulate transmitter release, such as Botulinum Toxin A and B. This project involves the preparation and evaluation of fluorescent chemical sensors for catecholamines with a view toward the fluorescent detection of neurotransmitters. These sensors are related to the NeuroSensor class of probes, which have already been used to detect norepinephrine in isolated chromaffin cells. The major advantage of the proposed sensors is that they will be selective for just one type of neurotransmitter. In addition to these selective sensors, pH dependent variants will be produced and tested for analyte binding as well as pH sensitivity. Here, the sensors will bind the neurotransmitter, but only fluoresce upon undergoing the pH jump typically associated with exocytosis. The sensors will be used to study mechanisms of exocytosis via fluorescence imaging in combination with amperometric measurements.

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Professors Timothy Glass and Kevin Gillis of the University of Missouri are studying how neurotransmitters are released into synapses. Neurotransmitters are critical to the regulation of the nervous system and control a number of functions such as learning, memory, sleep, and movement. Understanding the machinery of synaptic release, and the chemical activity of neurotransmitters is vital to understanding both normal and atypical neuronal processes. Furthermore, understanding the basic mechanisms of synaptic vesicle fusion and transmitter release via exocytosis is of broad significance because it will not only aid in the development of therapies for diseases where release of neurotransmitters is compromised, but it will also advance our understanding of FDA-approved treatments that modulate transmitter release, such as botulinum toxin A and B. As part of the broader impacts of this work, a new robotics camp for underserved high school students will be held. It has been demonstrated that activities such as these camps enhances the likelihood that participants pursue STEM (science, technology, engineering and mathematics) coursework during their secondary education.<br/><br/>This project involves the preparation and evaluation of fluorescent chemical sensors for catecholamines with a view toward the fluorescent detection of neurotransmitters. These sensors are related to the NeuroSensor class of probes developed in PI Glass’s lab, which have already been used to detect norepinephrine in isolated chromaffin cells. Sensors will be developed that produce fluorescence enhancements upon exocytosis. The sensors will be used in combination with novel transparent electrodes developed in PI Gillis’ lab to study mechanisms of exocytosis via fluorescence imaging in combination with amperometric measurements. These combination experiments will measure both release and retention of catecholamine from individual vesicles to test the hypothesis that the amount of catecholamine released from a vesicle is modulated by the stimulus intensity. This project is expected to result in the development and application of a new set of important chemical biology tools for the study of neurochemistry with potentially broad scientific impact.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.