John Warrick - US grants

A grant has been awarded to the University of Richmond under the direction of Dr. Roni Kingsley to acquire new and upgraded electron microscopy (EM) equipment for the use of students and faculty conducting independent and course-related research in the Biology, Chemistry, and Psychology departments. The following equipment will be acquired: a variable pressure scanning electron microscope (SEM) with digital imaging, equipment for digital imaging for an existing transmission electron microscope (TEM), a new ultramicrotome and diamond knives for TEM sample sectioning, and a sputter coater to coat SEM samples. The research to be conducted using this instrumentation is diverse, yet the common problem to be addressed throughout all of the projects is achieving state-of-the-art imaging for research and teaching. The images are so compelling while generating essential information. The goal of this project is to use the instrumentation to conduct engaging and innovative research with undergraduate students. A key to the success of the sciences is the continued accessibility to instrumentation to support the research done with students. Electron microscopy is an essential element in, and connection between many disciplines. Therefore, the EM laboratory will be a focal point of interdisciplinary research.

The acquired EM equipment will enhance the research programs of a number of faculty members, as well as opening up new pedagogical opportunities. It will be used for studies across several science disciplines and sub-disciplines including cell physiology and biochemistry (Kingsley, Aprille, Bell), genetics (Warrick), developmental biology (Radice), neural and behavioral biology (Kinsley), systematic botany (Hayden), nanotechnology (Leopold), and immunology (Stenger). Digital imaging will enhance the research programs of all EM users, plus this instrumentation will open up new areas of collaborative research. The paperless images generated by digital imaging may be reviewed in real time by a researcher at the electron microscope and colleagues in a different lab, or building, or institution. Similarly, images can be reviewed with individual student researchers or with an entire class of students. Compared to traditional photography, this is an excellent way to teach from imaged material: immediate feedback, less time and tedium in developing and printing, and much less expensive in materials. The variable pressure mode of the new SEM will allow researchers to study hydrated specimens and specimens in their natural states. The new equipment for TEM and SEM preparation will expedite all research programs and facilitate the training of student researchers.

The acquired EM upgrades and additions will create a truly multidisciplinary research facility. It will allow for greater collaborations and make EM far more accessible to students. The participation of students in independent and course research fosters analytical and critical thinking, and better prepares them for graduate and professional schools and the high technology work force. UR is a primarily undergraduate institution and undergraduates are involved in all phases of the projects described in this proposal. The involvement of students in research as part of their undergraduate education is an important part of the process of recruiting and training bright young people for careers in science. Students from the greater Richmond area involved in the University's ongoing outreach programs will also use the acquired instrumentation.

This award provides support to The University of Richmond (UR) for a suite of neuroscience and animal behavioral-recording equipment, which includes a fluorescence microscope with motorized stage and image analysis (IA) capabilities; motorized cryostat; histological processing system; tissue-sectioning Vibratome; neuron tracing software; automated behavioral analysis system with touch screen; acoustic startle boxes; and activity monitors. This complementary and high-throughput equipment will allow UR and affiliated students and faculty to make further advances in understanding the development of the brain. Several different combinations of investigators will use the requested equipment: research faculty and postdoctoral fellows at UR in Neuroscience, Psychology and Biology; faculty, graduate and undergraduate students from UR and affiliated universities and colleges (Randolph-Macon College, in Ashland, VA; Virginia Union University, a Historically Black College in Richmond, VA; Dickinson College, Carlisle, PA); and students and faculty from international institutions.

These faculty and students will design behavioral experiments that can be analyzed by the latest objective automated behavioral recording devices, including basic and complex social interactions. The cryostat and Vibratome and automated immunohistological apparatus will allow faster, more efficient, and more reliable processing of brain tissue. Automated IA and neuronal identification and 3-dimensional drawing features, as well as new double- and triple-labeling immunofluorescent techniques will provide precise neuronal localization of important neurochemicals. In sum, this equipment will allow the investigators to conduct many detailed, comprehensive, and complementary behavioral, and multiple antigen-labeling studies of maternal and paternal brains - studies that heretofore were difficult to perform well, if at all.

The addition of this apparatus will significantly increase the numbers of students -- broadly defined, on and off-campus, international, etc. -- that can be exposed to, and gain experience in, basic research and instruction in the neurosciences. In the process, these investigators can answer valuable questions, train a significant number of future scientists, and share the information with the scientific and lay communities alike, which include local and national media, and metro-Richmond K-12 schools, museums, and hospitals.

[unreadable] DESCRIPTION (provided by applicant): Machado-Joseph Disease (MJD) is a dominantly inherited neurodegenerative disease. It is caused by an expansion of a normally occurring polyglutamine repeat. Proteins with expanded glutamine repeats form aggregates which are toxic to certain neurons in the brain, eventually killing them. The mechanism by which these expanded polyglutamine proteins kill specific brain cells is not well understood. To develop new drugs and therapies to stop the disease, we need to find targets for research. We have a Drosophila model of MJD which closely recapitulates the human disease. We propose to use our model to find targets for future therapies. It is our hypothesis that the proteins expressed in fly brains expressing the mutant protein will be different from fly brains expressing the normal form of the protein. Our hypothesis is based on three observations. First, genomic studies show there are differences in gene expression between model organisms expressing mutant and normal forms of polyglutamine disease genes. Second, protein aggregates formed by mutant proteins co-localize with regulators of gene activity. This may alter the numbers and levels of proteins that are eventually produced. Third, Ataxin-3, the protein coded by the MJD1 gene, has been shown to be involved directly and indirectly in gene regulation. Mutation and co-aggregation of Ataxin-3 may change its regulatory activity. However post-transcriptional, translational, and post-translational controls ultimately determine what proteins will be found in the cellular environment and in what concentration. The differently expressed proteins represent potential targets for new therapies. We also suggest that modifiers of disease progression will also change the protein environment found in cells. Based on these observations, we propose three specific aims. 1) We will compare what proteins are expressed in the diseased state and non-diseased state of Drosophila brains expressing mutant and normal forms of the Ataxin3 (MJD1) gene. 2) We will compare the protein expression profiles of flies that express mutant Ataxin3 protein alone to those that have been rescued by modifiers of degeneration. 3) We will use the power of Drosophila genetics to observe how increasing and decreasing expression of the proteins we find in aims 1 and 2 influence neurodegeneration. The proteins identified in aims 1 and 2 that influence neurodegeneration in aim 3, are likely involved in the disease mechanism. These identified proteins are good targets for future therapies. [To observe these proteins, we will use the ICAT (isotope-coded affinity tags) technique. ICAT combines multidimensional liquid chromatography with mass spectroscopy to identify and quantify proteins found in complex mixtures.] PUBLIC HEALTH RELEVANCE: The mechanism of brain degeneration by inherited polyglutamine diseases like Machado- Joseph Disease is unknown and there are no cures or therapies. In order to find targets for cures and therapies, we propose to find what proteins are being expressed in brain cells with the disease compared to brain cells without the disease. The differences in proteins found represent potential targets for cures and therapies. [unreadable] [unreadable]