Christopher S. von Bartheld - US grants

DESCRIPTION: Neurotrophic factors are messengers for communication between neurons. They regulate neuronal differentiation and may be instrumental in the formation, stabilization and plasticity of synapses. Targeting of neurotrophic factors to their proper intracellular destination is essential for trophic signaling. The application's central aim is to elucidate pathways of internalized neurotrophins and to reveal mechanisms of their axonal transport and release from synapses in the visual system. The proposed studies will utilize an in-vivo system in which the transport of trophic factors can be quantified. The developing visual system (retinotectal and isthmo-optic projections) of chick embryos is a unique model which allows the controlled introduction of iodinated trophic factors into the posterior chamber of the eye and the study of retrograde and anterograde transport to distant targets. Experimental techniques have been designed to determine the mechanisms of release at the axon terminus and the function and significance of axodendritic transfer of neurotrophins. Specifically, the proposed studies will identify the source of neurotrophin-3 in the retina and use crosslinkers and antibodies to determine which receptors bind neurotrophins during anterograde and retrograde transport. The organelles in which internalized neurotrophins are sorted and in which anterograde transport and release takes place will be characterized at the ultrastructural level. The hypothesis that neurotrophins are co-localized with neurotransmitters or with neuropeptides in synaptic vesicles will be tested. The mechanisms of release at the axon terminus will be explored by measuring neurotrophin content in synaptosomes after treatment with pharmacological substances. It will be further examined if anterogradely transported neurotrophins promote synaptogenesis. The combination of molecular, pharmacological and ultrastructural approaches in an advantageous model system will allow the investigator to answer questions about trafficking of neurotrophins which are crucial to an understanding of how these factors may regulate neuronal survival as well as synaptic plasticity in the developing visual system and other parts to the brain. Neurotrophins are important regulators during the development of the visual system as well as potential therapeutic agents in degenerative retinal disease and after optic nerve injury.

DESCRIPTION (Adapted from applicant's abstract): Strabismus is a misalignment of the visual axis, which can lead to severe deficiencies such as loss of central vision from one eye, known as amblyopia. Strabismus is relatively common in the general population with estimates of 5-6 percent. The etiology of strabismus is multifactorial. Current therapies for restoration of visual alignment include muscle weakening by surgical recession or pharmacological denervation with botulinum toxin and muscle tightening by surgical resection. In the proposed research project, the trophic regulation between eye muscles an innervating oculomotor neurons will be explored with the long-term goal to supplement surgical treatment of strabismus with a pharmacological treatment targeted at trophic interactions. Injections of trophic factors or trophic antagonists into selected eye muscles may restore balanced eye movements by mimicking intrinsic trophic mechanisms. The proposed studies will test in an animal model how trophic manipulations of oculomotor neurons and eye muscles can adjust the strength of these muscles, increase the survival of oculomotor neurons during development, increase numbers of collateral axonal branches of oculomotor neurons, and maintain axon collaterals and endplates. Studies will determine which trophic factors are produced in the eye muscles, which functions they have on muscle mass, muscle strength, nerve sprouting, and maintenance of axons or endplates. Additional studies will determine whether the muscle-derived factors are transported retrogradely to the oculomotor neurons and support the survival of these neurons. The time course of trophic interactions between eye muscles and their nerves will be explored with the goal to understand and manipulate the trophic responses which are induced by denervation with botulinum toxin or in chronically paralyzed muscle such as the avian genetic mutant, crooked neck dwarf (cn/cn). These studies will focus on four trophic factors, brain-derived neurotrophic factor (BDNF), glial cell-line-derived neurotrophic factor (GDNF), and the insulin-like growth factors (IGF I, II), and, added in the resubmission, cardiotrophin-1 (CT-1). Additional trophic factors will be screened for their potential to modify the strength of eye muscles. A combined pharmacological, molecular, physiological and morphological approach including the ultrastructural level will provide a meaningful assessment of the prospects for a trophic, pharmacological treatment of strabismus and other eye muscle disorders as a supplement to current resection and denervation procedures.

Neurotrophic factors are messengers in the communication between neurons. They regulate neuronal differentiation and function. Internalization, intracellular transport, signal transduction and eventual degradation are essential steps in trophic signaling. Recent studies have shown that internalized neurotrophins can be sorted into either degradative or recycling pathways. The application's central aim is to elucidate how internalized neurotrophins are sorted into distinct intracellular pathways. This research will be done primarily at the Nencki Institute in Poland as an extension of NIH grant # RO 1 EY 12841. The proposed studies will utilize the developing visual system of chick embryos as a unique invivo model system which allows the introduction of radiolabeled trophic factors into a compartment and the quantification of intracellular pathways. Experiments will focus on the comparison of different subcellular pathways after internalization of neurotrophins by dendrites (retinal ganglion cells) and after internalization exclusively by axon terminals (isthmo-optic neurons). These experiments will show how the route of uptake (dendritic vs. axonal) influences the subsequent signaling and degradation pathways. Autoradiography at the ultrastructural level will be used to identify the subcellular distribution of neurotrophins. Distribution profiles will be compared between neurotrophins. Receptor binding of invivo- transported neurotrophins will be determined by crosslinking and immunoprecipitation with receptor-specific antibodies. The organelles in which internalized neurotrophins accumulate and their pathways and subcellular destinations will be compared and experimentally manipulated by competition with heterologous neurotrophins and inactivation of tyrosine kinase receptors. The combination of molecular, pharmacological and ultrastructural approaches will allow us to answer questions about trafficking of neurotrophins which are crucial to an understanding of how these factors may regulate events as diverse as neuronal survival and synaptic plasticity. Neurodegenerative diseases have been related to deficits in trophic support. Knowledge about the normal trafficking, sorting and recycling of neurotrophic factors will help us to understand pathologic conditions and how exogenous neurotrophins may be used as therapeutic agents.

[unreadable] DESCRIPTION (provided by applicant): Quantitative morphology is important in biomedical research. In order to quantify particle numbers in tissues, such tissues have to be sectioned. The optical dissector counting technique has particular advantages in developmental studies, because it is not affected even when particle size changes between groups, unlike conventional profile counting techniques. It was recently discovered, however, that the optical dissector can be significantly biased when tissue sections are differentially compressed in the z-axis. This finding has fueled an already contentious debate about how to appropriately count particles in tissue sections. Improving the optical dissector particle counting technique would help investigators to obtain valid, accurate, and largely unbiased data, and an objective bias assessment would resolve much of the debate on cell counting techniques. A simple and efficient technique has been developed which allows one to assess quantitatively the differential distortion and distribution of particles in the z-axis of tissue sections. Data on paraffin and plastic sections show that significant differential compression occurs, and thus that the optical dissector is biased for most if not all tissue sections if used as currently recommended. The proposed work will examine systematically and quantitatively the extent to which sections are affected by differential compression, for all five types of tissue sections commonly used (cryosections, paraffin sections, methacrylate plastic sections, celloidin plastic sections, and vibratome sections). A simple new methodology will be optimized to assess differential section compression. Strategies will be designed to improve the optical dissector counting technique and to minimize biases, by predicting the bias and placing counting boxes in ways that minimize the resulting bias. Counts of cells obtained with the optical dissector from vibratome, cryo- and celloidin plastic sections will be calibrated by comparison with 3D serial reconstructions, and the biases will be compared with those of profile counting methods. Finally, and most importantly, investigators will be provided with simple and easy-to-use protocols for cell counting using an improved optical dissector method, and clear, practical guidance will be given about the precise advantages and disadvantages of different counting techniques, based on calibrated analyses. The proposed work will allow researchers to perform easy and simple calibration and bias assessments on their particle counts when they use the optical dissector. This advance in research methodology is urgently needed to improve the validity and the quality of data which are currently obtained in studies in embryology, drug testing, and developmental biology, many of which are funded by the NIH, and in particular the NICHD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Quantitative morphology is important in biomedical research. In order to quantify particle numbers in tissues, such tissues have to be sectioned. The optical dissector counting technique has particular advantages in developmental studies, because it is not affected even when particle size changes between groups, unlike conventional profile counting techniques. It was recently discovered, however, that the optical dissector can be significantly biased when tissue sections are differentially compressed in the z-axis. This finding has fueled an already contentious debate about how to appropriately count particles in tissue sections. Improving the optical dissector particle counting technique would help investigators to obtain valid, accurate, and largely unbiased data, and an objective bias assessment would resolve much of the debate on cell counting techniques. A simple and efficient technique has been developed which allows one to assess quantitatively the differential distortion and distribution of particles in the z-axis of tissue sections. Data on paraffin and plastic sections show that significant differential compression occurs, and thus that the optical dissector is biased for most if not all tissue sections if used as currently recommended. The proposed work will examine systematically and quantitatively the extent to which sections are affected by differential compression, for all five types of tissue sections commonly used (cryosections, paraffin sections, methacrylate plastic sections, celloidin plastic sections, and vibratome sections). A simple new methodology will be optimized to assess differential section compression. Strategies will be designed to improve the optical dissector counting technique and to minimize biases, by predicting the bias and placing counting boxes in ways that minimize the resulting bias. Counts of cells obtained with the optical dissector from vibratome, cryo- and celloidin plastic sections will be calibrated by comparison with 3D serial reconstructions, and the biases will be compared with those of profile counting methods. Finally, and most importantly, investigators will be provided with simple and easy-to-use protocols for cell counting using an improved optical dissector method, and clear, practical guidance will be given about the precise advantages and disadvantages of different counting techniques, based on calibrated analyses. The proposed work will allow researchers to perform easy and simple calibration and bias assessments on their particle counts when they use the optical dissector. This advance in research methodology is urgently needed to improve the validity and the quality of data which are currently obtained in studies in embryology, drug testing, and developmental biology, many of which are funded by the NIH, and in particular the NICHD. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Understanding protein function requires knowledge about a protein's localization and trafficking. This is particularly important in neurons due to their complex morphology and polarized shape. Tagging proteins with either isotopes or fluorescent tags to trace them as biomarkers has become a widely used tool in biomedical science. However, when proteins are modified by a tag, normal protein interactions, trafficking and signaling may be compromised, and rigorous validation is required in order to have confidence in the data obtained with the biomarker. Currently, there is great enthusiasm and expectations for the use of quantum dots (QDs), because they have bright fluorescence, differential spectra, superb resolution, and they are electron dense, allowing for direct ultrastructural localization. However, QD tags are comparable in size to small proteins, and recent evidence indicates that QD conjugation to proteins can alter the function and trafficking of tagged proteins. The planned collaboration between two labs with unique and complementary expertise provides the opportunity to explore and answer these questions. We propose to compare the behavior of eight QD-tagged proteins from several different protein classes with the corresponding "normal" protein, minimally modified by radio-iodination. Our proposal entails three approaches that are novel in their combination: comparison of protein trafficking with a "gold standard" calibration, examination in two advantageous in-vivo model systems, and evaluation at highest resolution - the ultrastructural (electron microscopic) level. This work will result in a step-by-step methodological paper that will provide guidelines for users of QD-tagged proteins as biomarkers how to validate their QD-tagged proteins. We will also determine optimal conjugation schemes and QD sizes that are most suitable for use as biomarkers. While we are most interested in this question for neuronal protein trafficking and signaling, and use of QD-labeled molecules as diagnostic and therapeutic tools, the expected results should be relevant for a wide range of tissue types and cell biological questions extending beyond biomarker use in neuroscience. PUBLIC HEALTH RELEVANCE: Quantum dot-labeled proteins have great promise in basic research and they are proposed as diagnostic as well as therapeutic biomedical tools. Prior to their use as biomarkers, validation has to occur in in-vivo model systems, to identify changes in trafficking, kinetics, signaling and receptor binding that may impose inherent limitations and compromise optimal utility.

Signaling across cell membranes is fundamentally important for normal cellular function and for understanding many diseases. Signal transduction and molecular trafficking are intertwined in ways that make it impossible to truly understand mechanisms of either in isolation. We propose to create a Center of Biomedical Research Excellence (COBRE) at the University of Nevada, Reno (UNR) in the area of cell biology of signaling across membranes. This COBRE will make significant progress in the emerging fusion of the fields of membrane trafficking and signal transduction by examining how molecular signaling is achieved across membranes, both between cells and between subcellular compartments. Five junior faculty with outstanding research accomplishments will use diverse and uniquely advantageous model systems where critical aspects of signaling and transport functions can be examined by molecular, genetic and structural approaches. The proposed research topics will utilize interdisciplinary approaches in the field of signal transduction and molecular trafficking, including growth factor signaling, regulation of gene expression, nuclear and cytoplasmic transport, secretion, endocytosis, apoptosis, and axon guidance. The proposed COBRE is a comprehensive effort that will integrate high-quality biomedical research and expertise in different departments and colleges at UNR under one theme and program. This will bridge the traditional physical and administrative division between the Colleges of Science and Medicine on the upper and lower campus at UNR, and foster interactions that lead to synergism and enhanced biomedical research capacity. The COBRE will provide an efficient mentoring program for junior faculty that will expand already existing training programs at UNR, and support the research of selected young faculty by teaming with clinical mentors and consultants to establish disease focus. The COBRE will provide enhanced infrastructure and technical support for Nevada's new generation of biomedical researchers with truly outstanding credentials and promise for major achievements in biomedical research.

DESCRIPTION (provided by applicant): Major neurological and psychiatric diseases have been reported to be associated with significantly changed glia densities, but considerable confusion surrounds the question of the true numbers and ratios of neurons and glial cells in the brains of humans and animals. Traditionally and per textbook knowledge, it was thought that glial cells outnumber neurons by a ratio of between 10:1 and 50:1. Recently introduced new counting techniques have challenged this belief and postulate a ratio of approximately 1:1, reducing the number of glial cells in the human brain from previous estimates by as much as 4.9 trillion (from 5 trillion to a mere 100 billion). The new, much lower estimates of glial cells are primarily based on a novel methodological approach, the isotropic fractionator (IF). However, this technique has not been calibrated or validated. It is therefore uncertain whether it produces true numbers or may possibly be biased towards larger particles (neuronal nuclei) and may underestimate smaller ones (glial nuclei). The fractionator technique homogenizes whole brains (or dissected parts thereof), then takes samples of the cell nuclei in solution, and uses antibody labeling to distinguish neuronal nuclei from other nuclei such as glia in sample solutions. While conceptually elegant, it remains to be calibrated against known numbers and ratios of neurons and glial cells. Therefore, we propose a series of calibrations that will unambiguously determine the presence or absence of any biases in the isotropic fractionator (IF) technique. We will probe a variety of human and non-human primate brain regions (primarily white matter tracts) to determine whether the fractionator accurately estimates numbers of glial cells. The same brain regions will be examined by measuring DNA content (which can directly reflect cell numbers), and by histology, using systematic random sampling to independently estimate glial numbers and ratios. Preliminary data indicate that the histological approach is most suitable to provide a gold standard for comparison. Modern stereological sampling techniques will identify the ratios of cell types at the ultrastructural level. This work will clarify true neuron-glia ratios in the bain and other parts of the CNS. Validation of the new fractionator method will be crucial for understanding neurological and psychiatric diseases with reported imbalances of glia cell densities, such as schizophrenia, autism, bipolar disease, and depression.

The prevalence of horizontal strabismus differs dramatically between studies for reasons that are not clear. Our preliminary data show that ethnicity is a major factor: some populations have much more esotropia than exotropia, while other populations have much more exotropia than esotropia. Older literature suggested that differences in the interpupillary distance may be responsible for ethnic differences in strabismus, but this has never been systematically explored. Because of the largely undefined ethnic variations, the global prevalence of strabismus is currently unknown. Furthermore, the prevalence within the same ethnicity has never been longitudinally compiled and compared. This lack of information about local and global prevalence makes it impossible to determine trends and to compare them with trends of major risk factors and thus define underlying causes or predispositions for strabismus. This impedes the planning of health care that will be needed in the future. Here, we propose to solve these problems by conducting secondary data analyses. Aim 1: Compile ALL relevant studies and publish a systematic review of the prevalence of horizontal strabismus throughout the world, taking into account ethnic variations and methodology bias. This will provide a much-needed reference guide and allow to estimate the true global prevalence. Aim 2: Examine the distribution of three orbital parameters that are ethnically distinct: interpupillary distance, proptosis, and interorbital width, and discern which parameters (or combination of parameters) best explains the ethnic variation of horizontal strabismus prevalence and patterns. This will provide an evidence-based framework to understand the current ethnic variations and their evolutionary history. Aim 3: Determine the trends (over the last 50-150 years) in prevalence of horizontal strabismus and compare them with local trends for major risk factors of strabismus. This will identify effective risk factors in local populations ? which is relevant for planning purposes and for targeted health care initiatives. Our multidisciplinary team of investigators will move the field forward by establishing population-based prevalence numbers for each of the major ethnicities. This is important to provide a comprehensive reference work for future studies. The true prevalence in different countries and ethnicities is important for health care planning purposes. The second aim will inform which key anatomical features of the orbit underlie ethnic differences, with evolutionary, developmental and clinical implications. This will provide a novel conceptual framework of the impact of orbital parameters for strabismus. Finally, consideration of a much larger number of studies and populations throughout the world (~500 vs. ~30 in previous reviews) will allow us to define trends in prevalence and to compare such trends with those of major risk factors of strabismus within those same populations. This will inform which risk factors (e.g., maternal smoking, Cesarean section) likely affect prevalence rates in distinct populations.