Colin J. Barnstable - US grants

The long term objectives of this project are to understand the molecular basis of neural structure and development. This is essential not only for an understanding of the normal functioning of the central nervous system but also for an understanding of the wide variety of developmental and degenerative abnormalities that can affect neural tissue. This proposal seeks to characterise a series of cell-type specific molecules that have been identified with monoclonal antibodies and to use culture systems to study the mechanisms controlling the expression of these molecules. Monolayer and aggregate cultures of rat retina and other CNS areas will be set up from animals of various ages. The expression of defined cell-type specific molecules by particular cell types has already been characterized in these culture systems and is probably not dependent upon factors extrinsic to the retina. Expression of these molecules will be monitored after various treatments that modify the composition and possibilities for cellular interactions. Modification of cell density, removal of cell types by antibody-mediated cytotoxicity and growth of single cells in microcultures will all be carried out. These experiments will be interpreted in terms of the role of particular cell types upon the differentiation of other cell types. Some cell types have been found to take on a characteristic morphology in culture. The role of substrate adhesion, cell density and cellular interactions will all be examined to determine whether this is controlled by extrinsic factors or is a part of an intrinsic developmental program. The cell-type specific molecules will be characterised by standard biochemical methods to measure molecular weight and patterns of glycosylation. More detailed analysis will involve isolation of cDNA clones from existing plasmid and bacteriophage libraries. The cloned DNA fragments will be used both for biochemical analysis and for developmental studies to define the times of gene expression in vivo and in vitro. The cDNA libraries will also be screened to detect both cell-type and developmental-stage specific gene transcripts. These will provide the starting material for the studies of nucleotide sequences that may play a role in the regulation of gene activity during neural development.

The long term objectives of this project are to gain an understanding of the cellular and molecular mechanisms responsible for the development and function of the mammalian visual system. Knowledge of these mechanisms is essential to any understanding of congenital abnormalities in visual system development and the wide variety of degenerative diseases that can affect both the visual system and other areas of the central nervous system. To achieve these objectives it is necessary to have methods that allow the identification of the cell types and the individual molecules of the visual system. Two recent developments of molecular biology, namely the introduction of monoclonal antibodies and recombinant DNA molecules, have provided these methods. Antibodies that can identify the major subclasses of cells in the rat retina have already been produced. The objectives of this proposal are to produce further antibodies that recognise more discrete subclasses of cells of retina and visual cortex, and to use cloned cDNA fragments to study the heterogeneity and development of cell types in the visual system by using in situ hybridisation. To prepare the monoclonal antibodies, cells or membranes enriched in the cell type of interest will be used to immunise mice. Spleen cells will be taken from mice mounting an adequate immune response and fused with a mouse plasmacytoma. In addition spleen cells will be immunised by in vitro incubation with antigen and then fused. Hybrids will be selected by standard techniques and those secreting antibodies detected by a combination of radioactive binding assays and immunocytochemical assays. The antibodies will be used to study the in vivo development of cells of the visual system by immunocytochemical analysis of tissue from animals of various ages. The antibodies will also be used to purify defined populations of cells by combinations of affinity separations and fluorescence-activated cell sorting. Purified cells will be used both for further immunisations and for studies in tissue culture. Monolayer cultures of cells from retina or visual cortex will be prepared and the cell types identified by labelling with specific antibodies. By using anatomical and electrophysiological methods the nature and specificity of synaptic interactions between identified cells will be studied.

The purpose of the proposed experiments is to provide a detailed analysis of the functions of different subpopulations of neurons in the visual cortex which use the inhibitory neurotransmitter gama- amino butyric acid (GABA). We have produced two monoclonal antibodies, VC1.1 and VC5.1 that identify unique cell surface molecules restricted to subsets of GABAergic neurons in the visual cortex. We now propose to use these and new antibodies to study the functions of subpopulations of GABAergic neurons and to investigate the role of the cell surface molecules themselves in the formation of identified cortical circuits. To achieve these goals, we shall use immunological methods in combination with biochemical, anatomical, immunohistochemical and functional approaches. We shall define some of the biochemical characteristics of the cell surface molecules recognized by VC1.1 and VC5.1 and purify these molecules for functional assays. We shall determined whether the VC1.1 immunoreactive/GABAergic neurons in the visual cortex have other biochemical or anatomical features which provide clues to their function in cortical circuits. We shall determine whether extrinsic factors regulate the levels of VC1.1 or VC5.1 immunoreactive molecules by studying their expression in visual cortex following subcortical lesions or in cortical development and test the role of these molecules on cell survival, process outgrowth and synaptogenesis in vitro. As a foundation for future physiological analyses of the contribution of GABAergic neurons to the response properties of cortical neurons, we propose to immuno-ablate the VC1.1 or VC5.1 /GABAergic subpopulation of cortical neurons at different times during development and in mature animals. In other experiments, we shall obtain purified populations of VC1.1. positive neurons using fluorescence activated cell sorting and use these as immunogens to produce additional antibodies against subsets of cortical interneurons. Ultimately, these studies will aid further molecular and cellular analyses of cortical microcircuitry and lead to a more complete understanding of how cortical functions are disrupted by disease or congenital birth defects.

The long term objectives of this project are to gain an understanding of the cellular and molecular mechanisms responsible for the development and function of the mammalian visual system. Knowledge of these mechanisms is essential to any understanding of congenital abnormalities in visual system development and the wide variety of degenerative diseases that can affect both the visual system and other areas of the central nervous system. To achieve these objectives it is necessary to have methods that allow the identification of the cell types and the individual molecules of the visual system. Two recent developments of molecular biology, namely the introduction of monoclonal antibodies and recombinant DNA molecules, have provided these methods. Antibodies that can identify the major subclasses of cells in the rat retina have already been produced. The objectives of this proposal are to produce further antibodies that recognise more discrete subclasses of cells of retina and visual cortex, and to use cloned cDNA fragments to study the heterogeneity and development of cell types in the visual system by using in situ hybridisation. To prepare the monoclonal antibodies, cells or membranes enriched in the cell type of interest will be used to immunise mice. Spleen cells will be taken from mice mounting an adequate immune response and fused with a mouse plasmacytoma. In addition spleen cells will be immunised by in vitro incubation with antigen and then fused. Hybrids will be selected by standard techniques and those secreting antibodies detected by a combination of radioactive binding assays and immunocytochemical assays. The antibodies will be used to study the in vivo development of cells of the visual system by immunocytochemical analysis of tissue from animals of various ages. The antibodies will also be used to purify defined populations of cells by combinations of affinity separations and fluorescence-activated cell sorting. Purified cells will be used both for further immunisations and for studies in tissue culture. Monolayer cultures of cells from retina or visual cortex will be prepared and the cell types identified by labelling with specific antibodies. By using anatomical and electrophysiological methods the nature and specificity of synaptic interactions between identified cells will be studied.

The vision research group in the Department of Ophthalmology and Visual Science has collaborated to organize a core facility in order to share common resources and facilitate collaborative interactions between investigators. The proposed facility consists of six modules: anatomy, chemistry, machine shop, pharmacology, photography, and tissue culture. Each module supports an on-going shared effort including department members and vision science colleagues from outside the department.

The overall objective of this Core Grant remains to enhance vision research at Yale University. With five key modules, this grant will aid the ongoing research projects of individual investigators by providing facilities and expertise that cannot be supported by individual research grants. The modules will also serve to promote and facilitate collaborative interactions between investigators and will encourage pilot projects from which specific research proposals can be developed. Finally, the modules of this Core Grant will serve as an invaluable resource for trainees of the Yale Visual Sciences Training Program which sponsors both pre- and post-doctoral fellows. The specific modules proposed in this application include on serve module, a machine shp which serves an obvious function. Four resource modules are proposed. A tissue culture model will provide complete facilitates for the initiation and maintenance of small and large scale cultures of both primary cells and cell lines including hybridomas. The facility is also a designated area approved for work with viruses for introduction of genes into cells. A molecular biology module will provide and maintain specialized shared equipment such as scintillation counters, ultracentrifuges and chromatography systems. In addition it will provide Vision Center Investigators with oligonucleotide, DNA sequencing and real-time PCR resources. An imaging and image analysis module will provide a variety of imaging systems, including confocal microscopy, together with analysis workstations with a wide range of software.

The overall objective of this core grant is enhance vision research at Yale University. By providing five key support modules, this grant will enhance the ongoing research projects of individual investigators by providing facilities and expertise that cannot be supported by individual research grants. The modules will also serve to promote and facilitate collaborative interactions between investigators and will encourage pilot projects from which specific research proposals can be developed. Finally, the modules of this core grant will serve as an invaluable resource for trainees of the Yale Visual Sciences Training Program which sponsors both pre- and postdoctoral fellows. The specific modules proposed in this application include a machine shop and a photography module, both of which serve obvious functions. A tissue culture/hybridoma module will provide complete facilities for the initiation and maintenance of small and large scale cultures of both primary cells and cell lines. It will also provide a service for production and bulk growth of monoclonal antibodies. A pharmacology/tissue preparation module will provide eye tissues and cell preparations of reproducible quality for a number of investigators. A chemistry/molecular biology module will provide and maintain specialized shared equipment such as scintillation counters, ultracentrifuges FPLC and HPLC systems. In addition it will serve as a resource for Vision Center Investigators by providing vectors and bacterial strains as well as oligonucleotides for PCR, sequencing and binding studies.

The long term goal of this project is to understand at the molecular level the genetic and epigenetic factors responsible for cell determination and differentiation in the mammalian Central Nervous System. This knowledge is essential for understanding and treating many diseases caused by mutation or injury that result in abnormal brain development and for manipulating cell phenotypes in neural regeneration and replacement therapies. This project builds on past findings and has four specific aims. First, a continued analysis using transgenic mice will test the hypothesis that a specific DNA element found in many photoreceptor specific genes, RET 1, is both sufficient and necessary to direct photoreceptor expression of genes. Using specific DNA sequences, consisting of either RET 1 alone or the opsin promoter in which RET 1 has been mutated, linked to a lac Z marker gene, the spatial and temporal expression of the marker will be examined using X-gal histochemistry. If expression is detected in any cells other than photoreceptors, these will be examined for the RET 1 binding protein and for negative regulators that normally block expression of opsin. Second, the protein binding to the RET 1 element has been purified and it is now proposed to clone it and study its structure and activity in more detail. The cDNA sequence will categorize this molecule as a member of a new or an existing family of transcriptional regulators, the genomic sequence will identify promoter elements controlling its expression and antibodies will allow a precise description of the cellular extent of its expression and will provide tools to isolate associated proteins. These experiments will provide both new information about neuronal transcriptional regulators and probes to study molecular events closer to the time of cell birth and commitment. Third, retinoic acid and basic fibroblast growth factor have been shown to affect the binding activity of nuclear proteins interacting with the RET 1 sequence, and it is now proposed to study the ways in which these factors influence binding and thus provide insights into the molecular mechanisms by which they can influence neuronal development. Fourth, the hypothesis that transcription factors expressed during earlier phases of forebrain and retinal development are essential for terminal differentiation of neurons such as photoreceptors, and expression of cell type -specific molecules such as opsin, will be tested using a culture system in which a retina is formed by transdifferentiation of the adjacent retinal pigment epithelium. Together these specific aims represent a focussed effort to define specific and general rules governing the formation of neural cell types and the transcription of cell-type specific genes of known function in the mammalian Central Nervous System.

Cyclic GMP is an important second messenger in many tissues including the Central Nervous System. It is the central hypothesis of this proposal that cGMP plays an important role in regulating synaptic efficacy in the developing and mature mammalian visual system and that the actions of cGMP are mediated both by a class of cyclic nucleotide gated cation channels and by cGMP-dependent protein kinases. The overall hypothesis of this proposal will be tested with three specific aims. In the first, the levels of expression and cellular distribution of molecules involved in both cGMP metabolism and cGMP actions will be measured during normal development and in dark-reared animals. The results of these experiments will indicate whether cGMP acts in the same way in all cortical neurons, whether cGMP is used as a messenger at all stages of development and whether patterned visual input alters the cGMP second messenger system. The second specific aim will determine whether cGMP levels in visual cortex are regulated by neural activity by measuring the concentrations of cGMP in cortical slices following treatment with selected neurotransmitter agonists and antagonists. These experiments will focus on glutamate, acetylcholine and noradrenaline because these have been implicated in the regulation of cGMP levels and in visual cortical plasticity. The third specific aim will test directly whether cGMP can modulate membrane properties and synaptic interactions of identified visual cortical neurons. Direct and indirect effects of cGMP on membrane conductances will be measured by recording from cells identified by retrograde transport or antibody labeling. The effects of cGMP on responses to glutamate receptor agonists will be measured to determine whether receptor sensitivity or desensitization is altered. Finally, the ability of cGMP to alter synaptic interactions between cortical neurons will be measured. Using a series of agonists and antagonists selective for cGMP-dependent protein kinases of cyclic nucleotide gated cation channels, experiments will be carried out to determine the pathway by which cGMP exerts its effects. Overall, this project will elucidate the functions of an important second messenger system in visual cortex, particularly the ways in which cGMP can alter the efficacy of synaptic interactions between cortical neurons. The information gained from these experiments will improve understanding of normal information processing and of important developmental abnormalities that affect visual cortex such as amblyopia and strabismus.

tissue resource /registry; tissue /cell culture; monoclonal antibody; hybridomas; biomedical facility; vision; health science research;

DESCRIPTION (provided by applicant): Glaucoma is a disease characterized by visual field loss as a result of the death of retinal ganglion cells. Although increased intraocular pressure remains the most clearly defined risk factor for glaucoma, it is becoming clear that a wide range of other factors can also lead to ganglion cell loss. Pharmacological or surgical regulation of intraocular pressure can stabilize many patients but for some there is still a progressive loss of vision. Thus, there is an urgent need to develop new rational strategies to slow or prevent neuronal loss occurring in glaucoma. There is abundant evidence that the eye, like other regions of the CNS, contains endogenous neurotrophic/neuroprotective factors that function to limit cell injury. It is the basic premise of this proposal that these neuroprotective mechanisms can be exploited to prevent much of the cell death associated with diseases such as glaucoma. This proposal focuses on one neuroprotective molecule, CNTF, a factor that has already been shown to have potent neuroprotective effects in a number of CNS regions including the retina and is a leading candidate for slowing the progression of ganglion cell loss. In our preliminary data we present evidence that CNTF supports the survival of purified rat RGCs in low density cultures and that its downstream effector STAT3 prevents RGCs from degenerating in ischemia- reperfusion injury. We now propose a series of experiments to test if RGCs can be prevented from dying in the presence of toxic levels of glutamate by CNTF. In a first aim we will define the pathways used by CNTF to exert its protective action. Second, we will examine whether Muller glia can respond to CNTF and provide synergistic protection to RGCs by the secretion of additional neuroprotective factors or a range of other responses. Finally we will test whether the protective pathways activated by CNTF lead to a reduction in reactive oxygen species generation by mitochondria through the activation of mitochondrial uncoupling proteins. PUBLIC HEALTH RELEVANCE: Glaucoma is a blinding disease that affects over 65 million people worldwide. There is still not a good understanding of the basic biochemical mechanisms which cause the death of retinal cells and subsequent loss of vision. This research will identify some of these biochemical pathways. The results of this research will lead to the identification of target molecules for which new therapeutic drugs can be designed.

[unreadable] DESCRIPTION (provided by applicant): High-resolution structural analysis of cells and tissues is crucial to a wide variety of projects at the Penn State College of Medicine. Such studies include characterization of changes in cellular junctions in diabetes, altered synaptic structures in disease and gene knockouts, structure of melanosomes, ultrastructural analysis of zebrafush mutants, structural analysis of extracellular matrix and of chromatin, and mechanisms of viral budding. This proposal requests funds to replace our aged and inadequate Philips 400 electron microscope with a new JEOL JEM-1400 instrument. The upgrading of electron microscopy instrumentation will enable to better serve the needs of many funded investigators, of whom 14 are listed as the major users group in this proposal. Although our current instrument has provided many valuable images that have been used in publications and to support grant proposals, its effectiveness is greatly diminished by its substantial down time, complexity of use and lack of key features such as tilt to generate 3D images. Also it is complex to set up and use, which has limited access to the facility manager and a few highly trained individuals. To overcome these problems investigators have been increasingly utilizing the microscopes elsewhere, such as the State College campus (a ninety mile drive) or have been resorting to other techniques that do not have the same resolving power, such as confocal microscopy. In choosing the requested equipment we have consulted extensively with colleagues at other institutions and representatives of both JEOL and Philips. The requested JEOL JEM- 1400 microscope has excellent resolution and good contrast of biological samples, is very user friendly and has excellent digital image capability. The requested instrument will provide crucial support to our major users group's projects, but more importantly will aid the development of new research projects in our larger research community. Many of the projects supported by the requested electron microscope provide the essential intellectual underpinnings of the understanding of many important diseases. As such they are a key step in translating knowledge into better and more efficient health care for everyone. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The objective of this application is to purchase a new confocal microscope for the Penn State Hershey College of Medicine Core Imaging Facility. Though our core facilities are available to all investigators in the College of Medicine, this request is submitted to meet the specific needs of a group of NIH-funded major users. This new microscope will permit three types of study that cannot be accomplished with our current facility. First, because the microscope will be configured specifically for live-cell studies, we can meet the rapidly growing need for more sophisticated equipment dedicated to this experimental approach. The configuration will enable precise execution of time lapse studies, fluorescence resonance energy transmission (FRET) and fluorescent recovery after photobleaching (FRAP). Second, the microscope will also have a tandem scanner to provide both high resolution and high speed scanning options. These features will be vital for high resolution imaging experiments and will allow dynamic studies of ion flow and mitochondrial status. Third, the instrument will allow the simultaneous excitation and detection of up to five different fluorophores. The availability of this new microscope will improve the ability to perform intensive live-cell imaging projects focusing on a variety of health issues including viral infections, cance, diabetes and its complications (primarily diabetic retinopathy and muscle protein synthesis), heart disease, degenerative diseases of the central and peripheral nervous system (including Parkinson's disease, Alzheimer's disease, restless leg syndrome and eye diseases such as glaucoma). The projects will use the equipment to image rapid events in live cells or explant tissue transfected with fluorescent protein markers, or labeled with metabolic dyes, to determine changes in protein expression, trafficking and redistribution in response to pharmacological, environmental or genetic manipulations. PUBLIC HEALTH RELEVANCE: The confocal microscope requested will have broad relevance to human health because it will enable us to study rapid interactions between molecules in living cells, in real time and thus aid determination of their function in health and disease. This instrument will enhance a wide variety of public health projects being conducted at the Milton S. Hershey Medical Center, including those related to cancer, diabetes, heart disease, viral infection and neurodegenerative diseases.

The goal of this project is to test the hypothesis that the mitochondrial uncoupling protein UCP2 expressed in neurons and astrocytes can provide effective protection for neurons of the hippocampus in a model of seizure-induced cell death. Uncoupling proteins are powerful controllers of metabolism and the production of reactive oxygen species in the mitochondria. In previous work we demonstrated that UCP2 could protect mouse and primate neurons from a variety of excitotoxic and oxidative insults. Most relevant is our previous finding that overexpression of UCP2 in all cells gave significant protection to hippocampal neurons in the pilocarpine-induced seizure model proposed here. UCP2 also decreases neuronal cell death in various other CNS regions and protects them from the excitotoxic effects of glutamate agonists. We recently showed that the ability of the cytokine LIF to protect astrocytes from peroxide induced death was dependent on a STAT3-mediated induction of UCP2 RNA. Astrocytes are known to respond to oxidative stress, such as that seen in seizure-induced hyperactivity, by several changes including decreased ability to take up glutamate and to release cytotoxic cytokines such as TNFalpha. Our hypothesis is that increasing UCP2 will decrease reactive oxygen species and prevent these astrocyte responses. In Aim 1 we will test the hypothesis that conditional knockout and conditional overexpression of UCP2 in eother neurons or astrocytes will increase and decrease respectively the amount of cell death in the pyramidal cells of the hippocampus. We will use a tamoxifen-activated Cre-recombinase expressed under the control of eother a GFAP or a Thy-1 promoter. When crossed with mice containing a floxed UCP2 gene or a transgene cassette including a floxed segment that prevents expression, these mice will allow the selective deletion or overexpression of UCP2. After tamoxifen treatment, these mice, and untreated littermate controls, will be subjected to pilocarpine-induced seizures and 24 hr later the extent of cell death in the hippocampus measured. The natural expression of UCP2 is controlled at both the transcriptional and translational levels. We have previously shown that neuroprotective factors can increase the levels of UCP2 RNA but not protein. Oxidative stress rapidly increases translation of this RNA to increase UCP2 protein. In Aim 2 we will explore this further and test the hypothesis that the translational regulation is mediated by specific microRNAs. We will first identify the spectrum of candidate regulatory microRNAs by identifying those that alter expression following pilocarpine-induced seizures in ways that correlate with changes in UCP2 expression. We will test the effect of increasing or decreasing the level of a specific microRNAs on UCP2 expression. Together these studies will provide insights into the mechanism of action of a key regulator of oxidative stress and point to ways of exploiting this molecule as a valuable therapeutic target.

Abstract Epigenetic remodeling of gene expression is a powerful therapeutic approach whose potential has yet to be fully exploited. A number of degenerative diseases of the retina are good candidates for this approach and this proposal focuses Retinitis Pigmentosa (RP), for which there are both excellent animal models and an available patient population, but no treatments. In RP, mutant genes lead to the death of rod photoreceptors and a secondary death of nearby cone photoreceptors that leads to blindness. Based on preliminary data we have proposed the hypothesis that selective pharmacological manipulation of histone modifying enzymes will alter the epigenetic landscape and lessen the impact of deleterious mutations, allowing extended survival of rod photoreceptors in RP. To test this hypothesis we propose three specific aims. First, we will extend our preliminary findings to more fully define the optimum time course and dose of LDS1 inhibitors (TCP and GSK2879552) that can block rod degeneration in the rd10 model of RP. We will quantitate photoreceptor survival by immunocytochemistry, quantitative PCR, and OCT. We will also measure visual function using ERG. We will use a second model of RP, the rd3 mouse, to test whether the effects can be generalized to multiple forms of RP. Second, we will examine whether HDAC1 specific inhibitors can provide protection to rods in the same models of RP. In a second series of experiments we will test whether use of LSD1 and HDAC1 inhibitors together act synergistically to provide greater protection than either drug alone. Photoreceptor survival and function will be monitored in the same way as in Aim 1. Third, we will analyze whether the epigenetic modifiers act to alter expression of the mutant genes and gene in the same network, or have broader effects on processes such as apoptosis and inflammation. We will use RNA-seq methods to give an unbiased measure of changes in gene expression induced by the LSD1 and HDAC1 inhibitors. Key changes will be verified by qPCR. We will also measure changes in cytokines in the retina and the vitreous to provide a quantitative estimate of levels of pro- and anti-inflammatory cytokines, and changes in the activity of key signal pathways. Additionally, we will test whether changes in transcript or protein levels are due to direct changes on the gene epigenetic status, by carrying out ChIP-seq for H3K4me2 (LSD1 substrate) and H3K9ac/12ac (HDAC1 substrate) as measures of promoter accessibility. These experiments will provide insights into the mechanisms of protection and by analyzing the cellular networks most altered will provide new targets for even more specific therapy. Overall, these experiments will provide important information about pathways that can prevent damage in the retina and also offer a novel therapeutic approach that can combat RP and other retinal degenerations.