Thomas E. Hughes - US grants

Gamma-amino butyric acid (GABA) is an important inhibitory neurotransmitter used by many neurons in the retina. The GABAergic circuitry in the retina, however, is poorly understood. This is because the postsynaptic, GABA-ceptive cells have been difficult to identify. Recently, the GABA-A receptor has been purified and antibodies have been raised against it. These antibodies label many amacrine cells, a few bipolar or interplexiform cells, and a group of large, perhaps Y-like, ganglion cells in the squirrel monkey retina. The goal of this proposal is to use the well studied cat retina, a series of double labeling experiments, and electron microscopy to better identify these cells. 1. Identification of the GABA-ceptive amacrine cells: The size, density, and distribution of the receptor-positive amacrine cells will be examined. To better identify these cells, double labeling experiments will be conducted with the antibody against the receptor and antibodies against neurotransmitters (or their synthetic enzymes) known to exist in amacrine cell populations. These include: GABA, glycine, dopamine (tyrosine hydroxylase), and acetylcholine (Choline acetyltransferase). If receptor- positive interplexiform-like cells exist in the cat retina, their size, density and distribution will also be determined. 2. Identification of the GABA-ceptive ganglion cells: The size, distribution, and density of the receptor-positive ganglion cells in the cat's retina will be determined. Retrograde tracers will be injected into the superior colliculus, dorsal lateral geniculate, or medial terminal nucleus to double label the receptor-positive cells and thereby identify their central targets. By determining the number, size, and density of the GABA-ceptive ganglion cells, as well as the destination of their axons, it will be possible to identify these cells in terms of the X,Y, and W cell classification scheme. 3. Synaptic relationships of the GABA-ceptive neurons: Double label experiments with two different fluorophores will be conducted to examine the spatial relationship between the GABAergic telodendria and the receptor bearing processes in the inner plexiform layer. Electron microscopy will be used to study the ultrastructural localization of the receptor and the synaptic contacts may be the receptor-positive cells. Double labeling experiments, in which the receptor-immunoreactivity is labeled with peroxidase and the GABA containing profiles are labeled with colloidal gold, will be used to identify the GABAergic synapses.

The objective of this contract is to test chemicals for mutagenicity in Salmonella. The chemicals are tested, under code, in a series of Salmonella tester strains, both with and without metabolic activation. The results of this testing are used, in combination with other information, to evaluate the toxicity, and potential carcinogenicity, of the chemicals. The results of the testing are distributed throughout the Program, to other Federal agencies, and to other public and private organizations, and are published in the peer-reviewed scientific literature.

The cDNAs encoding many of the neurotransmitter receptors have been cloned recently. Consequently, we can now begin to identify both the retinal cells that contain a transmitter and those that bear the receptors for the transmitter. A fundamental observation is that the ionotropic-type receptors are quite diverse; for each neurotransmitter there are many different receptor subunits and an even greater number of potential subunit complexes. It is possible that for each transmitter used in the retina there are many different receptor-subtype specific synapses. The goal of the proposed work is to explore this heterogeneity by defining how the Kainate/AMPA-type glutamate receptor subunits are expressed and combined by the cells of the retina. Glutamate is a neurotransmitter fundamental to retinal function, and in situ hybridization experiments in our laboratory have revealed that the mRNAs encoding the seven cloned subunits (GluR1 through GluR7) are expressed in the retina. 1). Tools: the generation of a) glutamate receptor subunit-specific antisera and b) subunit-bearing cells: Subunit-specific antibodies must be created to study the actual receptor subunit proteins. To do this, the most dissimilar portions of each of the cDNAs encoding GluR1 through GluR7 will be subcloned into plasmids for bacterial overexpression. The resulting fusion proteins will be used as antigens to immunize rabbits and goats. Western blots of the fusion proteins will be used to identify immune responses. Kidney cells will be transfected with each of the receptor subunits to provide independent verification and validation of the subunit- specificity of the antisera in each of the following experiments. 2). Anatomy: the immunohistochemical localization of the glutamate receptor subunits in the retina: Subunit-specific antisera will be used to immunohistochemically localize the receptor subunits. Light and electron microscopy will be employed to define the cellular localization of the receptor immunoreactivity and to identify as best as is possible the receptor-bearing cells. Double labeling strategies will be used to determine which sets of receptor subunits are co-expressed. 3). Chemistry: the characterization of the receptor subunit proteins and the complexes they form: First, Western blots will be used to analyze the receptor subunit proteins in the crude membrane fraction of retina. Then, the rabbit antisera will be used to immune precipitate solubilized receptor complexes from the retina. These will then be fractionated on gels, blotted and probed with the goat subunit-specific antisera. The goal will be to define the sets of subunits that associate with one another.

Much of what we know about the neural retina is constrained by our available methods. For example, our anatomical catalogues of retinal cell types, the distribution within them of the neurotransmitter glutamate, and their expression of the glutamate receptor subunits are based entirely on images of dead tissue prepared for histology. Our physiology/pharmacology of the actions of glutamate in the retina is limited by the difficulty of reliably accessing particular cell types and is complicated by the action of receptor antagonists at more than one synapse. The idea of this proposal is that it should be possible to express flourescent proteins in transgenic mice to label defined cell types for studies of living retina, to follow and measure glutamate receptor subunits in retinal cells, and to determine the consequences of expressing mutant glutamate receptor subunits that act as dominant- negative elements. Specific aim one is to determine whether it is possible to express and detect a reporter enzyme fused to the jellyfish Green Fluorescent Protein (GFP) in the living retinae of transgenic mice. Specific aim two is to use GFP-tagged glutamate receptor subunits to ask: can we localize, measure, and follow fluorescent receptor subunits in retinal neurons. Specific aim three will test the idea that mutant receptor subunits, which act as dominant-negative elements, can be used to eliminate defined sets of glutamate receptors from a retinal cell type. The significance of the proposed work is that it would, if successful, provide a new means of studying the dynamic properties of retinal neurons as well as testing hypothesis of synaptic circuitry and function.

DESCRIPTION (provided by applicant): We have created a simple reporter system for the presence of Cre recombinase. A strong promoter with widespread activity normally drives the expression of a red fluorescent protein, but Cre recombinase can catalyze a recombination event that replaces the red fluorescent protein with a green one. We have tested this system in cell lines and now propose to test it in transgenic lines of mice. Specifically, we will screen founders for the presence of red fluorescent signal and then cross these with a line that expresses Cre recombinase in endothelial cells to determine whether the recombination even can occur at the genomic level in a transgenic mouse line. In addition, we have will modify the reporter system by replacing the DsRed sequence with two other sequences that may improve the utility of this reporter. These are straightforward goals that should only take a year to accomplish and the potential benefit is the production of reagents and mouse lines that may be quite useful for many vision scientists.

[unreadable] DESCRIPTION (provided by applicant): This is an R21 proposal to develop and test a technology that may have a large impact on the development of new probes for the nervous system, and which may serve as a bridge between what we know about the genome and what we want to know about the genes it encodes. We have created a Tn5 transposon that inserts GFP randomly into the proteins, producing tribrid fusion proteins that are fluorescent. Our tests of three proteins show that virtually all of the transposed insertions into coding sequences produce fluorescent proteins if the insertion is in the correct orientation and reading frame. Moreover, 1 in 6 of these proteins continue to function. Our first aim is to improve this tool, and test it more rigorously. This will involve rebuilding the transposon with different colored fluorescent proteins positioned in two ways. 1) Opposing orientations (a double-headed arrangement) will be tested to create a transposon that produces a fluorescent protein regardless of the orientation it lands in. These improvements will double the number of fluorescent proteins that can be recovered in any one screen. If successful, this tool should empower the field to more quickly develop genetically decodable sensors of activity as well as optimized fluorescence energy transfer pairs that can be used to make kinetic measurements in living neurons. This tool will be tested on two pairs of proteins that form heteroligomers to see if it can generate fluorescence energy transfer (FRET) pairs. The second aim of the proposal is to generate a transposon carrying CFP and YFP in a head-to-tail configuration. This is potentially a FRET cassette that could be used to scan a protein for regions that move up on activation. This tool will be tested on two proteins where we have good information about their structure and can control their activation.

[unreadable] DESCRIPTION (provided by applicant): This R21 proposal is to test a novel approach. The idea is compelling, and the approach could have a significant impact on many fields of basic research and biosensor development. Or it may not be feasible. The three proposed aims are designed to be direct tests of feasibility. The idea involves splitting a fluorescent protein and inserting the two fragments into different domains of a protein involved in cell signaling. The idea is that if the fluroescent protein is created betwee two subunits, or two different domains, it may be susceptible to relative movement of the proteins such that changes in fluorescence occurs. The goal is to create better reporters of cellular signaling in the nervous sytem. Such probes would offer us better temporal and spatial resolution. Moroever, they would enable us to optically record signaling activity in defined, genetically accessible, neural networks. The three aims of this proposal involve scanning a signaling molecule with a transposon tagging system that can insert fluorescent protein fragements into the target protein. In theory, a GFP should be formed at surfaces of the signaling protein that are close to one another. The hope is that these "split" GFPs may be more sensitive to the sort of distortion that will occur when protein domains move apart from one another. To test this idea, we will extensively scan a G protein subunit and two voltage-gated ion channels. The approach, experimental design, and scope of the project should suffice to show us whether this approach is worth further development. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A New Modular Tool Set for Live Imaging and Manipulating the Nervous System Project summary. The nervous system contains the most complex and heterogeneous set of cell types in the body. Modern approaches in imaging, and genetically encoded reporters based on the Green Fluorescent Protein (GFP), have made it possible to explore some of the dynamic processes involved in the development, function, and pathogenesis of the nervous system. However, the complexity of the nervous system has made it difficult to precisely target expression to particular sets of neurons. The objective of this proposal is to create and test prototypes of an entirely new generation of genetically encoded, modular fluorescent molecules for labeling and imaging specific cell types and for manipulating the genes that they express. Specific promoters for many cell types of the nervous system are not available, barring genetic access to these cells. Using an innovative combinatorial strategy, this project will provide access to these cells and enable researchers to specifically target gene expression and gain new insight into signal transduction, protein-protein interaction, and the dynamic processes of neuronal function and pathogenesis. Aim one is to create a modular pair of labeling proteins, the Fly and the Hook, that are tied together by leucine zippers. This modular system will provide access to neurons that have eluded labeling and purification to date. Aim two is to create a bi-functional, modular system for both labeling neurons and manipulating their genomes with complementing proteins that produce both fluorescence and Cre recombinase activity. A New Modular Tool Set for Live Imaging and Manipulating the Nervous System Project Narrative The development and function of the nervous system, as well as the death of neurons in disease and injury, are dynamic processes that are poorly understood. The goal of this project is to develop new fluorescent molecules for genetically labeling and manipulating gene expression in specific neurons in the living brain. If this project is feasible, these molecules would lead to new understanding of the dynamic processes that underlie the pathogenesis of neurological diseases such as epilepsy, stroke and Alzheimer's. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Project Summary/Abstract Drug discovery depends crucially upon reliable assays for biological activity. Live cell assays provide a rich environment for measuring biological activity. Coupled with genetically encoded fluorescent biosensors, live cell assays have the potential to provide read-outs with unprecedented specificity for particular signaling pathways. Although widely used for basic research applications in living cells, genetically encoded fluorescent biosensors have had little impact on drug discovery because of difficulties in measuring and interpreting fluorescence intensity read-outs, including poor signal to noise ratios, variability in cell expression, and interference from fluorescence emitted by compounds. This Phase 1 project will demonstrate the feasibility of a new strategy that combines highly specific biosensors with extremely fast fluorescence lifetime measurements to produce the speed, sensitivity and specificity needed for high throughput screening applications. This approach employs an alternative fluorescence measurement based on fluorescence lifetime that is much faster than time-correlated single photon counting (TCSPC), yet also more precise. It operates in non-imaging mode which makes for simple data interpretation and minimizes background fluorescence. It goes far beyond the expected incremental improvements to image-based technologies. Our preliminary data demonstrates the tremendous potential for robust live cell assays when lifetime methodology is applied to measuring genetically encoded fluorescent sensors. Our specific aims will accomplish the vital proof of principle steps and set the direction for our long term objectives of producing a robust live cell drug discovery platform within 5 years. PUBLIC HEALTH RELEVANCE: New assays for biological activity are urgently needed to develop safe and effective drugs that provide better treatment outcomes and improved human health. This proposal addresses the technical challenges associated with using fluorescent live-cell assays and has strong potential to reduce the cost and improve the reliability of drug discovery processes.

DESCRIPTION (provided by applicant): Neuronal electrical activity is the central underpinning of nervous system function. While understood as essential for over a century, the tools to study circuit level neurophysiology have remained largely unchanged in 50 years. The advent of molecular biology has dramatically advanced neurobiology by allowing molecular characterization of the nervous system but has not translated into significant gains in neural electrophysiology. Opto-molecular methods have revolutionized our study of neuronal connectivity, development, gene distribution, calcium signaling and recently, targeted neuronal activation (i.e. optogenetics). A glaring exception to this light-based revolution is the use of optical methods to monitor electrical activity. Intracellular calcium levels and metabolic signals are often used as a surrogate marker of electrical activity, however they are temporally delayed, do not detect subthreshold events and more often than not fail to capture the relevant suprathreshold activity. The PIs laboratories, as part of a multi laboratory collaboration have been developing genetically encoded voltage sensors based on fusions of green fluorescent protein orthologs and voltage sensing domains. Our grant members have published most of the significant advances in genetically-encoded voltage sensors in recent years. Our most recent probes, Arclight and ElectricPK significantly improved the signal size and response speed of fluorescent voltage probes. The current application will continue this successful collaborative search for voltage probes. We are seeking probes which combine large F/ V signal sizes, a range of useful response speeds and red-shifted fluorescence spectra. During this previous funded period time, we discovered that by altering the voltage sensor domain, the linker length, the fluorescent protein and by introducing point mutations in the fluorescent protein, we could develop probes with vastly superior signal size and response kinetics. We also confirmed, however, that a purely empirical step (i.e. large scale screening of single, incrementally-modified constructs) is required to make dramatic improvements in response properties. We will employ a staged evolution approach involving successive rounds of directed and random sequence modification followed by direct testing in mammalian cells. The current experiments will be an advance over all previous studies in two important ways: i) we will create vastly greater numbers (20x) of potential probe (thousands) using domain swapping and site directed / random mutagenesis and ii) the larger numbers of constructs will be prescreened by an automated, robotic microfluorimetry method which evaluates the fluorescence signal size and speed in electrically-active mammalian cells. Finally, all successful candidates will be validated for in vio functionality in Drosophila circadian neurons and rodent somatosensory/barrel cortex.

An award is made to Montana State University to develop a scanning laser microscope that incorporates fast active focus control and adaptive aberration correction to provide high-quality three-dimensional microscopic images within thick living tissue or intact animals. Optical microscopy of intact tissues, coupled with the explosive advance of fluorescent markers that can be genetically encoded to specific cell types and structures, is an incredibly powerful tool for fundamental research into biological development and function. To realize the potential of this approach, however, it is necessary to improve the spatial and temporal resolution of deep-tissue imaging techniques. Microscopy of intact tissue suffers from low resolution, low contrast and poor efficiency due to deterioration of the beam focus deep in the specimen. The new microscope uses two types of deformable mirrors to control both the depth of focus and the quality of the images produced. In addition to the new microscope optics, the project also develops the necessary control algorithms and user interface to create an instrument that will be devoted to both research and research training in developmental biology and neuroscience.

The proposed instrument will facilitate research that is addressing fundamental questions about how nervous systems develop and function. While the project is specifically constructing a scanning laser microscope, the underlying technology can bring many of the same benefits to wide-field microscopes in the future. Active and adaptive optics have other potential applications, including small-format cameras and endoscopes and optical data storage read/write heads capable of high-speed focus and aberration correction with no moving lenses. The technology is therefore broadly relevant. In addition to its scientific impact the project embraces integration of research and education, training two engineering graduate students and providing cross-disciplinary research experience for several undergraduate engineering and bioscience students. A new course module will be developed that features the active/adaptive microscope as a platform to teach advanced biophotonic methods.

DESCRIPTION (provided by applicant): The Northern Lights collaboration for better 2-photon probes will combine the expertise of four very different investigators and laboratories from Montana and Canada with complementary interests and skills to tackle a fundamental problem in imaging of the activity of the living brain. It is now well agreed that genetically encoded, fluorescent biosensors are uniquely suited to provide the specificity and versatility necessary to image the activity of defined cells in the brain. It is also clear that to image cells in thick slies or whole brains it will be necessary to turn to 2- photon (nonlinear) imaging, which penetrates deeper into living tissues and causes less damage. Furthermore, 2-photon signals provide additional molecular-level information that 1- photon signals lack. Many years have gone into developing better fluorescent proteins and biosensors for linear imaging. Unfortunately, directly optimizing probes for the 2-photon nonlinear properties has been beyond the reach of biosensor engineers because of the overwhelming complexity of the current state-of-the-art 2-photon characterization. This is a critical impediment for the BRAIN initiative, because robust 2-photon probes with high brightness and stability are needed. Here we describe a three year program to develop a new process and new instruments using available ultrafast lasers and advanced experimental designs that will empower biosensor engineers involved in the BRAIN project to evolve the next generation of multi photon probes.

Project Summary Montana Molecular is in the business of developing and marketing genetically encoded biosensors for academic research and drug discovery in living cells. Our biosensors provide robust detection of crucial second messenger signaling, including DAG, PIP2, cAMP and Ca2+ on both fluorescence microscopes and the large installed base of automated plate readers, with metrics that match or surpass those of traditional biochemical and luminescence based assays. We have an established customer base, with over 100 early adopters in academia and industry. Our customers and industry partners have indicated a growing need for assays capable of targeting specific populations of living neurons and for ?assay-ready? iPSC derived neuronal lines for use in academia and secondary drug screens. Genetically encoded assays for GPCR signaling, developed by Montana Molecular, fit seamlessly with these needs. They can be used for kinetic measurements in living cells, can be targeted to specific populations of cells in complex tissue, and have been independently validated on a wide variety of fluorescence microscopes and automated plate readers. To meet the needs of our customers and to penetrate the growing human iPSC market, we propose two specific aims. In Aim 1 we will engineer a new baculovirus vector that circumvents the payload limits and safety considerations of Lentivirus and AAV. In Aim 2, we will partner with iPSC developers in both academia and industry to create the first assay-ready iPSC derived neurons stably expressing biosensors for the detection of multiple facets of GPCR signaling.

! Project Summary The objective of this SBIR Phase II project is to optimize and validate a prototype assay for ligand bias to be used in screening drugs that target seven transmembrane G- protein coupled receptors (GPCRs). The assay is based on genetically-encoded fluorescent biosensors that report signaling with a change in fluorescence intensity. Aim 1 of this project involves engineering and testing to increase the change in fluorescence intensity of biosensors for ? arrestin1 and ? arrestin2. This involves using an established workflow and screening process to iteratively improve linkers in between a very bright green fluorescent protein and ?-arrestin. This proprietary process consists of randomly mutagenizing key amino acids to create low diversity libraries of candidate biosensors that are screened for improved fluorescence properties. Aim 2 involves optimizing assay parameters and validating the multiplex assay comprised of the optimized green fluorescent biosensor for ? arrestin, co-expressed with existing red fluorescent biosensors for diacylglycerol (DAG) or cyclic AMP. The red sensor indicates G-protein mediated activity while the green sensor indicates arrestin activity. The assay will be validated with a select panel of GPCRs and small molecule and peptide agonists. GPCRs, are one of the largest family of drug targets. Their activity is mediated by both G proteins and ?-arrestins which activate a network of distinct signaling pathways. Depending upon the GPCR and cell type, biased agonism can be therapeutically beneficial or produce unwanted or harmful side effects (Violin and Lefkowitz, 2007). Quantifying bias in GPCR signaling could help to identify new therapeutic drugs that avoid adverse effects. Detecting agonist bias early in the screening process, in cell types that are relevant to disease, has the potential to reduce the risk and cost of drug discovery.

Summary/Abstract Many diseases of the brain involve cellular stress. Early in life, the Zika virus triggers stress in neural progenitor cells causing microcephaly. Much later in life the mutant proteins produced in Parkinson's and Huntington's disease, as well as ALS, and Alzheimer's all produce cellular stress leading to the slow neurodegeneration that occurs over many years. Typically this degeneration is studied by counting dead cells, in animal or cellular models of the disease, which is an insensitive measurement of chronic, slow, cellular stress. Our goal is to create a genetically encoded, fluorescent biosensor that lights up living, stressed cells long before they are destined to die, giving scientists sensitive new tools to identify and potentially rescue stressed neurons. Aim one will test a series of multicolored sensor prototypes designed to measure cellular stress as well as second messenger signaling. These sensors will be tested in standard cell lines. Comparison of the sensor responses in cells expressing normal or disease forms of neurodegenerative proteins will be used to identify the best possible sensor designs. The broad suite of mutant proteins being examined will give us a first glimpse of whether one specific sensor is most sensitive, or whether different sensors are best suited to study different stressors and diseases. Aim two will test whether these tools can detect stress in neurons. The first goal will be to detect stress caused by overexpression of the normal and mutant proteins from aim one in neuronal culture as well as in neurons in rat brain slices. The second goal will be to use iPSC derived motor neurons from a patient with ALS, as well as an isogenic cell line in which the SOD1 mutation from the patient has been repaired. If the sensor can detect the difference, this will be a significant improvement over existing technologies.

SUMMARY/ABSTRACT The goal of our predicate SBIR Phase I award is to develop genetically-encoded fluorescent assays to detect cell stress in models of neurodegeneration. Typically this degeneration is studied by counting dead cells, in animal or cellular models of the disease, which is an insensitive measurement of chronic, slow, cellular stress. Our goal is to create genetically encoded, fluorescent biosensors that light up living, stressed cells long before they are destined to die, giving scientists sensitive new tools to identify and potentially rescue stressed neurons. Our aim is to create cell stress based assays that can be adopted into neurodegenerative research and drug development platforms. To successfully bring these assays to market a thorough understanding of the customer?s unmet needs are crucial. The I-Corps program will provide unique training and customer interactions that will greatly enhance our ability to successfully bring these products to market. Our current strategies for commercialization will be focused on developing multiple platforms for assay deployment. For example, the assay will be delivered in viral vectors as well as made available for stable cell line production and integration into animal models of neurodegeneration. The I-Corps program would grant us a deeper understanding of our current marketplace, the needs of our customers, and strategies to implement our commercialization plan. Upon completion of the I-Corps program our commercialization strategy will be reevaluated and modified based on customer feedback and strategies learned during the program.

Millions of Americans today have an opioid use disorder (OUD). Millions more misuse opioids, and the crisis continues to grow. The goal of this proposal is to speed the discovery of non-addictive analgesics by providing drug discovery teams with simpler, more robust, more quantitative, assays for agonist bias. Driven by the urgency of the problem we are seeking Fast Track support to create new assay and analytic tools for drug discovery in OUD research. Our goal is to optimize and test new assays for agonist bias at particular receptors that couple to both the Gi and ?-arrestin signaling pathway, and create new tools to improve the analysis of structure/activity relationships. There are good reasons to search for biased agonists to the receptors identified in the NIDA ?top ten? list of medication development priorities. Biased agonists could activate beneficial signaling pathways while avoiding those that cause adverse effects. Finding these biased agonists is difficult: current assays for detecting bias, while established and validated, suffer from drawbacks that are limiting translatability to animal models and clinical studies. These include entirely different sets of experimental conditions for measuring the different signaling pathways being compared and different time courses of the response being measured. The latter results in time-dependence of the bias measurement which complicates predictions of in vivo efficacy and complicates SAR tables by adding extra variables. Our new assay will simultaneously measure the kinetics of Gi and ?-arrestin signaling in living cells. This project will involved creating new tools as well as re-purposing ones we have already developed to study non-OUD drug targets. The assay will be optimized for use on standard fluorescence plate readers, and a data analysis toolbox will be developed to simplify quantification of agonist bias based on kinetic measurements. Phase I will complete the initial validation studies on the NOP opioid receptor, with goal of demonstrating assay reliability and sensitivity milestones. Phase II will optimize the assay for D3 dopamine, CB1 cannabinoid and OPRM1 opioid receptors and develop the analysis toolbox for deployment on standard plate readers and software packages commonly used in drug discovery. In the second half of Phase II, assays with detailed protocols will be ready distribute to researchers who are developing new drugs for OUD.