James Bryant Hurley - US grants

The first objective of the proposed research is to define the function of a novel retinal guanyl nucleotide binding protein recently identified in bovine retinas. A cDNA clone encoding this protein was selected from a retinal cDNA library using probes that were designed to detect the alpha subunit of transducin, the photoreceptor G protein. This cDNA clone, referred to as T-alpha-II, encodes a protein that is 80% homologous to the transducin alpha subunit. T-alpha-II mRNA is expressed only in the retina, but the location and function of T-alpha-II product are unknown. Retinal mRNA corresponding to this clone is to be localized by in situ hybridization to bovine retina sections. Peptides are to be synthesized that correspond to amino acid sequences specifically found in the novel alpha subunit. Antibodies are to be raised against these peptides and used to localize the novel alpha subunit by immunocytochemical analysis of retina sections. These antibodies are to be used to purify the novel alpha subunit and the properties of the purified protein are to be compared with properties of the transducin alpha subunit. The second objective of the proposed research is to define structure-function relationships within the alpha subunit. A mammalian cell expression system is to be used to make proteins that have been altered by oligonucleotide directed site-specific mutagenesis. The normal and altered proteins are to be purified from cell extracts by affinity to specific antibodies or by conventional methods and then analysed. Sequences that are thought to be involved with GTP binding and hydrolysis as well as with protein-protein interactions are to be examined by this method.

Rod and cone photoreceptors of vertebrates retinas hyperpolarize in response to light. Rods are very sensitive, but slow to respond. Cones are insensitive but quick. Phototransduction in both rods and cones occurs via G-protein mediated light stimulated cyclic GMP hydrolysis. Many phototransduction proteins, including opsin, transducin and phosphodiesterase catalytic and inhibitory subunits, are products of different genes in rods and cones. The first aim of this proposal is to identify kinetic differences between rod and cone phototransduction enzymes and to explain, at a molecular level, the physiological differrnces between rods and cones. Cone photorecpetors are to be isolated, cone phototransduction enzymes individually purified and directly compared biochemically with their rod counterparts. Recoverin is a recently identified Ca4++-dependent regulator of photoreceptor guanylate cyclase. As part of the comparison of rod and cone phototransduction enzymes, recoverin is to be characterized and the existence of rod and cone-specific recoverins is to be investigated. THe second aim of this proposal is to determine physiological consequences of expressing normal and mutant forms of phototransduction enzymes in rod photoreceptors. Several lines of transgenic mice carrying transducin transgenes have been produced. Retinas from these mice are to be characterized biochemically and electrophysiologically to determine how altered transducin activity affects the photoresponse. Other phototransduction enzymes are also to be expressed as potential dominant mutations in these proteins are identified. The overall aim of this proposal is to identify factors that determine the speed and sensitivity of the photoresponse.

Rod and cone photoreceptors of vertebrates retinas hyperpolarize in response to light. Rods are very sensitive, but slow to respond. Cones are insensitive but quick. Phototransduction in both rods and cones occurs via G-protein mediated light stimulated cyclic GMP hydrolysis. Many phototransduction proteins, including opsin, transducin and phosphodiesterase catalytic and inhibitory subunits, are products of different genes in rods and cones. The first aim of this proposal is to identify kinetic differences between rod and cone phototransduction enzymes and to explain, at a molecular level, the physiological differrnces between rods and cones. Cone photorecpetors are to be isolated, cone phototransduction enzymes individually purified and directly compared biochemically with their rod counterparts. Recoverin is a recently identified Ca4++-dependent regulator of photoreceptor guanylate cyclase. As part of the comparison of rod and cone phototransduction enzymes, recoverin is to be characterized and the existence of rod and cone-specific recoverins is to be investigated. THe second aim of this proposal is to determine physiological consequences of expressing normal and mutant forms of phototransduction enzymes in rod photoreceptors. Several lines of transgenic mice carrying transducin transgenes have been produced. Retinas from these mice are to be characterized biochemically and electrophysiologically to determine how altered transducin activity affects the photoresponse. Other phototransduction enzymes are also to be expressed as potential dominant mutations in these proteins are identified. The overall aim of this proposal is to identify factors that determine the speed and sensitivity of the photoresponse.

DESCRIPTION (provided by applicant): Energy metabolism is essential for viability and function of retinas. Earlier studies of retinal metabolism revealed a predominance of aerobic glycolysis and higher rates of energy consumption in darkness than in light. New technologies have been developed since those pioneering studies. We're using mass spectrometry and other state-of-the- art techniques to investigate retinal energy metabolism in ways that were not possible in previous studies. Symbiotic metabolic relationships between neurons and glia are important for function and survival of neuronal tissues. In the retina photoreceptors die when Muller cells are ablated and the metabolic state of Muller cells changes when photoreceptors degenerate. One of the important functions of glia is to synthesize glutamine. In brain a metabolic cycle known as the Astrocyte Neuronal Lactate Shuttle explains the metabolic relationship between glia and neurons. However, the unusual morphology and metabolic requirements of the outer retina create unique metabolic requirements for photoreceptors and Muller cells. We have found evidence that aspartate produced by retinal neurons may play an important role in the relationship between neurons and Muller cells in retinas. Our first aim is to test the hypothesis that an aspartate/glutamine cycle shuttles carbons between neurons and Muller cells in retina. Metabolic demands of vertebrate retinas are different in darkness and in light. In darkness the demand for ATP is high as photoreceptors use it to fuel active ion pumps. In light, ATP demand is lower, but metabolites must be diverted for production of reducing power to fuel regeneration of rhodopsin and to synthesize phospholipids and other metabolic building blocks. During the previous funding period we made the novel observation that the metabolism of purine nucleotides is strongly influenced by illumination. Our second aim is to identify the mechanism by which purine metabolism is regulated by darkness and light. We will identify the mechanism by which darkness and light influence purine metabolism in retinas.

DESCRIPTION (provided by applicant): Light adaptation is the ability of a photoreceptor to make biochemical adjustments that enable it to detect changes in illumination even in the presence of what would otherwise be a saturating stimulus. A cyclic GMP-mediated enzymatic pathway is used to detect light in rod and cone photoreceptors. One of the biochemical reactions that initiates light adaptation is a Ca2+-mediated feedback pathway that regulates synthesis of cyclic GMP by guanylyl cyclase. The aims of this application are to: (i) understand at a structural level how photoreceptor membrane guanylyl cyclases are regulated by Ca2+ and Ca2+-binding proteins; (ii) understand at the molecular level how mutations in a photoreceptor guanylyl cyclase cause a dominant degenerative disease of the retina, Autosomal Dominant Cone-Rod Dystrophy; and (iii) understand at the physiological level how regulation of cyclic GMP synthesis determines how a photoreceptor adapts to light and how well it returns to the dark state following illumination. These studies will provide fundamental information about how photoreceptors detect light and they will provide insights into the molecular basis of inherited diseases of the retina.

[unreadable] DESCRIPTION (provided by applicant): Photoreceptors are among the most metabolically active cells. They have extraordinarily large mitochondria and consume oxygen at exceptional rates. Energy production by glycolysis and oxidative phosphorylation must keep pace with energy consumption in these highly specialized neurons. Photoreceptors consume oxygen slightly faster in darkness than in light, but the processes that consume energy are qualitatively very different. Mutations that create an imbalance of energy production and consumption under specific conditions may introduce stresses that cause oxidative damage and cell death. The aim of this proposal is to establish a quantitative model to understand production and consumption of energy in photoreceptors. Specific aim 1 is to develop methods to quantify the rates at which various biochemical reactions consume high energy metabolites in photoreceptors. In specific Aim 2 we will use those methods, together with specific genetic mutations in mice, to investigate the processes in photoreceptors that produce and consume energy. In aim 2 we also will determine how energy production is regulated. Specific aim 3 focuses on the role that the calcium-binding protein, recoverin, plays in energy metabolism. Understanding how energy is produced and consumed in these highly metabolic neurons will provide information fundamental to understanding photoreceptor function. These studies also will provide much-needed insights into why specific classes of inherited mutations cause stress and retinal disease. [unreadable] [unreadable] [unreadable]

Antigen Presentation; Biogenesis; CRISP; Cell Communication and Signaling; Cell Signaling; Cereals; Complex; Computer Retrieval of Information on Scientific Projects Database; Core Assembly; Data; Endosomes; Funding; Grain; Grant; HIV Budding; Homology Modeling; Hybrids; Individual; Institution; Intracellular Communication and Signaling; Investigators; Journals; Magazine; Membrane Protein Traffic; Membrane Traffic; Modeling; Models, Structural; NIH; National Institutes of Health; National Institutes of Health (U.S.); Organelles; Organism-Level Process; Organismal Process; Origin of Life; Paper; Pathway interactions; Physiologic Processes; Physiological Processes; Process; Proteins; Publications; Receptosomes; Research; Research Personnel; Research Resources; Researchers; Resources; Scientific Publication; Series; Signal Transduction; Signal Transduction Systems; Signaling; Solutions; Source; Structural Models; Structure; United States National Institutes of Health; base; biological signal transduction; experiment; experimental research; experimental study; gene product; pathway; research study; simulation; trafficking

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Membrane trafficking pathways are essential for normal physiological processes such as signal transduction, antigen presentation, organelle biogenesis, and many others, and for pathophysiological processes such as HIV budding. Membrane trafficking via endosomes is carried out by a series of multi-protein complexes, including the ESCRT complexes and the retromer complex. We have obtained a series of crystal structures of the cores of these assemblies, and in combination with the structures of individual domains and homology modeling, models for the complete structures can be generated. The models have been assessed by comparison to hydrodynamic studies of multiple truncation constructs, providing constraints on the solution structures of the intact complexes. We have applied coarse-grained Monte Carlo simulations using residue-based potentials with rigid domains and core structures to model the assemblies.

DESCRIPTION (provided by applicant): Photoreceptors are different from neurons in the brain because they are exposed to electromagnetic radiation from the environment. Membranes with the most accumulated exposure to radiation are shed from the tip of the photoreceptor outer segment and replaced by addition of newly synthesized membranes at the base of the outer segment. This type of anabolic activity occurs daily. The high demand for anabolic metabolism during these periods of renewal is reminiscent of the metabolic demands of cancer cells. Recent findings in cancer research have identified important metabolic adaptations of cancer cells that enhance their growth. Specific biochemical mechanisms divert glycolytic intermediates away from energy production and instead into anabolic pathways that support growth. Cancer cells carry out aerobic glycolysis, prefer glutamine as a fuel for their mitochondria and they express a unique form of pyruvate kinase, PKM2 that can be regulated by tyrosine phosphorylation. By reducing PKM2 activity cancer cells divert glycolytic intermediates away from energy production to more anabolic roles like phospholipid synthesis. We have carried out preliminary studies that suggest photoreceptors use some of the same types of adaptations that cancer cells use to enhance their anabolic activity. The goal of this project is to extend discoveries from cancer metabolism research toward an understanding of photoreceptor metabolism. We will explore the novel hypothesis that photoreceptor cells regulate anabolic activity by mechanisms similar to those used by cancer cells. The outcome of this project will be a framework with which to understand how energy metabolism influences photoreceptor function and survival.

Photoreceptors, the neurons that initiate vision, must adapt to survive in a hostile cellular environment. In the retina they are exposed to damaging light radiation, experience 100-fold fluctuations in intracellular Ca2+, are located near blood vessels with high levels of O2, and use ATP faster than most other types of cells in the body. To ensure optimal function and survival, photoreceptors must have a robust system for maintaining healthy mitochondria. This would involve a regulated balance between mitochondrial clearance, biogenesis, fusion and fission. Here we propose a comprehensive analysis of the circadian regulation of these processes and how they relate to changes in mitochondrial structure and metabolic function. We exploit advantages of both the mouse and the zebrafish models and our expertise studying metabolism and photoreceptor biology. In Aim 1 we will define daily and circadian changes in photoreceptor mitochondria function and structure. In Aim 2 we evaluate mitochondrial clearance and biogenesis and in Aim 3 we examine cellular triggers underlying mitochondrial mobility. In summary, we are asking several questions that are all directed toward understanding mitochondria homeostasis. We will examine mitochondrial biogenesis, clearance, morphology, number, factors influencing motility and metabolism at different times of day both in light and dark. This will provide a broad and impactful overview of how these processes are coordinated to optimize photoreceptor health and function.

Molecular Biology Component: Services and Shared Instrumentation: Project Summary The main objective of the Vision Research Core is to enhance the productivity and efficiency of the research programs of the UW vision scientists, with special priority given to investigators holding NEI R01 grants. The core achieves this objective by: (1) giving investigators and their laboratory personnel access to resources that are outside the resources of individual R01 grants, (2) giving investigators and their laboratory personnel access to technical expertise that is outside the scope of individual labs, (3) providing training of laboratory personnel to enhance the capacity of individual labs, and (4) providing a culture that promotes collaboration. The Molecular Biology Services and Shared Instrumentation Module achieves this goal by ? providing a centralized service for genotyping model organisms ? providing assistance and training in immunohistochemistry, immunocytochemistry, in situ hybridization, developing PCR assays for genotyping, cryopreservation of zebrafish, metabolomics sample preparation, and apoptosis assays ? by providing access to a variety of instruments that are used on a daily basis by multiple labs to avoid unnecessary duplication of equipment such as gel documentation systems, ultracentrifuges real time PCR machines, and spectrophotometers ? by providing access to a dedicated core cell/tissue culture facility

Project Summary/Abstract Metabolic features of photoreceptors, Müller cells and retinal pigment epithelium cells are strikingly different. Metabolic relationships between these cells are important for retina function and survival. A key component of this metabolic ecosystem is the extraordinary efficiency with which photoreceptors in the outer retina convert glucose to lactate. Highly efficient glycolysis of glucose to lactate in the presence of abundant O2 and abundant mitochondria is referred to as ?Aerobic glycolysis? or the ?Warburg effect?. It is a metabolic feature of retinas and also of many types of cancer cells. The molecular mechanism responsible for the Warburg effect is unknown. We will use strategies described in the following three specific aims to identify the molecular mechanisms that enhance aerobic glycolysis in retinas. In the first specific aim of this proposal we will measure metabolic flux in live cells to reveal which steps in glycolysis are enhanced in retinas. Our preliminary findings show that the steady state concentrations of glycolytic intermediates in retinas is very low and that flux through those intermediates is very fast. Our preliminary findings suggest that steps that involve production and consumption of NADH appear to be enhanced. In the second aim we will compare retinal and RPE proteins using mass spectrometry without and with cross-linking to determine how retinas favor reduction over oxidation of pyruvate. These experiments will demonstrate the relative enrichment of glycolytic vs. mitochondrial proteins in the retina and RPE. The cross-linking studies will identify protein-protein interactions between the enzymes that catalyze reactions in the glycolytic pathway. In the third aim we will use in vitro assays of glycolytic enzyme activity to determine how retinas favor reduction over oxidation of pyruvate in mitochondria. Assaying production of lactate from glucose in the presence of enzymes that can compete for consumption of glycolytic intermediates will test the hypothesis that glycolysis is enhanced by channeling of glycolytic products and substrates between enzymes.

Project Summary/Abstract Metabolic fuels from the choroidal blood must pass through the retinal pigment epithelium (RPE) to reach photoreceptors in the retina. The retina and RPE have unique specialized metabolic features that facilitate this flow of nutrients. In previous studies we showed that the RPE minimizes glycolysis so that more glucose can reach the retina. More recently we showed that the retina, which is hypoxic in the eye of a living animal, transfers electrons from mitochondrial respiration to fumarate to make succinate instead of transferring the electrons to oxygen to make water. We also showed that succinate made and released by the retina can fuel oxygen consumption by the RPE. We have proposed a model for energy metabolism in the vertebrate eye in which succinate transfers reducing power from the hypoxic retina to the oxygen rich RPE. After succinate is oxidized by RPE cells, its carbons can be recycled from the RPE back to the retina to accept more electrons and transfer them to oxygen in the RPE. We reported recently several lines of evidence that support this model for a succinate-mediated metabolic cycle in the eye. The evidence so far is based on ex vivo analyses of healthy, functioning retina and RPE/choroid living tissues. However, it also is important to establish to what extent this metabolic cycle occurs in vivo, i.e. in the eyes of living animals. Recently we established an experimental protocol in which we infuse 13C labeled metabolic fuels including succinate and malate through catheters into the jugular veins of mice. We then measure the time course and steady state levels of incorporation of 13C into metabolites in the retina and RPE/choroid. In Aim 1 of this proposal we will confirm our initial findings that the succinate cycle occurs in vivo and we will optimize the infusion protocols. In Aim 2 we will use in vivo infusion to show how circadian and diurnal cycles influence metabolic flux between the retina and RPE. In Aim 3 we will explore strategies to exploit the succinate cycle to slow degeneration of photoreceptors in mouse models of retinal degeneration.

Molecular and Cellular Biology Core Module ? Project Summary The main objectives of the Vision Research Core are to enhance the productivity and efficiency of the research programs of the UW vision scientists and to facilitate collaboration among investigators. The Molecular and Cellular Biology Module enhances productivity and efficiency by providing access to shared equipment and facilities that are used daily by multiple labs. This enhances efficiency by avoiding unnecessary duplication of expensive equipment and enhances the capabilities of individual labs by providing access to expensive equipment that would be beyond the resources of the individual labs. Shared instruments include Sanger DNA sequencing, quantitative real-time PCR, gel documentation systems, MilliQ water purification system, ultracentrifuges, a variety of tabletop centrifuges, shakers for bacterial cultures, a spectrophotometer and a cell culture facility. In addition to providing access to equipment and facilities, the core also maintains the equipment in good working order. Core Module staff provide technical expertise in the use of the module?s equipment, and in a variety of molecular and cellular biology methods. Interactions among investigators using the shared resources available in this module stimulates collaboration.