Joshua J. Rosenthal - US grants

Understanding the regulation ion channel is critical to understanding basic mechanisms of cellular excitability. Defective ion channel loci, and misprocessing of their gene products, have been linked to a rapidly expanding list of disorders, including cystic fibrosis, cardiac arrhythmias, and certain forms of epilepsy. RNA editing is an important regulatory mechanism for ion channels at the synapse. This proposal draws extensively from biophysical analysis of voltage-dependent K+ challenge go answer a question of fundamental biological importance: is RNA editing also important at the level of the action potential? The focus of these studies is the delayed rectifier K+ channel of the squid giant axon (SqKv1A). Thus far, 12 codons which are edited have been identified. These sites are scattered in a variety of functionally important channel domains, and preliminary data suggest that they modify physiological properties. The aim of this work is to define the functional effects of editing in the channel's score and tetramerization (T1) domain. An unknown combination of 5 core editing sites effect deactivation and steady-state voltage sensitivity. The relevant sites will be identified, and the biophysical basis for their actions will be examined on a single channel level. Position 87 in the T1 domain, which is edited from arginine to glycine (R87G), has a tremendous effect on regulating functional expression in Xenopus oocytes. R87 G homotetramers express at 100 fold lower levels than their edited counterparts. Because this position is partially edited in the giant axon, it's effect on heterotetramer expression will be examined. To accomplish this, monomer specific tags will be developed. In the future it is intended that these studies will be extended to the native system. For this reason the giant axon is ideal: it is identifiable, extensively studied, and its dimensions permit high resolution voltage clamp on the macroscopic, gating, and single channel levels.

All modern biology is based on the principle that genetic information is stored in genes and realized in proteins. Surprisingly, recent genome sequencing projects indicate that drastically different organisms, such as humans, flies and worms, carry a more or less common set of genes. What then is the genetic basis of complexity? RNA editing, a process that changes and increases genetic information, could obviously play an important role, however its biological significance remains poorly understood. One form of editing, mediated by the hydrolytic deamination of adenosine (A) residues in mRNAs, is prevalent in the nervous system of all metazoans. By changing A to Inosine (I), which is read by the ribosome as guanine (G), codons can be mutated and protein structure and function changed. In mammals, relatively few mRNA substrates for A-I editing have been identified, most encoding proteins involved in synaptic transmission. More recent investigations, however, have identified a surprisingly large number of substrates in Drosophila and Loligo, suggesting that editing in invertebrates is a particularly robust process. Many of these examples are ion channel transcripts. Editing permits multiple proteins from a single gene. How and when do different organisms edit? Which mRNAs are targeted and how is protein function changed?
In terms of behavior, cephalopods are the most sophisticated invertebrates. Experiments outlined in this proposal will compare how K+ channel mRNAs, expressed in the giant axons of four closely related species of squid, are edited. These species were chosen because their habitats span a large thermal gradient, and physiological studies have determined that their potassium conductance varies according to temperature. Earlier investigations identified fourteen editing sites in a K+ channel mRNA from Loligo opalescens. These edits influence channel function in diverse ways, regulating voltage-dependent gating, overall K+ conductance, and subunit tetramerization. Preliminary evidence suggests that the position, and A-I conversion frequency, of some of these sites can vary between species. During the present funding period, experiments will examine the molecular basis, and functional consequences, of species-specific edits, using a broad range of approaches. Molecular techniques will be used to map editing sites and the associated A-I conversion frequency. Biochemical and molecular techniques will be used to identify the critical secondary structure that surrounds edited adenosines and regulates their deamination. Biophysical techniques, including both macroscopic and single channel recordings, will be used to study how the amino acid changes caused by editing sites affect channel function. Taken together, these approaches will be used to investigate how the pattern of editing changes between species, the biochemical and molecular properties underlying these differences, and how species-specific edits affect the physiological properties of K+ channels. These data are important because they provide a window on how A-I editing influences the evolution of nervous function.

The long term objective of this work is to understand, at a molecular and biophysical level, how RNA editing regulates ion channels and transporters. RNA editing by adenosine deamination is a posttranscriptional process that is essential for proper nervous system function. Although many mRNA substrates have been shown to be edited by this mechanism, very little is known about the functional consequences of these modifications. This proposal focuses on the Na/K pump as a model membrane protein because it plays a central role in the regulation of ion homeostasis. The enzymatic conversion of adenosine to inosine (A -> I) in pre- mRNAs allows multiple protein products from a single gene, thereby extending the genomic capability of an organism. These conversions are not random, but are targeted to functionally important domains within the protein. For example, in the GluRB subunit of the glutamate-gated ion channel, important for fast excitatory synaptic transmission in the central nervous system, editing underlies the conversion of Q to R in the channel's pore. This change, which is essential for survival, renders the receptor impermeable to calcium ions. Edited substrates are identified by comparing genomic and cDNA sequences for the same gene. This process is greatly simplified in squid where editing is extensive. This proposal takes advantage of high level editing in squid to identify mutations that are functionally relevant.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A major long-term objective of the University of Puerto Rico Medical Sciences Campus is to expand and improve the environment for molecular neuroscience. To help meet that goal, this project will establish a Neurogenetics Core Facility at the Institute of Neurobiology (Specific Aim 1). This facility will be used to enhance the research capabilities of the present faculty and will serve as a resource for the further development of the Institute. A key feature of this aim will be to recruit a neurogeneticist to head the facility. Funds will be used to remodel lab space, obtain major instrumentation, support collaborations in the areas of neurogenetics or neurogenomics, and for the recruitment process. They will also be used to develop collaborative research teams between the Institute of Neurobiology and outside researchers. The operation of the Molecular Neurobiology Core Facility, established under the last RCMI initiative, will also be continued (Specific Aim 2). This facility is adjacent to the space that is being proposed to house the Neurogenetics Core. It has been used extensively by the participants in this proposal and is considered a vital asset for their research. All molecular biology at the Institute centers on this facility, and it is anticipated that it will also be an essential resource for investigation in neurogenetics. In addition, improvements will be made in communications between the Institute and research groups outside of Puerto Rico (Specific Aim 3). This will be accomplished by improving the internet connections, video conferencing capabilities and by implementing a mechanism to support collaborations with outside scientists. Taken together, these changes will add powerful new research and training capabilities to the Institute of Neurobiology, as well as enhance existing capabilities. They will also help create better links between current research and public health. These changes are required to meet the challenges of an evolving environment for Neuroscience research.

Human activities are altering the environment at an alarming rate. A multidisciplinary approach is essential to understand the complex interplay between molecular, cellular, and behavioral responses by organisms under these increasingly stressful conditions. The nervous system is the interface between an organism and its environment.

The Puerto Rico Center for Environmental Neuroscience (PRCEN) will combine neuroscience (the study of the nervous system and behavior) and environmental science (the study of local ecosystem environments) to tackle environmental issues in Puerto Rico's tropical setting. The Center will combine neuroscientists from the Institute of Neurobiology and the Dept. of Anatomy of the University of Puerto (UPR) Medical Sciences campus and environmental scientists from the Environmental Sciences Program and the Depts. of Biology and Chemistry of the UPR Rio Piedras. The alliance will bring together cutting-edge techniques normally associated with cellular and molecular neuroscience with expertise in local ecosystems and environmental science to create a novel field that will require participants to move outside of their comfort zones and learn about entirely new areas of research.

Objectives of the center will be to: (1) establish research programs in the new field of environmental neuroscience, (2) enhance research productivity through faculty and infrastructure development, (3) increase the numbers of minority students attaining advanced degrees in interdisciplinary science, and (4) generate community understanding of the work being done in the Center.

The research subprojects focus on four local ecosystems: terrestrial, freshwater rivers, estuaries, and marine systems. The habitats under study are intimately connected: contaminants in the mountains make their way into rivers, pass through the estuaries, and end up in the sea.

The oceans subproject is designed to understand the consequences of environmental pressures on tropical corals, using state of the art molecular-cellular techniques.

The estuaries project will focus on the blue crab, which supports one of the largest fisheries industries in the United States. This project will use high resolution monitoring to track the presence of contaminants and other environmental stressors, and correlate the resulting environmental data with physiological monitoring of heart and endocrine functioning in this crab.

The freshwater studies will monitor contaminants in three representative Puerto Rican rivers. Four animal models (zebrafish, mosquitofish, and two types of prawn) will be exposed to pollutants found in the three rivers, and a range of physiological and behavioral parameters will be examined.

Finally, the terrestrial project will use sophisticated molecular biology and electro-physiology to examine the nervous systems of fruit flies from different habitats in Puerto Rico. The standard laboratory-reared fruit fly (Drosophila) is a prized and widely-used model system in neurobiology laboratories throughout the world. However, there is a paucity of studies examining this animal in the wild, especially with respect to the specific habitats in which they are living.

Intellectual Merit
The conceptual linchpin of the PRCEN is that the nervous system is the interface between an organism and its environment; a multidisciplinary approach is essential to understand the complex interplay of molecular, cellular, organismal, and ecosystem dynamics faced by organisms under the increasingly stressful conditions created by human impacts on the environment. We refer to this approach as environmental neuroscience. The program will be unified by the central hypothesis that a full understanding of the consequences of pollution and climate change requires dialogue between investigators monitoring environmental conditions and organismal biologists using that information to determine how environment affects function.

Broader Impact
The PRCEN center will change the way we look at environmental problems, and will create a new category of scientists prepared for the environmental challenges developing from human activities. The Center will impact a large number of minority students by tapping into the collective student population of over 19,000. Our undergraduate participants will integrate closely with ongoing NSF sponsored mentorship initiatives such as the Lewis Stokes Alliance for Minority Participation, the Research Experience for Undergraduates Program, and the Undergraduate Research Mentoring Program. Our graduate students will have access to broad training here, and will also be given the opportunity to take courses and train stateside at places like the Marine Biological Laboratory in Woods Hole, MA. Finally, our studies will integrate with local organizations such as the San Juan Bay Estuary Program to coordinate community outreach targeting K-12 education.

DESCRIPTION (provided by applicant): There is currently no way to correct disease-causing mutations in the nervous system without altering the physiological level of the endogenous mRNA. This is a serious challenge because haplo-insufficiency or two-fold over-expression is often sufficient to cause neurological disorders. An example is Rett Syndrome, caused by mutations in the Mecp2 gene. Mecp2 gene duplication, as well as loss-of-function, results in severe disease. We propose to meet the challenge by harnessing the natural ability of RNA editing enzymes to site-specifically fix mutations in endogenous mRNAs. As a target for gene therapy, mRNA offers advantages over DNA. Messenger RNA is cytoplasmic, a readily available substrate, and unlike DNA in which 'mistakes' will be maintained, mRNAs turnover, replenishing the therapeutic target. Our new approach, Site Directed RNA Editing (SDRE), offers enormous untapped potential for correcting mutations, particularly those affecting the nervous system, and for exploring fundamental biological questions. RNA editing, which occurs through adenosine or cytidine deamination, is a natural process. When it occurs within the coding sequence of an mRNA specific codons can be re-coded to produce an altered amino acid sequence. For example, excitatory neurotransmission absolutely depends on the editing of a single adenosine within AMPA-type glutamate receptor mRNAs. Recognizing the power of this activity, we engineered a hybrid modular adenosine deaminase. When used in combination with a small antisense guide RNA we can site-specifically target any chosen adenosine. A similar strategy will be employed to create a site-directed cytidine deaminase. Unlike established therapies that focus strictly on regulating gene expression, SDRE can also fine-tune protein function. Inherited mutations that underlie diseases due to amino acid substitutions or premature stop codons can be corrected, and second-site suppressor mutations that restore function can be selectively introduced. We will demonstrate the power of SDRE within the context of neurobiology, but importantly, it applies to any biological system.

DESCRIPTION (provided by applicant): The University of Puerto Rico (UPR) Medical Sciences Campus proposes to establish a COBRE Center that will significantly strengthen the research infrastructure of the institution and that will impact biomedical investigation throughout the island. The specific aims of this COBRE Center for Neuroplasticity at the University of Puerto Rico are to: 1) Foster development of junior investigators into competitive researchers working on projects with direct biomedical significance. An intensive mentoring program will be implemented with the goal of creating a culture of research comparable to that of major research-intensive universities. 2) Provide a Neuroimaging and Electrophysiology Facility (NIEF) that will offer state-of-the-art instrumentation, training, and expertise to the neuroscienc community of Puerto Rico. The NIEF will be located in the Institute of Neurobiology, but will incorporate ultra-high-end instrumentation in the new Biomolecular Sciences Building (BSB) after the COBRE has become well-established at the Institute; 3) Partner with IDeA Networks of Biomedical Research Excellence (INBRE) entities in Puerto Rico; 4) Support programmatic activities that increase interdisciplinary collaborations at the basic research level. The major strengths of the proposed COBRE include: 1) the PI, who is both an accomplished researcher with extensive funding and advisory activities within the NIH, and an experienced administrator with a history of successful initiation of research and training programs; 2) the team of young, aggressive and highly committed scientists; 3) enhanced mentorship possibilities provided by the current level of intellectual and scientific accomplishment present at the Institute of Neurobiology; 3) rational planning for the future by deployment of the new BSB as a centralized hub for integrated and collaborative research in the future of the COBRE Center for Neuroplasticity; 4) the full commitment of the UPR Administration toward assuring the stated goals of the project. This UPR COBRE Center should define pathways and benchmarks for basic and translational research across the UPR system for the next decades.

Squid and their relatives (other cephalopods such as octopuses) have the ability to change skin color with chromatophores, microscopic muscular organs that are under control of the nervous system. All work on the cellular mechanisms of chromatophore control in squid has focused on three related species that inhabit relatively shallow coastal areas that have prominent features like seaweed, rocks and coral on the ocean floor. Skin-color changes in these species are associated with camouflage, signaling between individuals of the same species and threat displays with other species. The deeper open ocean presents a radically different environment that is also inhabited by many squids, primarily of different taxonomic families from the one commonly inhabiting coastal waters. An important open-ocean family includes the Humboldt squid (Dosidicus gigas). There is little light in the ocean at depths inhabited by these squid during daytime, and visual features such as coral and rocks are non-existent. Novel color-change behaviors in Dosidicus include repetitive whole-body "flashing," used for signaling between individuals of this species, and chaotic "flickering" that may underlie camouflage in the open ocean. Although these dynamic behaviors contrast with the more static patterns typical of coastal species, squids of both families employ temporal and spatial patterning to varying degrees. It is therefore likely that basic mechanisms for controlling the chromatophore network are the same in most, if not all, squids. "Vertical" control from the brain to the chromatophore muscles is known in the coastal squids, and may account for most chromatophore-based behaviors in those species, but behaviors like flickering in deeper-water species may be more influenced by processes within the skin itself that permit changes in chromatophores to spread from one to another without directly involving the nervous system. This hypothetical pathway would define a "horizontal" or distributed control system in the periphery that would permit autonomous behavior within the chromatophore network. This issue is the primary significance of the project. Understanding the fundamentals of horizontal control of chromatophores has the potential of being transformative to the field, because the current paradigm is that all control is directly exerted by the brain. Horizontal control is relevant to blood delivery to local tissues by circulatory systems, gut function and nervous system micro-circuits in vertebrates. Therefore, results from this project would also influence understanding of local control more broadly. From a wider perspective, results of this project will provide insight into the interactions of distributed (horizontal) and top-down (vertical) control mechanisms, a subject relevant to the general ability of complex systems to generate non-predictable, emergent phenomena. This concept is of fundamental interest to a broad sector of society, ranging from engineering to economics to politics.

An integrated approach will permit testing the hypothesis that control of the chromatophore network in squid involves peripheral mechanisms that are distinct from the neuronal motor-control pathway that descends from the brain. Spontaneous chromatophore activity that is independent of canonical neural control will be isolated by experimental manipulations in coastal loliginid squid (Doryteuthis opalescens), including chronic denervation and pharmacological block of neuronal activity with tetrodotoxin. In addition, a comparative approach will take advantage of an oceanic ommastrephid species, Dosidicus gigas, in which spontaneous, tetrodotoxin-resistant chromatophore activity is extremely prominent. Relevant methods involve molecular transcriptomics, cellular electrophysiology, immunohistochemistry with confocal microscopy and high-resolution electron microscopy. Specific aims are: 1) identify molecular and physiological properties of relevant ion channels and receptors that control excitability in the radial muscle fibers that operate individual chromatophore organs; 2) define structural, molecular and physiological features of coupling mechanisms between muscle fibers of neighboring chromatophores that define an excitatory transmission pathway within the skin; 3) elucidate the inhibitory role in controlling spontaneous chromatophore activity played by serotonin; 4) carry out parallel experiments in Dosidicus, a member of a family of ecologically important squid in which cellular studies of chromatophores have never been carried out. This project will support undergraduate and graduate student training, and includes significant efforts to involve students from groups underrepresented in STEM.

Project Summary Alternating Hemiplegia of Childhood (AHC) is a devastating neurological disorder that is characterized by bouts of paralysis and is often accompanied by developmental abnormalities. Mutations within the ATP1A3 gene, which encodes a neuronal isoform of the Na+/K+ ATPase, are the most common cause of AHC. AHC is rare, and at present there is no cure. Two factors significantly contribute to the lack of progress in the development of treatments. First, as with most rare genetic disorders, there are insufficient resources and limited financial motivation in the private sector. Second, the mechanisms by which the mutations cause the disease are not understood. AHC mutations are dominant recessive, and it is unclear how mutant Na+/K+ ATPases interfere with wild type versions to create physiological deficits that are higher than expected. For any genetic disorder, the most direct treatment would be to correct the underlying mutation. In theory, this could be accomplished by editing the gene or the messenger RNA that it encodes. For neural disorders, gene editing is not practical because the most advanced systems using CRISPR technology don?t work well in neurons. In addition, they are difficult to deliver in vivo because they are based on bacterial components which will likely generate immunological complications. Recently, new systems for editing mRNAs, called site-directed RNA editing (SDRE), offer distinct advantages for the treatment of genetic diseases. First, they can operate in neurons, and they are based on enzymes that occur naturally in humans. Another advantage is that they are relatively simple, being composed of a small oligonucleotide guide RNA coupled to a human RNA editing enzyme. Because genetic information is encoded the same way between different RNAs in different cells, it can be edited in much the same way wherever it is expressed. This make SDRE a semi-generic approach for different genetic disorders. In this work, SDRE components will be optimized to efficiently and selectively correct the most frequent mutation that underlies AHC (ATP1A3 D801N). Top guide RNAs will be identified from pools of billions of randomized candidates through an iterative selection procedure. These will then be tested in cells in combination with different versions of engineered RNA editing enzymes. These reagents will then be packaged into virus particles so that they can be efficiently delivered to cells. Simultaneously, the mechanisms by which the ATP1A3 D801N mutation alters Na+/K+ ATPase function will be studied, both in enzymes that contain the mutation and in wild type enzymes. These experiments will provide a better understanding of the physiological basis of AHC and help provide estimates of the proportion of mutants that must be corrected to offset functional deficits. Taken together, the development of SDRE reagents coupled with a clear understanding of the aberrant physiology caused by AHC mutations will allow us to begin to develop the first therapeutics for this condition.