Ronald G. Gregg - US grants

The Molecular Biology Core will be used by subprojects 1, 2, 4, and 6. The core will provide services that will greatly facilitate the research proposed by subprojects 1 and 6, and more importantly, the core will allow subprojects 2 and 4 access to methodologies absent from there own laboratories. Access to such a core is essential because implementation of these technologies in each investigators' laboratory would be prohibitively expensive. For example, Dr. Coronado (subproject 2) wishes to obtain a reliable and abundant supply of certain toxins, from the Mexican breaded lizard Heloderma horridum horridum, that will be used as tools to dissect the function of the ryanodine receptor in cardiac muscle. Services provided by the Molecular Biology Core will include isolating the toxin cDNAs, determining the DNA sequence and constructing expression vectors that will permit the production of the toxins in bacteria. The expression and purification of these recombinant toxins will be done by the Protein Biochemistry Core (Core B). Dr. Coronado does not have either the equipment or the expertise to allow this to be done in his own laboratory. However, the use of the two core services will enable this project to be completed in a timely manner.

L-type voltage dependent calcium channels (VDCCs) are critical components in the regulation of cardiac function. The kinetic characteristics of these channels, which are important in controlling heart rate and force of contraction, are defined by the primary sequence of the four subunits that make up each channel. The objectives of this proposal are to determine the intracardiac distribution of the three beta subunits of L-type VDCCs present in the heart, and to determine the role of the beta subunit in the regulation of the L-type VDCC present in working myocardium. The specific aims are; 1) to isolate and characterize murine cDNA and genomic clones from the beta2 and beta3 subunit genes, 2) to determine the distribution of the beta1, beta2 and beta3 subunits by in situ hybridization and immunohistochemical detection in heart sections. 3) use gene targeting, to produce a mouse that lacks the beta subunit that is predominantly expressed in working myocardium, and 4) to characterize the effect of this beta null mutation on the L-type VDCC of cardiac myocytes and on the physiology of the intact animal. Specifically modified beta subunits will be introduced into either beta null myocytes or beta null animals to determine their effect on the regulation of the cardiac L-type VDCC.

DESCRIPTION (provided by applicant): Vision begins when light is converted to an electrical signal in the photoreceptors. Increases in light decrease release of the neurotransmitter, glutamate, from cone and rod photoreceptor terminals, and decreases in light increase its release. These changes in synaptic glutamate concentration are detected by two classes of bipolar cells that then transmit the signal vertically through the retinal circuit to the ganglion cells. The neurotransmitter changes also are detected by horizontal cells that provide lateral transmission in the form of feedback and feedforward inhibition. There are two classes of bipolar cells, hyperpolarizing (HBCs) and depolarizing (DBCs). HBCs utilize ionotropic glutamate receptors and hyperpolarize in response to a light flash. DBCs utilize a metabotropic glutamate receptor, mGluR6, that signals to TRPM1, depolarizing in response to a light flash. Defects in transmission in DBCs results in complete congenital stationary night blindness (cCSNB). Mutations in GRM6, NYX, TRPM1 and GPR179 cause cCSNB. The mechanism by which mGluR6 signals TRPM1 is largely unknown. The long term goal of this project is study the molecular interactions between two recently discovered retinal components, GPR179 and Cav1.1, and the other DBC signal transduction components. The specific aims are: 1) Determine the role of GPR179 in DBC signalplex assembly/function, 2) determine the role of Cav1.1 in DBC signalplex assembly and function, and 3) Determine functional and trafficking interdependence of DBC signalplex components. At the completion of this project, we will have characterized the function of newly discovered proteins (GPR179 and Cav1.1) critical to signal transmission in DBCs. Further, we will have identified new candidate genes for congenital stationary night blindness.

[unreadable] DESCRIPTION (provided by applicant): The objective of this proposal is to accelerate the identification of zebrafish genes discovered during mutagenesis screens. This proposal is in response to PAR-02-142 to develop "Tools for Genetic Studies in Zebrafish." A major focus of this PA is to use novel screens to identify mutants of interest. However, an essential part of this effort is to identify the genes responsible for the novel phenotypes generated. While thousands of mutants already have been described, only 167 genes are listed in the zebrafish database (ZFIN) for which mutations are known (although there are probably some not listed). The major reason for this is the time consuming nature of the positional cloning strategy that must be used. The major bottleneck in gene discovery by positional cloning is the need to do whole genome linkage mapping. This takes considerable resources and time, as well as technical expertise. This goal of this proposal is to eliminate this bottleneck by setting up a high throughput mapping facility. The usual laborious task of screening sometimes hundreds of markers to find one that is linked to the mutation of interest will be accomplished in 2 days. This will be done using fluorescence sequencers to automate genotyping of SSLPs (short sequence length polymorphisms) that span the genome. The mapping will be done in two main stages. First, an approximate location will be determined using bulk segregant analyses. Second, once closely flanking markers are identified 500-1000 individual mutant embryos will be genotyped to identify the region containing the mutant gene. Third, for a small number of mutants the genes will be identified. The core will map a minimum of 100 mutants per year and identify the gene in 5-10, although both these numbers are likely to increase dramatically once the genome sequence is completed. The service will be modular so that investigators can have the initial mapping phases done by the facility, and then complete the mutant gene isolation in there own laboratories. This project will have considerable impact because it will greatly accelerate the discovery of genes that already have been shown to be models of human diseases. The facility is committed to full and open access and all data pertaining to mapping will be made available at publication. Given the importance of zebrafish as a model organism this facility will have an immediate impact on the rate at which mutant genes are discovered, which will provide new insights into the genetic underpinnings of important biochemical pathways involved in disease. [unreadable] [unreadable]

Abstract Congenital stationary night blindness (CSNB) is the clinical term for non-progressive retinal disorders that impair rod-mediated vision. The complete form of CSNB (cCSNB) is caused by defects in depolarizing bipolar cell (DBC) signal transduction and in patients has been linked to mutations in NYX, GRM6 or TRPM1. Because the flow of all visual input is transferred from the outer to the inner retina via bipolar cells, it is critical to understand the mechanism of signal transduction in DBCs. Recent work has defined several, but not all, players in this cascade. In this project, we will identify another key protein. In Aim 1, we will identify the gene and mutation that underlies a new mouse model of DBC dysfunction, nob5. The nob5 gene locus is distinct from all other known models of DBC dysfunction and therefore its identification will add another protein to those known to be critical for DBC function. These studies will use next generation sequencing and positional cloning to map and clone the gene responsible for the nob5 phenotype. In Aim 2, we will define the nob5 phenotype with respect to retinal function, using electroretinography and whole-cell patch clamp recordings from rod and cone DBCs and cone hyperpolarizing bipolar cells, and the morphology of the synapses between photoreceptors and DBCs, using confocal microscopy and immunohistochemistry. At the completion of this project, we will have identified a new protein that is required for normal DBC function and which can be used to screen patients with cCSNB.

Retinal ganglion cells (GCs) integrate excitatory and inhibitory inputs and perform computations that encode diverse features of the visual scene. The output of these functionally and morphologically diverse GCs establish visual signaling streams, maintained throughout the visual pathway. Ultimately, they create our perception of the size, shape and color of objects and their relationships in space. They establish relationships of objects in time, so that we know when it is stationary or moving, and when moving, its direction and velocity. To this end, there is a general division of labor between the retina's inhibitory neurotransmitter systems. GABA, and its receptors, modulate spatial vision, whereas glycine, and its receptors, modulate temporal vision. There are five glycine receptor subunits (one ? and 4?s). The GlyR? subunits combine with a single ? subunit to make functional receptors with diverse biophysical properties. Our work shows that all GCs expresses one or more GlyR??s and the composition differs across GC type. We hypothesize that variety enhances the diversity of inhibitory functions across and within GC types and are used to encode the visual scene. However, the specific function of GlyR? subunits is almost completely unknown. We know that the responses (and transmitter release) of the GABA and glycinergic amacrine cells presynaptic to GCs are modulated by other glycinergic amacrine cell input. This means that until GlyRs can be selectively eliminated in GCs or ACs, we cannot disambiguate the role of direct GlyR? inhibition to GCs from GlyR? modulation in the upstream circuit that provides input to the GCs. We developed a novel AAV-shRNA knockdown (KD) approach that eliminates expression of individual G lyR? subunits in GCs, while leaving their upstream expression intact. We propose to use this approach to define the role of GlyR? direct inhibition in 7 identified GC types and by extension the role of isolated GABA inputs in those same GCs. In Aim 1 we ask if a single GlyR?, GlyR?1 expressed by the 4 functionally diverse ?GCs modulates the same or different aspects of their visual responses. In aim 2 we ask if two different GlyR? subunits, expressed in the same GC, increase the diversity of inhibition to modulate different aspects of the visual response in a single GC type. We use electrophysiological assays to characterize the spiking responses of the GCs, underlying currents and the relationships between the kinetics of excitatory and inhibitory input with the postsynaptic response. This proposal addresses the novel concept: that diverse glycine subunit specific inhibition interacts with presynaptic inputs to controls visual computation.