Cheryl M. Craft - US grants

The major goal of this project is to define the structure of the retinal/pineal gene, N-acetyltransferase (NAT) and to examine NAT gene expression in visual and neural transduction. The primary objective of this five year research proposal is to answer the question: Is the retinal/pineal NAT enzyme a key component of a circadian driving oscillator and is the retinal/pineal NAT enzyme controlled by environmental cues at the molecular level? This project will investigate one specific phase of this endogenous rhythm and the entrainment by the light:dark cycle of the rate=limiting enzyme that synthesizes melatonin. The pineal is an excellent system to study all steps in neurochemical transduction, including ligand-receptor interactions, second messenger regulation, and selective regulation of specific genes. Two characteristics for studying molecular mechanisms of pineal neurotransduction are (1) innervation by sympathetic fibers involving alpha- and beta-receptors; and (2) high amplitude circadian rhythms in biochemical activities, including some that are rapidly responsive to known molecular triggers. Although the retina shares similar proteins and biochemical pathways with the pineal, the role of melatonin in the retina is not understood. The experiments will characterize the retinal/pineal tissue-specific genes coding for NAT. Normal tissue-specific expression will be analyzed in retinas and pineals of rats and inbred mice. Primary pineal cell cultures will also be used to study the role adrenergic regulation has in gene expression. The project will have three specific aims: 1. Isolate full-length cDNAs and characterize cDNA probes encoding the retinal/pineal enzyme, NAT, by screening a rat pineal expression library. 2. Generate antisera for immunological probes against recombinant fusion proteins and synthesized peptides identified from the translated cDNAs encoding NAT. 3. Apply these recombinant and immunological probes to localize, characterize and measure changes in circadian regulation and environmental modulation of their mRNA and protein for NAT. The experiments described will employ immunological and recombinant DNA tools to examine the influence of environmental cues on the genes expressed in the pineal and retina. The pineal and retina have been extensively studied by physiological, pharmacological and biochemical approaches. These studies will extend these studies concerning the fundamental molecular nature of circadian rhythms in the nervous system. Biological circadian rhythms are important not only in our daily lives, but in maintaining our health throughout life. Knowledge of the normal structure of genes expressed in the pineal and retina may lead to a better understanding the involvement of the melatonin synthesizing enzyme in visual and neural transduction.

[unreadable] DESCRIPTION (provided by applicant): Both rod and cone photoreceptors absorb light, triggering an amplification cascade, which produces membrane hyperpolarization through closure of selective ion channels. Radiating from the light-initiated event is a G-protein-coupled response of secondary activities, including rhodopsin or cone opsin receptor shut-off through a GRK1 phosphorylation and subsequent binding of either rod or cone arrestin. Cone photoreceptors are distinct from rods in morphology, light sensitivity, recovery rate, thermal stability, timing of outer segment shedding and resistance to programmed cell death by apoptosis. Characterization of the dynamic interactions and functions of these cone gene and their gene products may provide a basis for diagnosis, treatment or prevention of age related macular degeneration and other retinal rod and cone degenerations, thus preserving vision for currently untreatable forms of blindness. To address the distinct aspects inherent to the cone photo-transduction pathway and to accomplish our goals, experiments are designed to explore the function(s) of cone arrestin (CAR), its targeted G protein-coupled receptors (S and M opsin pigments) and other potential relevant partners in the cone synapse. Our working hypothesis, based in part on our ongoing biochemical and electrophysiological studies, support a role for CAR in regulating cone photo-transduction through binding to light-activated, GRK1 phosphorylated S and M opsins. We propose that when this X-chromosomal gene encoding CAR is genetically deleted with mouse knockout (KO) technology, a defective receptor shut-off will lead to a delayed recovery of cone photoresponses. To test this hypothesis, the specific aims and experimental design include 1) characterization of the morphological, biochemical and electrophysiological retinal phenotypes of the newly generated Car KO. Further experiments will explore these parameters in Grk1/Car double KO mice on two genetic backgrounds (transducin alpha -/- with normal rod morphology but no rod response and Nrl -/- with pure cone retina) compared to wildtype; 2) examine the effects of Grk1 S and M opsin phosphorylation and CAR binding on the cone visual retinoid cycle pathway; and 3) identification of other potential interacting cone synaptic partners for CAR and its alternatively spliced isoforms. Studies of the photo-transduction cascade and the molecular triggers for initiation and termination of high acuity vision are vital for sustaining lifelong vision. [unreadable] [unreadable]