Harris Ripps - US grants

DESCRIPTION (provided by applicant): The vertebrate retina is a highly ordered neuronal network in which electrical coupling through gap junctions influences every aspect of retinal function. Each of its various cell types make gap junctions with neighboring cells, and intercellular junctional communication is profoundly affected during light- and dark-adaptation by virtue of concomitant changes in the extracellular concentrations of ions, retinoids, and neurotransmitters. Many new retinal connexins capable of forming gap-junction channels have been cloned, and the discovery of hemi-junctional channels provides a new tool with which to investigate the pharmacological properties of the gap junction on retinal cells. Considering the ever-increasing number of human diseases associated with connexin mutations, it is highly likely that many retinal disorders of unknown origin will ultimately be linked to aberrations in the molecular structure of the connexins expressed in retinal neurons and glia. Clearly, it is important to gain a better understanding of the connexins expressed in retinal cells, their functional attributes, and their essential role in the complex microcircuitry of the vertebrate retina. The principal goals of the present application are to use molecular, electrophysiological, and imaging techniques to identify the connexins used by retinal neurons and glia, and to characterize the electrical and pharmacological properties of their gap-junctional channels and hemichannels in retinal neurons, in Xenopus oocytes, and in transfected cells. Single-cell PCR techniques will be used to identify the connexin content of subclasses of retinal neurons, and experiments will be performed to examine electrical coupling between native retinal cells and transfected cell lines expressing known retinal connexins. Chemical- and voltage-gating of neuronal hemichannels will be compared with the hemichannel properties of oocytes expressing retinal connexins. Domain swapping and site-directed mutagenesis will be used to determine regions of the connexin sequence that govern the formation of hemichannels. In addition, the assembly of connexin subunits in the formation of gap-junctional channels will be studied, and channel modulation by retinoic acid, pH, second messengers, and other agents will be investigated. The information gained from these studies, and on the photic regulation of connexin expression, will afford a better understanding of how gap-junction channels are regulated physiologically, provide insights into the identity of the connexins expressed by specific retinal cell types, and help to elucidate the role of direct cell-cell communication in neural function.

DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) constitutes a group of genetically-mediated, degenerative retinal diseases that display a broad range of phenotypes. There is appreciable heterogeneity in the pathologies that underlie the various forms of RP, but of the known cases, a substantial percentage arise as a consequence of mutations in rhodopsin or other rod-specific proteins. However, despite the fact that the genetic defect is expressed solely in the rod photoreceptors, otherwise healthy cone photoreceptors invariably die, resulting in severe visual impairment. The principal goal of this proposal is to test the hypothesis that the spread of the disease from dying rods to genetically normal cones is a form of "bystander" effect, mediated by the gap junctions that exist between these photoreceptor subtypes. On this view, agents that trigger the apoptotic process permeate the intercellular gap-junctional channels to carry proapoptotic signals from diseased rods to neighboring cones. If gap junctions are a significant factor in the non-cell-autonomous spread of photoreceptor degeneration, blocking transmission through these channels may provide a novel form of therapeutic intervention that would enable cones to be spared the fate of their neighbors. The experiments we plan to conduct will help to determine whether the cone photoreceptors of transgenic mice that express rod-specific gene defects exhibit a significantly higher survival when the gap-junctional protein (connexin36) that couples their rods and cones is disrupted. Cx36 "knockout" mice are available, and we propose to cross them with mice expressing various rod-specific mutations that result in rod-cone dystrophy. Cone structure and function will be assessed by a panel of histological, immunocytochemical, and electrophysiological methods. In addition, we plan to conduct a series of molecular biological and biochemical studies to examine further connexin expression in mammalian photoreceptors, and to identify proapoptotic agents that are able to permeate the intercellular channels.