This continuing project is the only one, to the PI's knowledge, which is aimed at understanding the mechanism by which the 70,000 Dalton (70 kDa) heat shock protein (hsp70), when administered by injection into the eye, interacts with the retina to protect photoreceptors from light damage. This information will be instrumental in elucidating methods by which hsp70 can be used to reduce or prevent retinal cell death caused by injury or disease. Hsp70 is one of a group of about eight proteins which have in common the property that they are synthesized in greater amounts in cells that are under metabolic stress. Metabolic stress in this context refers to a variety of conditions that pose a threat to a cell's ability to maintain its normal internal environment. An increase in a cell's hsp content, in particular, the pair of closely related 70 kDa hsps, can dramatically improve its changes of surviving an acute metabolic stress. This cytoprotective action of hsp70 is thought to be based on its ability to inhibit denaturation of other vital cytoplasmic and nuclear proteins. Hsp70 is a normal constituent of the frog, rat, and human retina. In the rat retina, elevation of hsp70 content induced by artificial fever correlates with a marked increase in photoreceptor resistance to light damage. Furthermore, this increased resistance to light damage also can be produced by direct administration of purified hsp70 into the vitreous humor of the eye. To determine how to best utilize the cytoprotective action of hsp70, we will examine first the way in which exogenous hsp70 administered by intravitreal injection interacts with the photoreceptors. A variety of approaches will be employed using the rat eye in vivo. The optimal conditions for uptake will be determined, as well as the fate of the added hsp70. For example, several of the proposed studies will examine whether hsp70 interacts with the plasma membrane of retinal cells or is internalized by them. If it is taken into the cells its intracellular distribution will be evaluated. The information obtained from these studies will be applied to subsequent investigations of the ability hsp70 injected into the vitreous of the rat eye to prevent light- induced photoreceptor degeneration. Light exposure in the albino rat is a convenient, noninvasive method to produce controlled levels of damage to the photoreceptors and is considered to be a general model for studying factors involved in photoreceptor degeneration. In addition to hsp70, however, other natural cellular constituents, like vitamin C (ascorbic), are known to protect photoreceptors from light damage as well. Since the best protection of photoreceptor against damage is likely to involve multiple cell defense systems, the combined effects of hsp70 and ascorbic acid administration on photoreceptor sensitivity to light damage also will be examined. As a further inquiry into the multiple defense mechanism hypothesis, the distribution of four other well-known hsps with molecular weights of 28, 60, 78, and 90 kiloDaltons, respectively, will be evaluated by immunohistochemistry within the normal and hyperthermically stressed rat retina. Taken together the multiple facets of this project have high potential to provide the ophthalmologist with a new approach to prevent loss of vision caused by retinal injury or disease.
