Joshua Dunaief, MD/PhD - US grants

Age related macular degeneration, the most common cause of blindness among people aged 50 and older in the United States, results in photoreceptor degeneration. Similarly, retinitis pigmentosa, retinal detachment and ischemic diabetic retinopathy all lead to photoreceptor death. The loss of photoreceptors is the ultimate cause of significant visual loss. The mechanism of photoreceptor degeneration in these diseases is poorly understood, but is known to occur through apoptosis. This programmed cell death is a highly ordered and regulated cellular suicide pathway that has been well defined in lymphocytes. This application proposes to draw from the rich knowledge of apoptosis in lymphocytes to elucidate mechanisms of photoreceptor cell death in the photic injury animal model of retinal degeneration. This model has been studied extensively at the cellular but not yet at the molecular level. Good evidence suggests that photoreceptor degeneration in this model occurs through apoptosis. The ability of anti-apoptotic genes expressed in transgenic mice to inhibit photic injury induced cell death will be tested. Specifically, the ability of anti-oxidant genes and anti-apoptosis genes that act upstream or downstream in apoptosis pathways to inhibit photoreceptor degeneration will be evaluated. Further, the intracellular localization of cytochrome c, a mediator of apoptosis, and the role of caspase activation will be probed. Caspases are proteases involved in a number of apoptotic pathways. These studies will define critical apoptotic pathways and suggest therapeutic interventions for the blinding disorders that result from photoreceptor degeneration. The proposed study is well within the realm of feasibility. The principal investigator has experience with molecular biology, transgenic mice and retinal histology. The mentor is an international leader in the molecular mechanisms of apoptosis, and the co- mentor has extensive experience with transgenic mouse models of retinal disease and gene therapy. This proposal should serve as a good launching pad for the PI's career devoted to understanding the molecular basis of photoreceptor degeneration.

Iron is necessary in the retina for oxidative phosphorylation, membrane biogenesis and retinol isomerization, but can also produce oxidative stress if improperly regulated, leading to cell death. This can contribute to retinal disease as follows: 1) Iron toxicity causes rapid retinal degeneration following direct entry of iron into the eye carried by an intraocular foreign body. 2) Human AMD retinas have more iron than age-matched controls, suggesting that iron overload may play a role in AMD pathogenesis. 3) Inherited defects in the ferroxidase ceruloplasmin (Cp) result in retinal iron accumulation and early onset macular degeneration. 4) Mice with mutation in Cp and its homolog hephaestin (Heph) have an age-dependent retinal iron overload and degeneration with a number of features similar to AMD, including subretinal neovascularization. The latter two points indicate that Cp and Heph are important for retinal health. Evidence from other organs suggests that Cp or Heph can cooperate with the plasma membrane iron transporter ferroportin (Fpn) to export iron from cells. The goal of this proposal is to increase understanding of the roles of Cp, Heph and Fpn in retinal iron homeostasis and their regulation by the secreted hormone hepcidin (Hepc). Hepc is produced in the retina (as well as the liver) and triggers internalization and degradation of Fpn. Hepc may serve as a message from retinal cells sensing iron excess (such as photoreceptors) to degrade Fpn and limit iron transfer from RPE and Muller cells. Our existing Cp/Heph double mutant and Hepc-/- mice indicate that these three proteins are critical for retinal iron homeostasis and health, but provide little information about the specific functions of the proteins within the retina. Conditional mouse knockout technology (lox/cre) affords the opportunity to determine how these proteins function within specific retinal cell types and how intercellular iron transfer is executed and regulated. In Aim1, the photoreceptor-specific functions of Heph, a possible iron release valve to prevent PR iron overload will be investigated using a Heph conditional knockout on a Cp-/- background. In Aim 2, the iron transport function of Fpn will be investigated using RPE and photoreceptor-specific conditional knockout mice. In Aim 3, the retinal function of Hepc will be investigated in knockout and conditional knockout mice. These studies are important because: 1) They will provide new information about the cell-type specific functions of Heph, Fpn and Hepc and the routes of intercellular iron transfer that control retinal iron homeostasis. 2) The conditional knockout mice are likely to provide models for several features of AMD, including subretinal neovascularization while avoiding the lifespan-limiting brain iron overload in our existing Cp/Heph double mutant mice.

DESCRIPTION (provided by applicant): Oxidative stress is implicated in a wide variety of diseases, including but not limited to age-related macular degeneration (AMD), diabetes, atherosclerosis, aging, and neurodegeneration. The biochemical markers of oxidative stress, including lipid peroxidation, DNA base hydroxylation, and protein modification, result primarily from reaction with the highly reactive hydroxyl radical, OH7, itself a product of iron-catalyzed reactions, with oxygen species. While iron is an essential and beneficial component of healthy cells, this deleterious reactivity suggests that any free iron in the cell will cause significant damage. Inhibiting metal-promoted oxidative stress is therefore a promising strategy for treating a number of diseases, especially those where normal metal ion homeostasis is impaired or where aberrant metal accumulation is documented, as is the case for AMD. Generalized iron chelation could protect against oxidative damage, but could also be detrimental if iron needed for metabolic processes is removed. We are designing chelating agents (prochelators) that are active only when needed: they selectively sequester and inactivate iron only in cells undergoing oxidative stress, thereby limiting oxidative damage in these cells. These chelators are chemical modifications of salicylaldehyde isonicotinoyl hydrazone (SIH), which we have found able to protect cultured retinal pigment epithelial cells (RPE) from death induced by a wide variety of insults. We propose herein to test in RPE cells the ability of several modified SIH molecules to decrease the labile iron pool selectively when cells are experiencing oxidative stress, and to determine whether the chelators increase cell viability. Preliminary results suggest that these prochelators are activated by oxidative stress both in the test tube and in RPE cells, and that they can protect cultured RPE cells from oxidant induced death. The goal of this proposal is to identify prochelators with the optimal characteristics: maximal RPE protection with minimal toxicity. These studies will lay the groundwork for future prochelator testing in mouse retinal degeneration models. It is hoped that these studies will lead to a new preventative agent for patients with early AMD.

PROJECT SUMMARY Iron plays a critical role in both the healthy and diseased retina. The long term goals of the proposed studies are to understand regulation of retinal iron flux, determine why iron accumulates in retinal disease, and discover how to protect against retina iron toxicity. Iron is necessary in the retina for oxidative phosphorylation, membrane biogenesis and retinol isomerization, but becomes a central producer of oxidative stress when improperly regulated. Iron toxicity is evident in retinal disease as follows: 1) Iron causes rapid retinal degeneration following entry into the eye carried by an intraocular foreign body. 2) Human AMD retinas have more iron than age-matched controls, suggesting that iron overload may play a role in AMD pathogenesis. 3) Consistent with this hypothesis, in the inherited disease aceruloplasminemia, loss of the ferroxidase ceruloplasmin (Cp) results in retinal iron accumulation and early onset macular degeneration. 4) Mice with knockout for Cp and its homolog hephaestin (Heph) have an age-dependent retinal iron overload and degeneration sharing features of AMD, including complement activation and subretinal neovascularization. The latter two points indicate that Cp and Heph are important for retinal health. Evidence from other organs suggests that Cp or Heph can cooperate with the plasma membrane iron transporter ferroportin (Fpn) to export iron from cells. Progress from the prior funding period indicates that Fpn is expressed on the ablumenal side of retinal vascular endothelial cells, Muller cells, and the basolateral membrane of the RPE. Since Fpn is the only known cellular iron exporter, this expression pattern suggests the route of iron flux. Mice with a mutated Fpn that is resistant to degradation triggered by the iron regulatory hormone Hepc, have retinal iron accumulation. These results suggest a local iron-regulatory axis within the retina mediated by Fpn and Hepc. Experiments proposed herein will utilize retinal cell type specific knockouts of Fpn and Hepc to determine the route of Fpn-mediated retinal iron flux and evaluate its regulation by Hepc. AMD and control retinas will be analyzed to determine whether Hepc/Fpn dysregulation contributes to the documented iron accumulation in AMD retinas. The role of serum iron levels versus local control of iron influx into the retina will be determined. The outcome will help focus AMD-iron clinical studies on either serum iron levels or local iron regulatory mechanisms within the retina.

PROJECT SUMMARY Iron plays a critical role in both the healthy and diseased retina. The long term goals of the proposed studies are to understand how inflammation associated with retinal disease promotes iron dysregulation, and discover how to protect against retinal iron toxicity. Iron is necessary in the retina for mitochondrial energy production, membrane synthesis and the visual cycle, but becomes a central producer of oxidative stress when improperly regulated. Iron toxicity is evident in retinal disease as follows: 1) Iron causes rapid retinal degeneration following entry into the eye carried by an intraocular foreign body. 2) Human AMD retinas have more iron than age-matched controls, suggesting that iron overload may play a role in AMD pathogenesis. 3) Consistent with this hypothesis, in the inherited disease aceruloplasminemia, loss of the ferroxidase ceruloplasmin (Cp) results in retinal iron accumulation and early onset macular degeneration. 4) Mice with knockout for Cp and its homolog hephaestin (Heph) have an age-dependent retinal iron overload and degeneration sharing features of AMD, including complement activation and subretinal neovascularization (which causes wet AMD). Recent evidence from non-ocular cells indicates that interleukin 6 (IL-6) can trigger a ?cellular iron sequestration response? to prevent microbes from accessing the iron they need for growth and replication. This, combined with the finding of elevated intraocular and serum IL-6 levels in AMD, suggest that IL-6 may promote retinal iron overload in AMD. Experiments proposed herein will utilize human iPSC-RPE cell culture, mouse models, and post mortem human tissue to define the IL-6 retinal iron sequestration response. An inhibitor of the IL-6 trans-signaling pathway will be tested. Following activation or inhibition of IL-6 pathways, changes in the levels of iron and its transporters will be assessed. These changes will be correlated with those in AMD versus normal post mortem retinas. It is expected that these studies will lead to development of novel anti-AMD approaches that diminish inflammation-induced iron accumulation.

1. Project Summary / Abstract The Penn Vision Clinical Scientist Program (VCSP) is designed to prepare clinician scientists to identify and prioritize important questions in vision research, formulate a comprehensive approach to address the questions, and develop skills to effectively lead the efforts of a research team to provide answers. The (VCSP is centered in the Department of Ophthalmology, School of Medicine. Candidates for the VCSP hold a clinical doctoral degree (MD, PhD, DO, OD, DVM or equivalent) and have completed their clinical training, usually through the fellowship level. Scholars initially engage in educational and research activities to lay the groundwork for submission after 2 years of an application for an independent K08, K23, or R01. Scholars are supported on the K12 for up to 3 years, or until award of their individual grant, whichever comes first. Up to 2 scholars are supported on the K12 at any given time. The program takes advantage of established educational programs within the University, the concentration of strong basic science, translational research, and patient- oriented research programs ongoing within the Department of Ophthalmology, and the breadth of expertise available through Penn's interdisciplinary institutes and centers. Scholars have access to formal educational programs and applied research experiences in a vast array of areas such as clinical epidemiology, single-center and multi-center clinical trials, health services research, bioethics, genetics, molecular biology, and neuroscience. Established investigators in basic science, translational research, and patient-oriented research serve as mentors to guide choices by scholars in educational programs and research projects. The Penn VCSP has two defined tracks, translational research and patient oriented research. Each track has a didactic training component and a component of supervised research with increasing independence. However, the specific content of each program is custom built for each scholar. Each scholar has a primary mentor and a mentoring team to advise on the scientific aspects of their research and on activities necessary for professional development, collaboration among investigators, and developing long-term research programs. Upon completion of the program, scholars have received superb training to become productive and successful independent researchers.