Jian-xing Ma - US grants

In a new application, an young investigator requests five years of support for studies of RPE65, an abundant microsomal associated protein. The major goal of this proposal is to determine if RPE-65 is the retinol isomerase, and if so, to characterize it's activity. Specific Aim 1 is to express human RPE-65 and determine the role of the recombinant protein in retinoid processing. Specific Aim 2 is to determine the amino acids in RPE-65 that are critical for its subcellular localization and retinoid processing activity. Specific Aim 3 is to clone salamander RPE-65 and determine its cellular localization. Specific Aim 4 is to examine the retinoid profile of RPE-65 in vivo using RPE-65 knockout mice that have been generated by a collaborator.

Kallistatin, a serine protease inhibitor (PI), inhibits kallikrein and kinin production and growth of human retinal capillary endothelial cells (RCEC). KS levels are decreased in retina, serum and vitreous of Db patients and in STZ-rats. The hypothesis formulated herein is that in diabetes, low KS leads to a weakened growth inhibition of EC in the retina, indirectly enhancing neo-vascularization. Aims are: 1: identify the causes and mechanisms of low KS in DM by testing the effects of high glucose, insulin and hypoxia on KS production in RCEC and hepatocytes, 2: determine if KS effects on cell growth occur via the kallikrein-kinin system or through a KS-specific receptor or other growth factors. These effects will be tested in the presence or absence of a kinin receptor antagonist. KS-R will be defined on RCEC and the effect of KS on the expression of growth factors will be examined. 3: Retinal neovascularization, induced by hyperoxia in KS-transgenic mice will help determine whether overexpression of KS can reduce or prevent retinal NV in these mice. Thus, KS will be examined with respect to its effects as an endothelial growth inhibitor. The mechanisms of KS regulation may provide evidence of new pathogenic factors in DR.

PROJECT SUMMARY/ABSTRACT Visual pigments consist of an apoprotein opsin and a chromophore, 11-cis retinal. Light-induced isomerization of 11-cis retinal to all-trans retinal initiates vision. Efficient regeneration of 11-cis retinal is essential for normal vision. The retinoid visual cycle refers to a multi-step process to regenerate 11-cis retinal. A key step in the visual cycle is the conversion of all-trans retinyl ester into 11-cis retinol, catalyzed by retinoid isomerase in the RPE. Previously, we identified RPE65 as the retinoid isomerase. Multiple recessive RPE65 mutations are known to cause retinal dystrophies. Recent genetic studies identified the first dominant point mutation of RPE65, D477G, associating with retinitis pigmentosa (RP) in human patients. Unlike the recessive mutations, the patients carrying a single copy of D477G develop progressive vision loss, suggesting a novel and yet to be understood mechanism for retinal dystrophy. This project will elucidate the pathogenic mechanism by which D477G impairs vision. We have recently generated a heterozygous D477G knock-in (KI) mouse. D477G KI mice displayed delayed dark-adaptation and decreased 11-cis retinal regeneration, suggesting that D477G may function as a dominant negative mutant that interferes with the isomerase activity of WT RPE65 and impairs the visual cycle. We will investigate the impacts of D477G KI on the visual cycle and retinal structure and function. We will also elucidate the mechanism by which D477G disturbs the visual cycle. We will investigate if D477G protein may physically interact with WT RPE65 and affect its conformation, leading to decreased enzymatic activity or stability of WT RPE65. These studies will not only reveal a new pathogenic mechanism for inherited retinal dystrophies, but also contribute to the understanding of structure and function of RPE65 and the visual cycle. Diabetic retinopathy (DR) is a major blinding disorder. The implication of the visual cycle in DR has not been investigated. Our preliminary studies demonstrated that generation of 11-cis retinal and visual pigments is deficient in diabetic rats, while opsin levels are unchanged. The consequent increase of free opsin may contribute to photoreceptor degeneration and visual impairment in DR. Further, we found that a visual cycle protein, interphotoreceptor retinoid-binding protein (IRBP) is down-regulated in diabetic retinas. An objective of this project is to investigate the causative role of the visual cycle dysfunction in visual deficits in early DR. We will determine if administration of a chromophore to diabetic mice can ameliorate vison loss in early DR. We will also induce diabetes in mice with ablation or over-expression of IRBP to evaluate the impacts of IRBP down-regulation on the impaired rhodopsin regeneration and visual deficits induced by diabetes. This study will explore an undocumented association of a disturbed visual cycle with DR and has potential to identify a new therapeutic strategy for DR.

DESCRIPTION (provided by applicant): This is a R21/R33 phase-combined proposal aiming to develop a new treatment for diabetic macular edema using peptide angiogenic inhibitors. Vascular leakage is an early feature of diabetic retinopathy and can result in diabetic macular edema. Over-expression of VEGF is a major causative factor leading to vascular leakage in diabetic retinopathy. Currently, there is no satisfactory treatment for macular edema which remains a major cause of vision loss in diabetic patients. Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. Our recent studies have shown that K5 significantly decreases vascular leakage in the retina in the experimental diabetes, laser-induced choroid neovascularization and oxygen-induced retinopathy rat models. The K5- induced reduction of vascular leakage requires only less than one-tenth of the dose needed for the inhibition of neovascularization. Furthermore, our preliminary data suggest that the K5-induced reduction of vascular leakage may be through blocking hypoxia-induced VEGF over-expression in the retina, primarily in Muller cells. We hypothesize that a sustained ocular delivery of K5 may induce a long-term reduction of vascular leakage in diabetic retina and thus, may have therapeutic effect on cyctoid macular edema (CME) secondary to cataract surgery and diabetic macular edema. In the R21 phase, we propose to first reveal the mechanisms for the K5-induced down-regulation of VEGF expression and identify the receptor or binding protein on the cell surface which mediates the K.5-induced reduction of permeability. As diabetic macular edema is a chronic complication of diabetes and requires a long-term treatment, we propose to develop a KS-polymer pellet to achieve a sustained release of K5. The ocular delivery routes of the K5 pellet will be optimized and the pharmacokinetics will be studied in rats. The long-term effect of the K5 pellet on vascular leakage will be determined in a diabetic rat model. The R21 phase will achieve the following goals: 1),to reveal the mechanism and identify the receptor mediating the K5 action, 2) to develop a sustained delivery system for K5 and 3) to prove the concept that a sustained delivery of K5 can induce a prolonged reduction of vascular leakage, The R21 phase will provide essential tools and information for starting the R33 phase. In the R33 phase, we will study the pharmacokinetics of K5 in ocular tissues and optimize the delivery route in normal dogs, With the optimized delivery route, the efficacy of K5 on reduction of vascular leakage will be confirmed in a dog model of vascular leakage induced by intravitreal injection of IGF-1. The possible toxicity of K5 to the retinal vasculature and retinal structure will be examined in both rats and dogs by histochemistry. The retinal function will be examined by ERG recoding. Although this project does not reach clinical trials, the proposed studies will obtain pre-clinical data such as pharmacokinetics, delivery route, efficacy and toxicity from more than one species, which are essential and useful for starting clinical trials. These studies will contribute to the development of a new treatment for CME and for diabetic macular edema. This new treatment will use natural human peptides and will be less invasive. This new therapy, if successful, can prevent vision loss from macular edema in diabetic patients.

SUMMARY Oklahoma is one of the states in the US with the highest prevalence of diabetes, with 10% of the population affected. Diabetes is particularly prevalent among Native American communities, where it affects up to 40% of older individuals in some tribes. Thus, diabetes and its complications constitute a major threat to the working- age and older population, and confer an immense social and economic burden on Oklahoma. The goal of this COBRE is to enhance diabetes research in Oklahoma. In Phase I and Phase II, our COBRE achieved its milestones. All of the COBRE-supported Junior Investigators have received independent grants, including eight NIH R01 grants, two R21 grants, and multiple grants from foundations such as the American Diabetes Association and the American Heart Association. Five of our Junior Investigators have been promoted to Associate Professor, and three of them have received tenure thus far. In addition, because of their success in research, two of them received endowed chair positions. Between them, the Junior Investigators have published 207 peer-reviewed papers, 135 of which received support from COBRE-funded Cores. In collaboration with the College of Medicine, the COBRE has recruited three NIH R01-funded and 1 K99/R00-funded diabetes researchers into The University of Oklahoma Health Sciences Center (OUHSC). The COBRE-funded Cores have provided support and service to multiple PIs and supported their publications and NIH-funded grants, which enhanced diabetes research in Oklahoma. In Phase III of the COBRE program, we plan to sustain and augment these successes. We will continue supporting and mentoring Junior Investigators who graduated from the COBRE. We will expand and improve Core facilities to serve diabetes research in Oklahoma and complete transition of the COBRE-funded facilities into institutional research Cores. We will further increase the critical mass of diabetes research in Oklahoma by promoting recruitment of diabetes researchers into OUHSC. Moreover, we will continue promoting collaborative and translational research. These efforts should contribute to continuous growth of diabetes research to achieve our goal of becoming a NIH P30-funded Diabetes Research Center (DRC). This Center should greatly improve prevention and treatment of diabetes.

DESCRIPTION (provided by applicant): Choroidal neovascularization (CNV) is a severe complication of age-related macular degeneration (AMD). Human genetic studies have recently revealed significant association of the very low-density lipoprotein receptor (VLDLR) and a wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6) genes with AMD in human patients. Our recent studies showed that VLDLR knockout mice develop typical CNV, and manifest most abnormalities of human AMD such as retinal inflammation, vascular leakage, and impaired cone ERG and cone degeneration. Wnts are a group of secreted, cystine-rich glycoproteins which bind to frizzled (Fz) receptors or to a co-receptor complex consisting of Fz and LRP5 or LRP6 (LRP5/6) and regulate expression of target genes, such as VEGF. The wnt signaling is known to mediate multiple biological functions including angiogenesis. However, the role of the wnt pathway in CNV has not been investigated. Our preliminary studies have provided the following evidence suggesting a pathogenic role of VLDLR and the wnt pathway in CNV: 1) LRP5/6 expression is significantly up-regulated in Vldlr-/- eyecups. 2) The down-stream effectors of the wnt pathway, glycogen synthase kinase-32 (GSK- 32) and 2-catenin, are both activated in Vldlr-/- eyecups. 3) VEGF expression is up-regulated in Vldlr-/- eyecups. 4) In cultured endothelial cells, down-regulation of VLDLR by siRNA activates the wnt signaling and VEGF over-expression. 5) DKK1, a specific inhibitor of the wnt pathway blocks the VEGF over- expression induced by the VLDLR siRNA and in the RPE of Vldlr-/- mice. The central hypothesis of this project is that VLDLR functions as a negative regulator of CNV, and the regulatory effect of VLDLR is through the wnt pathway. In this project, we will first determine if the RPE-derived VEGF over-expression in Vldlr-/- mice is essential for the development of CNV. We will generate VLDLR/VEGF double knockout (KO) mice by crossing the RPE-specific VEGF conditional KO mice with Vldlr-/- mice, and determine if the VEGF KO in the RPE attenuates CNV in Vldlr-/- mice. We will also investigate if the VEGF over-expression induced by VLDLR KO is through HIF-1 using RPE-specific HIF-11/VLDLR double KO mice. Second, we will test the hypothesis that the CNV in Vldlr-/- mice is mediated by the wnt pathway. The activation of the 2-catenin will be determined in primary RPE and retinal endothelial cells from Vldlr-/- mice. We will investigate if inhibition of the wnt pathway by DKK1 will prevent or alleviate CNV in Vldlr-/- mice. Further, constitutively active mutants of LRP5 and LRP6 will be expressed in the RPE of transgenic mice to determine if over-activation of the wnt pathway alone will induce CNV. We will determine whether LRP5 or LRP6 mediates the CNV in Vldlr-/- mice by generating LRP5/VLDLR double KO mice. Third, we will investigate how VLDLR regulates LRP5/6 gene expression. Two postulated mechanisms will be tested: 1) VLDLR and LRP5/6 may compete for binding with the same ligand. When VLDLR is deficient, more ligand could bind to LRP5/6 and induce the expression of the receptors. 2) VLDLR may interact with an intracellular signaling pathway, through which VLDLR down-regulates LRP5/6 gene transcription. We will delete the intracellular domain and the extracellular ligand-binding domain of VLDLR and determine if the ligand binding domain or the intracellular domain interacting with signaling pathways is essential for its regulatory effect on LRP5/6 expression. If the ligand binding domain is essential, we will identify the ligand binding to both VLDLR and LRP. On the other hand, if the intracellular domain is essential, we will identify the signaling pathway which could interact with VLDLR. The role of the VLDL-inducible factor-1 and DAB-1/Src kinase pathways will be investigated as the first candidates. This project will establish VLDLR as a new member of the wnt pathway and establish the roles of VLDLR and the wnt pathway in pathogenesis of CNV. These studies have potential to reveal a new pathogenic mechanism of CNV and a new drug target of future treatment of CNV in AMD. PUBLIC HEALTH RELEVANCE: Choroidal neovascularization is a severe complication of age-related macular degeneration and a major cause of blindness in aged population. This project aims to explore a novel pathogenic mechanism for choroidal neovascularization and to establish a new animal model of choroidal neovascularization.

PROJECT SUMMARY/ABSTRACT Diabetes-induced oxidative stress and chronic inflammation in the retina play a key pathogenic role in diabetic retinopathy (DR). Mitochondrial dysfunction and impairment have been identified as the major cause of oxidative stress and inflammation in DR. Peroxisome Proliferator-Activated Receptor ? (PPAR?) is a hormone- activated receptor and transcription factor. It is known to regulate lipid metabolism, and thus, PPAR? agonists are used clinically to treat hyperlipidemia. Recently, two independent, prospective clinical studies reported a surprising finding that oral administration of fenofibrate, a PPAR? agonist, has robust therapeutic effects on DR in type 2 diabetic patients. In the prior grant period, we have successfully demonstrated that the therapeutic effect of fenofibrate on DR is through a PPAR?-dependent mechanism. We have shown that PPAR? is down- regulated in the retinas of diabetic humans and diabetic animal models, and PPAR? has protective effects against DR. We have shown that PPAR? knockout (KO) exacerbated, while activation of PPAR? by fenofibrate alleviated retinal oxidative stress and retinal inflammation in DR models. This proposal will extend these studies and elucidate the mechanism responsible for the protective effects of PPAR?. Our preliminary studies showed that fenofibrate treatment decreased diabetes-induced acellular capillary formation and pericyte loss in the retina. Further, Seahorse analysis showed that PPAR? KO resulted in mitochondrial dysfunction in the retina and primary pericytes. Further, PPAR?-/- retina showed decreased mitochondrial DNA (mtDNA) copy numbers, suggesting impaired mitochondrial biogenesis and/or DNA repair. This project will address a novel hypothesis that diabetes-induced down-regulation of PPAR? expression is responsible for, at least in part, for diabetes-induced mitochondrial dysfunction, which leads to retinal oxidative stress and inflammation in DR. We will determine if PPAR? KO exacerbates, while PPAR? over-expression alleviates, mitochondrial dysfunction (basal OCR, maximal OCR and ATP production) and mtDNA damage as well as retinal oxidative stress, leukostasis, vascular leakage, acellular capillary formation and pericyte dropout in the retina of diabetic mice. We will also determine the impacts of PPAR? deficiency in pericytes on diabetes-induced mitochondrial damage, oxidative stress and pericyte apoptosis using pericyte-specific conditional PPAR? KO mice and primary PPAR?-/- pericytes. We will also investigate if PPAR? regulates mitochondrial biogenesis and function through the SIRT1/PGC-1? pathway using pericyte-specific SIRT1 KO mice and primary SIRT1-/- pericytes. These studies will elucidate a novel pathogenic mechanism responsible for mitochondrial damage and oxidative stress in DR and reveal a new therapeutic strategy for DR. These studies will also contribute to the understanding of the mechanism underlying therapeutic effects of fenofibrate on retinal inflammation and pericyte loss in DR.

DESCRIPTION (provided by applicant): Our recent studies have demonstrated that abnormal activation of the Wnt pathway plays key roles in pathogenesis of retinal inflammation, NV and fibrosis in both AMD and diabetic retinopathy. A genetic study has reported that variants of very low-density lipoprotein receptor (VLDLR) are associated with AMD. We have recently reported that VLDLR functions as a negative regulator of the Wnt pathway, as VLDLR knockout resulted in Wnt pathway over-activation, leading to AMD-like pathologies, such as retinal inflammation, vascular leakage and sub-retinal NV. Toward the mechanism by which VLDLR regulates the Wnt pathway, we have recently obtained the following preliminary data: 1) Expression of VLDLR and its soluble extracellular domain (VLDLRN) inhibits Wnt signaling. 2) The VLDLRN peptide decreases total LRP6 protein levels. 3) Co-immunoprecipitation assay showed that VLDLRN binds with LRP6, forming a VLDLR-LRP6 heterodimer. 4) VLDLRN peptide blocks Fz-LRP6 dimerization induced by Wnt ligand. Based on these observations, we hypothesize that VLDLR forms a heterodimer with LRP6, which blocks the Wnt ligand-induced LRP6 aggregation and LRP6 signalosome formation. This binding may represent a mechanism by which VLDLR inhibits Wnt signaling. To test this hypothesis, we will determine if binding of VLDLR to LRP6 blocks the Wnt ligand-induced LRP6 aggregation and formation of signalosomes. We will also determine if binding of VLDLR to LRP6 affects LRP6 endocytosis and stability. Further, we will define the sequence domains responsible for the interaction between VLDLR and LRP6 using deletion mutants and co-immunoprecipitation assays. The therapeutic potential of the sequence domain of VLDLR binding to LRP6 and inhibiting Wnt signaling will be explored by evaluating its efficacy on retinal inflammation, vascular leakage and NV. Recently, two independent large clinical trials reported that fenofibrate, a PPAR1 agonist which lowers VLDL levels in the circulation, has therapeutic effects on retinal vascular leakage and NV in type 2 diabetic patients. Our preliminary studies found that fenofibrate inhibits Wnt signaling and up- regulates VLDLR expression and its promoter activity. Therefore, we will test the hypothesis that inhibition of Wnt signaling through up-regulation of VLDLR expression by fenofibrate represents a mechanism for its beneficial effects on retinal inflammation, vascular leakage and NV. We will use VLDLR KO mice, PPAR1 KO mice and primary RPE and endothelial cells from these KO mice to determine if VLDLR and PPAR1 are essential for mediating the Wnt-inhibiting effect of fenofibrate. These studies will elucidate the mechanism by which VLDLR regulates the Wnt pathway and identify a novel, endogenous regulatory mechanism for this important pathway. The proposed studies will reveal the interactions between PPAR1 and the canonical Wnt pathway and contribute to the development of new treatment for AMD.

The objective of the Administration, Mentoring and Recruitment Core is to assist all of the COBRE team members by providing administrative service. In the first Phase of the COBRE, the Core has been responsible for budgeting, ordering, progress report submission for the entire COBRE, and organizing meetings of the External Advisory Committee and the Internal Advisory Committee as well as meetings of COBRE Promising Junior Investigators (PJIs). This Core has substantially reduced the workload of PJIs in their administrative work and enhanced collaborations between PJIs, thus contributing to the overall success of our COBRE. This Core will be responsible for the overall administration of the COBRE including: announcement of openings of COBRE PJI or Early Career Investigator (ECl) positions and pilot projects, selecting PJIs/ECIs and mentors based on the EAC's recommendations, establishing the mentoring program, overseeing the overall budget, organizing meetings of the EAC and lAC, organizing the seminar series, interacting with all the Core directors and PJIs, following the progress and milestones of PJI's projects, and submitting progress reports to NCRR. The Core will also work closely with the Harold Hamm Diabetes Center and the Provost the Oklahoma University Health Sciences Center and the Dean of the College of Medicine to recruit new investigators to Oklahoma. Finally, the Administration Core will continue to organize the Annual Diabetes Research Symposium to enhance collaboration.

PROJECT SUMMARY Diabetes research is recognized as one of the two strategic areas in the Research Strategic Plan of The University of Oklahoma Health Sciences Center (OUHSC). The major goal of this COBRE is to increase the critical mass of diabetes researchers and improve infrastructure for diabetes research at OUHSC. Our long-term goal is to apply for a NIH P30 Diabetes Research Center (DRC). In the past nine years of COBRE support, we have closely worked with the Harold Hamm Diabetes Center and reached our milestones of Phase I and Phase II. The COBRE has incubated eight NIH R01 grants, two R21s and multiple grants from research foundations such as the American Diabetes Association and the American Heart Association. The Junior Investigators supported by the COBRE have published 207 peer-reviewed papers. Five of the Junior Investigators supported by the COBRE have been promoted to Associate Professors. In addition, the COBRE has recruited four NIH-funded diabetic researchers into OUHSC. The Administrative Core has played a central role in leading COBRE operations and contributed to these achievements. The major goal of this Core in Phase III is to further expand these successes and assist the research Cores to become sustainable research facilities supported by a combination of user fees and institutional resources. In Phase III, the Administrative Core will continue to provide central management of the entire COBRE and coordinate COBRE activities and interactions among all of the COBRE components. The Administrative Core will also facilitate interactions of the Diabetes COBRE with other COBREs and research Centers at OUHSC and throughout the State of Oklahoma. This Core will make an effort to improve the diabetes research environment and facilitate collaborations in diabetes research. This Core will promote resource and data sharing strategies and ensure compliance with human subjects, vertebrate animals and biosafety regulations. This Core will greatly facilitate the continuous growth of the COBRE to achieve its goals.

PROJECT SUMMARY Diabetes represents a major threat to the health of the working population, and constitutes an immense social and economic burden. Rodent models of streptozotocin (STZ)-induced or genetic diabetes are commonly used in diabetes research. Diabetic animals have a high mortality rate and require intensive care and characterization. Diabetic complications tend to occur only long after the onset of diabetes, and long-term maintenance and monitoring of diabetic animals are labor-intensive and associated with high costs. The objective of this Core is to centralize the induction, breeding, monitoring, maintenance, and use of diabetic animal models, and to coordinate the sharing of diabetic animal tissues among the investigators. In the past nine years of this COBRE, the Diabetic Animal Core has provided service, diabetic animals, and animal tissues to 35 investigators, including the COBRE Promising Junior Investigators (PJIs), members of the Harold Hamm Diabetes Center (HHDC), and other diabetes researchers. This Core has provided support to 208 publications and 29 funded NIH grants on campus. The Core has greatly increased the efficiency of diabetes research using diabetic animal models and has reduced costs for PJIs and other diabetes researchers at the HHDC. The Core has become an essential and unique facility for diabetes research in Oklahoma. Considering the excellent service of this Core and rapid growth in diabetes research on our campus, the HHDC started to provide funds to subsidize this Core in Phase II of our COBRE. The HHDC is also committed to supporting the transition of this Core to an independent research core facility supported by the HHDC after the completion of the COBRE Phase III. In Phase III, we will further improve the Core services and start the transition of this Core to a HHDC-supported facility. In Phase III, we will induce diabetes by STZ injection in rats and mice, and breed genetic diabetic animals as requested by investigators. We will also monitor diabetes and collect ?clinical data? on diabetic animals. We will coordinate sharing diabetic animal tissues by different users and expand the diabetic animal tissue bank. Further, this Core will provide training or technical assistance for specialized assays of diabetic complications. Through these efforts, this Core will provide support for the Pilot Projects funded by the COBRE, greatly enhance diabetes research in Oklahoma and contribute to further growth of the HHDC. It will also attract more local basic scientists into diabetes research and facilitate recruitment of new diabetes researchers into Oklahoma. This Core will contribute to the development of new treatments for diabetes and its complications.

PROJECT SUMMARY / ABSTRACT Diabetic keratopathy is a complication of diabetes and a major cause of vision loss. There are no effective drugs that can prevent or reverse corneal defects related to diabetes. Two independent longitudinal clinical studies have shown robust therapeutic effects of fenofibrate, a specific agonist of Peroxisome Proliferator-Activated Receptor-? (PPAR?), on diabetic retinopathy. Our preliminary studies using diabetic human donor corneas and animal models suggest a role of PPAR? in maintaining corneal nerve integrity. In our preliminary studies, we have fabricated an innervated 3D in vitro human corneal model that demonstrates basic anatomical and physiological similarities to the corneal tissue in vivo. Using this novel model we began unravelling PPAR??s role in diabetic keratopathy. Significant downregulation of PPAR? expression was seen in cells from both T1DM and T2DM human donors, in agreement with decreased PPAR? levels as shown in diabetic human corneas. Our in vivo preliminary studies have shown that non-diabetic PPAR? knockout (PPAR?-/-) mice have decreased densities of the sub-basal nerve fibers and reduced corneal sensitivity, similar to what is seen in diabetic humans. Furthermore, to our surprise, aged, non-diabetic PPAR? knockout mice naturally developed more severe corneal ulcerations compared to that in age-matched WT mice. Treatment of diabetic rats with fenofibric acid, an active metabolite of fenofibrate, alleviates corneal nerve degeneration in diabetic rats. As shown by Seahorse analysis, mitochondrial function is impaired in PPAR?-/- retina. Based on these preliminary studies, we hypothesize that diabetes-induced down-regulation of PPAR? expression plays a key pathological role in diabetic keratopathy and represents a novel drug target. We propose the following studies to address the hypothesis. First, we will induce diabetes in PPAR?-/- mice and PPAR? transgenic mice over-expressing PPAR? in the cornea, to determine if PPAR? KO exacerbates while PPAR? over-expression alleviates diabetes-induced decreases of corneal nerve density and sensitivity. We will also treat diabetic mice with fenofibrate to determine if activation of PPAR? arrests progression of corneal nerve fiber degeneration. Second, we will determine if the neuroprotective effect of PPAR? is through attenuation of oxidative stress and inflammation, protection of mitochondrial functions and up-regulation of neurotrophic factors using PPAR?-/- mice and PPAR? transgenic mice as well as the innervated in vitro 3D human corneal model. Third, to translate the neuroprotective PPAR? function into a therapy, we will evaluate therapeutic efficacy of topical application of a proprietary fenofibrate eyedrop on diabetes-induced nerve fiber degeneration. This study has potential to identify a new function of PPAR? in the cornea. These studies have potential to establish a novel pathogenic mechanism for diabetic keratopathy and to lead to the development of a novel therapy.

PROJECT SUMMARY/ABSTRACT Light-induced isomerization of 11-cis retinal, the chromophore of visual pigments, to all-trans retinal initiates vision. Efficient recycling of 11-cis retinal through the visual cycle is essential for the formation of visual pigments and maintaining normal vision. A key step in the visual cycle is the conversion of all-trans retinyl ester to 11-cis retinol, catalyzed by RPE65, the retinol isomerase. Documented studies have shown that retinoids are largely stored as all-trans retinyl ester, the substrate of the isomerase RPE65, in lipid droplets (retinosomes) in the RPE. However, RPE65 is located in the ER and not near the lipid droplets. It is unknown how hydrophobic all-trans retinyl ester, the substrate of RPE65, is transported from lipid droplets to the ER. This represents an important knowledge gap in the visual cycle. A retinyl ester hydrolase (REH) may be present in lipid droplets to mobilize all-trans retinyl ester to provide the substrate for the RPE65 isomerase in the ER. Patatine-like phospholipase domain containing protein 2 (PNPLA2) is known as an adipose triglyceride lipase with an REH activity in the liver. Its function in the RPE and its association with the visual cycle have not been investigated. Our preliminary studies identified PNPLA2 in the RPE and in lipid droplets. Importantly, PNPLA2-/- mice showed declined ERG responses and delayed regeneration of visual pigments. PNPLA2 KO also resulted in reduced 11-cis retinal generation and increased levels of all-trans retinyl ester in the RPE, suggesting a decreased isomerase activity. These findings suggest a potential role of PNPLA2 in the visual cycle. We hypothesize that PNPLA2 functions as an REH in the RPE and mobilizes all-trans retinyl ester from lipid droplets to provide sufficient substrate for the RPE65 isomerase in the ER to generate 11-cis retinol. In this project, we will define the role of PNPLA2 in maintaining the visual cycle and normal vision. We will use RPE-specific PNPLA2 conditional KO mice to study the impacts of PNPLA2 KO on retinal function, retinal degeneration, visual pigment formation and regeneration of 11-cis retinal. We also plan to determine if co-expression of PNPLA2 together with RPE65 in the RPE of RPE65-/- mice by gene delivery will enhance the effect of RPE65 on restoring visual function, visual pigment formation and 11-cis retinal regeneration, compared to RPE65 gene delivery alone. We will also elucidate the mechanism by which PNPLA2 promotes the RPE65 isomerase activity and accelerates 11-cis retinal regeneration in the visual cycle. We will investigate if knockout of PNPLA2 in RPE cells will decrease, while overexpression of PNPLA2 will promote, isomerase activity of RPE65 and reduce lipid droplets in RPE cells. We will also determine if a PNPLA2 activator will promote, while a PNPLA2 inhibitor will inhibit the isomerase activity in RPE cells. These studies have potential to identify a missing component in the visual cycle and address an important knowledge gap in vision. This project will also identify a new function of PNPLA2 in the RPE. PNPLA2 has potential to improve the RPE65 gene therapy for retinal dystrophies caused by RPE65 mutations.