Muayyad Al-Ubaidi - US grants

DESCRIPTION (Adapted from applicant's abstract): The purpose of the proposed research is to determine the functional significance, catalytic and structural, of C-terminal palmitoylation and N-terminal glycosylation of vertebrate rhodopsin, the rod visual pigment. Polymerase chain reaction will be utilized to modify, by site-directed mutagenesis, the opsin glycosylation and palmitoylation sites and mutant opsins will be expressed in transgenic mice. The mutant opsins will also contain a three amino acid, bovine-specific C-terminal epitope "tag" which, in conjunction with an epitope-specific monoclonal anti-bovine opsin antibody, will permit identification and localization of the transgenic "bouse" opsin against the background of the normal mouse opsin, using conventional Western blotting and immunocytochemical methods. Initial experiments will be performed to determine the level of expression of "bouse" opsin (not containing modified palmitoylation and glycosylation sites) that does not by itself produce structural and functional alternations. This proposal aims at testing the hypothesis that palmitoylation of rhodopsin is involved in the deactivation of photoactivated rhodopsin. It also aims at testing the hypothesis that impaired N-terminus glycosylation of rhodopsin is sufficient to cause defective morphogenesis of the outer segment. These features will be studied using a combination of biochemical methods, light microscopic autoradiography, and light and electron microscopy and immunocytochemistry. Electroretinography and suction pipette single cell recording will be used to determine the effects of transgene expression on phototransduction and overall electrophysiological competence of the retina.

molecular biology; vision; nucleic acid chemical synthesis; biomedical facility; oligonucleotides; nucleic acid sequence; RNase protection assay; southern blotting; polymerase chain reaction; biomedical equipment purchase; northern blottings;

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The overall goal of this proposal is to investigate the role of protein tyrosyl-sulfation in normal retinal function. The intermediate aims include the identification of retinal proteins that are sulfated by tyrosylprotein sulfotransferases and determine how lack of either (or both) of these enzymes influence retinal function. There are two distinct TPST isoenzymes present in most tissues. These two enzymes are responsible for the post-translational tyrosine O-sulfation of cellular proteins. Neither these enzymes nor their targets have been studied in the retina or other ocular tissues. We have generated knockout mice for TPST-1 &2. These two mice, and the double knockout obtained by crossbreeding, exhibit different retinal defects in retinal development, function and aging. Lack of TPST-1 results in the retardation of functional development. Moreover, these mice demonstrate a slow retinal degeneration characterized by a gradual loss of scotopic and photopic electroretinography (ERGs) that become very apparent at 4 months of age (-30% loss). Histologic examination of P120 Tpstrl- retinas revealed a reduced number of nuclei in ONL (9 rows at P120 versus 11 rows at P50) and INL (4 rows at P120 versus 5-6 at P50). Unlike TpsWI - retinas, TpstZI - retinas never fully develop to the wild-type level. This very clearly demonstrates a direct role for TPST-2, or its protein products, in retinal maturity. In Aim 1, we will characterize the presence, localization and biochemical behavior of retinal TPST-1 and TPST-2 during development. These experiments will be performed in normal mouse retinas from embryonic day 10 to 2 years of age utilizing antibodies that are specific for TPST-1, TPST-2, and to sulfotyrosine. As part of this aim, we will identify the proteins modified by TPST-1/2 using classic immunoaffinity purification techniques and MS and MS/MS. In Aim 2, we will fully characterize retinal phenotypes caused by lack of TPST-1, TPST-2 or both. Characterization will be performed structurally at the light and electron microscopic levels, functionally by scotopic and photopic ERGs and suction electrode recordings, and biochemically by analysis of the sulfation of retinal proteins and expression of the targets of TPST-1/2 by SOS-PAGE. These studies will, for the first time, shed light on the role of TPST-1 and 2 in protein sulfation in the retina. Further, they will increase our understanding of retinal development and the mechanism(s) involved in retinal degeneration.

DESCRIPTION (provided by applicant): Research on retinitis pigmentosa (RP) and age related macular degeneration (AMD) has significantly advanced knowledge into their genetic causes. Although such findings triggered exploration of numerous therapeutic paradigms, we still come short in our understanding of the cause of the late onset of visual loss although the mutant protein is present from birth. It is often proposed that these cells survive due to effects f neuroprotective factors that significantly prolong the life of the sick cell. However, it is not clar as to why the patient starts to encounter visual difficulties all of a sudden? Here we propose an additional contributing hypothesis for the delayed onset of visual loss based upon two related findings. The first is the identification of CFH and fibulin 5 as sulfated proteins. Besides being present in drusen, mutations in both proteins associate with AMD. Our second is that we observe modulations in levels and types of tyrosine-sulfated proteins during retinal degeneration in two well-studied animal models of RP, the VPP and the rds+/- mouse models. Since sulfation is a post-translational modification that occurs on secreted and membrane-associated extracellular matrix (ECM) proteins, we hypothesize that the slow death of cells will lead to gradual deterioration of ECM. However, when a critical number of ECM-maintaining cells are lost, a breaking point is reached, suddenly accelerating the degenerative process. We propose two specific aims to address our hypothesis. In Aim 1, we are generating retina/RPE-specific conditional knockout mice for both of the enzymes responsible for protein sulfation, protein tyrosyl sulfotransferases 1 and 2. The double knockout (DKO) mice will be characterized functionally, structurally and biochemically from birth to 2 years of age for effects of lack of sulfation. To determine if the lack of sulfation of ECM proteins exacerbate the AMD phenotype, we plan to backcross these mice into an AMD transgenic model (cfhY402H). To address the effect of lack of sulfation of ECM proteins on the RP phenotype, we plan to backcross the DKO mice to two well-studied RP models representing the P23H mutation in the rhodopsin gene (VPP) and the haploinsufficiency of the retinal degeneration slow (rds+/-) protein. In Aim 2, we will evaluate the role of sulfation in the function of CFH and fibulin 5. The identified sulfated tyrosine(s) will be mutated to phenylalanines and in vitro analyses of 1) ability of unsulfated CFH to bind CRP (C-reactive protein), 2) its ability to self-dimerize and 3) its interactions with polyanions. For fibulin 5, we will test the ability of the unsulfated protein to interact with interins and ECM super oxide dismutase (SOD) 3. Subsequently, we will generate and characterize retina/RPE-specific conditional knock-in mice expressing either sulfation-deficient CFH or fibulin 5. Accomplishment of the proposed studies will open a new window on the role of ECM in degenerative process and direct attention to development of new therapeutic approaches focusing on protection of the ECM to prolong vision in patients and widen the window for interventions. This is of significance to over 10 million US Citizens of all ages and races that suffer from RP and AMD.

PROJECT SUMMARY Flavins are essential cofactors for wide-ranging metabolic processes; hence they are particularly critical in highly metabolically active tissues. The retina, in which levels of flavins are several folds higher than in blood, is an important example. The physiological significance of modulating levels of retinal flavins is underscored by the observation that riboflavin deficiency results in photosensitivity and degeneration, a process thought to result from lipid peroxidation. Unbound flavins are toxic so, in practice, flavins are virtually always bound to flavin binding proteins. Although tight regulation of flavin levels is clearly critical for maintaining retinal homeostasis, nothing is known about the mechanisms governing their uptake, regulation, or binding proteins. We have recently begun studying a novel, and highly relevant candidate retinal flavin binding protein called Retbindin (Retb). Retb has homology to chicken riboflavin binding protein (RBP), and we have shown in vitro and in retinal explant that Retb binds flavins. This, coupled with the importance of flavins in the retina, led us to hypothesize that Retb's function is tied to flavin regulation. We recently reported that Retb is exclusively expressed by rods, secreted into the interphotoreceptor matrix (IPM) and maintained via electrostatic forces at the interface between photoreceptors and retinal pigment epithelium (RPE) microvilli, a region critical for retinal function and homeostasis. This localization combined with its ability to bind flavins, implicates Retb as a potential carrier of flavins between the retina and the RPE. To further assess Retb's function in the retina, we generated a knockout mouse (Retb-/-), in which Retb sequence was replaced with eGFP. Electroretinography revealed an age- and dose-dependent decline in rod and cone responses at postnatal days (P) 120 and 240 and a concurrent degeneration of rods and cones. We also show flavin levels significantly reduced in P45 Retb-/- retina, prior to the onset of degeneration. In light of the potential pathological consequences of elimination of Retb, coupled with a complete lack of knowledge of Retb function, we propose to explore the role of flavins in the retina and determine how Retb could regulate their levels. Specifically, we propose three aims. First, we will determine the functional role of Retb in the photoreceptor cells by evaluating Retb-/- and Retb+/- animals at different ages and under various lighting/dietary treatments as well as identify Retb binding partners. Second, we will take a metabolomic approach to identify the metabolic pathways that are affected by the absence of Retb. Third, because we observed levels of Retb are significantly elevated in the retinas of animal models of retinal diseases, we will test the role Retb plays in the development and progression of retinal degenerations such as retinitis pigmentosa. We have also initiated the generation of a Retb over-expresser mouse model to be used to assess the capability of Retb in ameliorating disease progression. These experiments are highly significant, not just to further our knowledge of a poorly understood protein critical for retinal function, but also to explore the role of metabolic dysregulation in retinal homeostasis and diseases.

PROJECT SUMMARY We aim to advance our current DNA nanoparticle (NP) delivery platform and gene expression to develop safe and effective therapies targeting important photoreceptor-associated ocular disorders caused by defects in large genes. We are merging knowledge in molecular bioengineering, nanoformulation, material science, eye biology/physiology and chemistry to accelerate essential steps for the generation of effective non-viral gene delivery platform for eye diseases. The NPs consist of single molecules of DNA compacted with poly-lysine- PEG polycation and have a diameter of 8-11 nm. Their small size coupled with cellular uptake via cell-surface nucleolin, which efficiently traffics the NPs to the nucleus, accounts for their ability to transfect post-mitotic photoreceptors. Using NPs has led to efficient expression of large genes, an essential prerequisite for targeting hard-to-rescue diseases of the retina. Although upper gene size limitation has not been established, the largest size tested in the lung was 20 kb and in the eye was ~14 kb making NPs an ideal alternative to AAVs for delivery of large genes. We have showed that NP treatment led to efficient transfection of ocular cells including photoreceptors and retinal pigment epithelium, exerted no toxic effects on the eye even after multiple injections, distributed throughout the subretinal space, and mediated appreciable structural/functional rescue in mouse models of retinitis pigmentosa, Leber?s congenital amaurosis, Stargardt?s and diabetic retinopathy. Effective gene expression without toxicity has also been achieved in baboon eyes. These proof- of-principle studies confirms the potential clinical significance of this technology and highlights the value of a large capacity, but emphasized the need for prolonged high levels of gene expression. Our main goal is to enhance photoreceptor uptake of NPs to achieve long-term therapeutic levels of expression of large genes for full phenotypic rescue. We propose to accomplish this by targeted vector engineering to boost expression levels along with NP-delivery platform to enhance their delivery from the vitreous to photoreceptors, achieve pan retinal distribution, promote episomal stability in the nucleus and prevent epigenetic silencing. Subsequently, we will test these optimized NPs/delivery platform(s) for their ability to mediate full phenotypic rescue in a large gene disease model; specifically the Abca4-/- model of Stargardt?s associated with the lack of ABCA4 gene. ABCA4 is a large gene which has not been fully rescued by traditional viral vectors and as a result development of targeted therapeutics for Stargardt?s has lagged. We plan in aim1 to engineer vectors that can achieve therapeutic levels of expression in photoreceptors and in aim 2 to develop effective NP- delivery platforms to enhance photoreceptor uptake of NPs from the vitreous. Aim 3 will test the efficacy of the best delivery platform of the most effective NPs for therapeutically-effective levels of expression in photoreceptors of Abca4-/- mice before and after the onset of the disease. In summary, results from this application will facilitate the advancement of DNA NP use for ocular diseases associated with large genes.

PROJECT SUMMARY Our aims are focused on advancing current knowledge on the role of peripherin 2 (Prph2), also called retinal degeneration slow (RDS), in outer segment (OS) rim and disc formation, and in understanding the pathogenic mechanisms of PRPH2-associated disease. We use state-of-the-art technologies and our novel knockin mouse models to learn 1) how different mutations in Prph2 lead to different disease phenotypes; 2) what contributes to variability among patients carrying the same mutation; 3) what role the Prph2 partner, rod outer segment membrane protein 1 (Rom1), plays in these events; and 4) how we can shift PRPH2-associated severe phenotypes to milder ones. PRPH2 mutations lead to retinal diseases ranging from retinitis pigmentosa (RP) to a variety of macular degenerations (MD) including pattern dystrophy (PD), which often associates with secondary defects in neighboring tissues such as the retinal pigment epithelium and retinal/choroidal vasculature. In spite of the scientific progress so far, a therapeutic option suitable for clinical testing has not yet been developed. This disappointing outcome is further complicated by the diverse role of Prph2 in rods versus cones, poor genotype-phenotype correlations, vast intrafamilial and interfamilial phenotypic variability, the involvement of multiple tissues in the disease process, and the need for a precise dose of Prph2 to combat the devastating effect of haploinsufficiency. Thus a thorough understanding of Prph2-associated disease mechanisms, an absolute prerequisite for the development of effective therapies, requires precise knowledge of the differential role of Prph2 in rods versus cones and the processes that link photoreceptor defects with subsequent toxic effects in other tissues. Our findings suggest that rod-targeted disease (e.g. RP) arises due to haploinsufficiency while cone-dominant diseases exhibit a more complex and variable pathology associated with gain-of-function or dominant-negative effects. However, little is known about the link between these primary defects in photoreceptors and the blinding secondary sequellae or about what causes intrafamilial phenotypic heterogeneity. For the first time we have generated mouse models that will allow us to address these questions. In addition, we have developed an outstanding team of investigators comprising recognized field leaders in photoreceptor cell biology, with researchers skilled in understanding and evaluating choroidal and retinal vasculature. Our preliminary data support the hypothesis that Rom1 is a key modifier in cases where there is significant within-mutation disease variability. Aim1 will assess the role of Syn3B interactions with Prph2/Rom1 and in OS trafficking. Aim 2 will evaluate mechanisms of diversity in Prph2-associated disease phenotype. Aim 3 will address the role of Rom1 in modulating the disease pathology of Prph2 mutations. In summary, results from this application will facilitate our understanding of the role of Prph2 in rods and cones in health and disease states. Outcomes from these studies will help direct therapeutic strategies to overcome blindness associated with PRPH2.