Kang Zhang - US grants

DESCRIPTION (provided by applicant): Stargardt-like macular dystrophy (STGD3, MIM 600110) is an autosomal dominant form of juvenile macular degeneration characterized by decreased visual acuity, macular atrophy, and extensive flecks. Using a positional cloning approach, we identified ELOVL4 as the causal disease gene responsible for Stargardt-like macular dystrophy (STGD3, MIM 600110). ELOVL4 encodes a protein with sequence and structural similarities to the ELO family of proteins. The ELO family of proteins is involved in the elongation of long chain fatty acids (LCFA), suggesting that ELOVL4 may have a similar function. The long term objectives of this proposal are to characterize the biochemical properties of ELOVL4 and to elucidate molecular mechanism leading to macular degeneration due to ELOVL4 mutations in humans. ELOVL4 proteins are strongly conserved throughout vertebrate species. The exact enzymatic function of ELOVL4 and the pathogenic mechanisms by which mutant ELOVL4 causes macular degeneration are unclear. Our central hypothesis is that ELOVL4 is a key enzyme in a biosynthetic pathway that produces very long chain fatty acids which play an essential role in retina development and function. Guided by this hypothesis, we propose to define ELOVL4 function through a comprehensive analysis of fatty acid composition in vitro and in vivo, and to examine retinal development and pathogenesis in ELOVL4 transgenic, knockin, and knockout mice. To further understand ELOVL4 functions and pathogenic mechanisms giving rise to macular degeneration we will address the following specific aims: Specific Aim 1: To determine the enzymatic function of ELOVL4 Specific Aim 2: To determine the role of ELOVL4 in retinal development and pathology Specific Aim 3: To examine RPE dysfunction in ELOVL4 mutant transgenic mice Stargardt macular dystrophy is the most common juvenile macular degeneration and shares many important clinical and histopathological similarities with AMD including an abnormal accumulation of lipofuscin in the RPE, atrophy of the RPE and overlying photoreceptor cells, and loss of central vision. ELOVL4 is the first gene involved in the biosynthesis of long chain fatty acids implicated in any form of macular degeneration. The proposed study should lead to new insights into lipid metabolism in photoreceptor cells and reveal a novel pathway in the pathogenesis of macular degeneration. Our study should also provide insights into the formation of lipofuscin and provide new avenues for treatments for Stargardt macular dystrophy and AMD.

[unreadable] DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the most common cause of visual impairment of the elderly in the developed world. Despite the increase in its prevalence within the aging population, its etiology and pathogenesis are poorly understood and treatment options are limited. We and others have demonstrated that HTRA1 may play a major role in genetic susceptibilty to AMD. The objectives of this proposal are to further analyze the possible causative variant(s) in 10q26, define the normal function of HTRA1, and to elucidate the molecular mechanisms leading to macular degeneration. Our central hypothesis is that HTRA1 plays a key role in retinal development, angiogenesis, and extracellular matrix modeling. Guided by this hypothesis, we propose to conduct the following specific aims. Specific Aim 1: To identify all SNP variants associated with AMD in LOC387715/HTRA1 region and investigate their role in AMD in transgenic mice. Our preliminary results have defined a major disease haplotype spanning a 10 kb region in 10q26 that explains the major risk of AMD. This region contains previously identified SNP LOC387715/ARMS2 rs10490924 and HTRA1 rs11200638, already shown to be associated with AMD. We propose to identify all SNP variants in this region by comprehensive direct sequencing analysis. Newly discovered SNPs will be used in a case control association study to investigate their association with AMD in a large cohort. Functional studies will be performed in transgenic mice carrying these AMD associated SNPs. Specific Aim 2: To determine the role of HTRA1 in retinal development and pathology. HTRA1 is expressed in the retina and RPE. In order to differentiate the role it plays in the RPE independent of that in retina, we will generate conditional knockout mice which delete HTRA1 in either the retina or RPE. Pathology will be examined in the conditional knockout (KO) by ophthalmoscopy, histology, ERG, and HTRA1 expression will be determined by QPCR, western blot and immunohistochemistry (IHC). Loss of HTRA1 function will be determined by measuring the reduction of or inability for choroidal neovascularization (CNV) to take place in homozygous and heterozygous conditional KO mice. Specific Aim 3: To examine the role of HTRA1 in AMD pathogenesis in vivo. A. Inhibition of CNV by injection of HTRA1 antibody in a CNV model. Our preliminary data indicate that over-expression of HTRA1 may contribute to AMD pathogenesis in humans. To verify this, we plan to quantify HTRA1 expression and the extent to which an HTRA1 antibody can inhibit CNV in a laser induced CNV model. B. Transgenic mice expressing WT and mutant HTRA1 genes. HTRA1 is a multi functional protein. In order to delineate which function of HTRA1 (protease, TGF? inhibition, or IGF domain) is involved in AMD pathogenesis in vivo, we will generate transgenic mice expressing different HTRA1 mutations. Mutants will lack either protease activity (SA mutant), the PDZ domain (?PDZ), the Mac25 domain (?Mac), or constitutively active protease. We will quantify the development of AMD-like features in these HTRA1 transgenic mice by ophthalmoscopy, angiography, immunohsitochemistry, biochemistry, and histopathology and compare them to age-matched wild-type mice. The identification of genes that have substantial impact on the risk of the disease may define key molecular pathways involved in its pathogenesis. This may lead to therapies directed at the underlying cause and pre-symptomatic diagnostics to allow for earlier intervention and treatment. PUBLIC HEALTH RELEVANCE: This grant application focuses on investigation of a major gene (HTRA1) that causes age-related macular degeneration, the leading cause of blindness with a significant public health impact. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The retinal photoreceptors are the light-sensing cells that convert complex external visual stimuli to electrical and chemical signals. Degeneration of photoreceptors is the end point of the commonest causes of irreversible blindness including age-related macular degeneration and retinitis pigmentosa, affecting over 50 million people world-wide. In non-mammalian vertebrates such as fish, after retinal injury, resident Muller glia cells in the retina can proliferate, and differentiate into all retinal cell types including photoreceptors and restore visual functions. However, this regenerative potential is almost non-existent in mammals, with only very few new neurons generated after damage. We propose to develop and apply a high throughput screening to identify small molecules that will enhance Muller glia cells reprogramming and differentiation into retinal neurons in mammals, in vitro and in vivo. Identification, optimization, and characterizations of chemical tools for Muller cells reprogramming and differentiation will provide new avenues in developing cell-based therapy as well as conventional small molecule therapeutics for regenerative medicine, and facilitate new understanding of the transdifferentiation mechanisms. Our proposed research will facilitate the development of therapies to restore visual functions that have been lost in human patients with severe blindness.

DESCRIPTION (provided by applicant): Biomaterial enhancement of stem cell transplant efficacy for macular degeneration No effective therapies yet exist for advanced macular degeneration. Restoring vision in severe cases requires replacing both retinal pigmented epithelium (RPE) and photoreceptors because photoreceptors require functional RPE to survive. However, no approach to replace both cell types has yet been introduced. Further, previous attempts to replace photoreceptors have met limited success in rodent models, likely because of low survival rates and restricted distribution of transplanted cells. This project integrates solutions to both of these challenges: we propose to transplant human embryonic stem (ES) cell-derived primitive retinal stem cells (hpRSCs), a cell population that is more pluripotent than cell types previously transplanted to restore vision. We have shown that hpRSCs yield both RPE and photoreceptors in vitro and in vivo; replacing both cell types in vivo would yield longer-lasting visual improvements, as transplant-derived RPE would support transplant-derived photoreceptors. In order to develop a maximally effective therapy, we will deliver hpRSCs in an injectable hydrogel whose biochemistry and mechanics closely match those of the ocular extracellular matrix, which has recently been shown to uniformly distribute transplanted cells across the retina. This project stands to position ES cell-derived RPCs for translation into the clinic. The key to this advance is the collaboration between the PIs, bringin together a leader in macular degeneration models and the differentiation of stem cells into retinal neurons (Zhang) with a biomaterials research group (Almutairi). The Zhang group's expertise will ensure that this project advances development of HAMC for ocular transplantation significantly: previous work has not employed relevant animal models, so the impact of the material on transplanted cells' differentiation and functional recovery has not yet been assessed. Further, it has relied on cells that are easiest to derive, whereas this project will employ ES cell-derived RPCs, which are the only clinically viable option. By completion of this project, we expect to: 1. Assess the effect of HAMC delivery on survival and distribution of subretinally injected retinal stem cells 2. Define the effect of HAMC on the ability of retinal stm cells to differentiate into RPE and photoreceptors in vitro and in vivo 3. Identify the delivery method that maximizes visual improvement ensuing from subretinal injection of retinal stem cells in Royal College of Surgeons (RCS) rats

? DESCRIPTION (provided by applicant): The cornea is a specialized epithelium, which protects the eye and maintains transparency for normal vision. Diseases of the cornea or its stem cells (limbal stem cells) affects millions of people worldwide and result from a spectrum of etiologies, ranging from genetic defects to infection, inflammation, or trauma. Vision loss often occurs as the result of transformation of the transparent cornea into a skin-like epithelium. Recent work from our labs and others now reveal that this critical transition may be precipitated by the loss of PAX6 expression. Downregulation of PAX6 causes the loss of cornea features and gene expression and the ectopic activation of skin-like epithelium. Conversely, introduction of PAX6 into skin cells induces many cornea-like features. These studies indicate a critical role of PAX6 for maintaining cornea epithelium, but the mechanisms, which determine cornea vs. skin identity, are still unknown. Our hypothesis is that PAX6 contributes to cornea identity through tissue-specific DNA regulatory elements called enhancers. Enhancers are responsible for coordinating tissue-specific gene expression and for the development of specific cell types. Enhancers are also thought to be of considerable medical importance as genetic variation in and epigenetic regulation of enhancers is thought to play a major role in susceptibility to disease. Despite their biological and medical importance, little is known about the enhancers of the cornea and disease. Advances in chromatin biology and high-throughput sequencing provide new, powerful tools to identify tissue-specific enhancers of the cornea and have the potential to expand our knowledge of cornea disease. Here we propose to (1) identify enhancers, which distinguish cornea from skin epithelium via ChIP-seq; (2) determine how tissue-specific enhancers are regulated in the cornea by chromatin analysis and reporter studies in cultured cells, animal model transplants; and (3) determine the role of PAX6 in the regulation of tissue-specific enhancers. To accomplish these goals, an interdisciplinary approach is needed. The combined expertise and resources of two labs (eye, skin) provides an ideal path to identifying cornea- vs. skin-specific regulation and will result in a better understanding of how ectopic skin features become activated in the cornea. This major effort will result in the identification of genome-wide enhancers in the cornea, critical to stem cell biology and translational studies, and the generation of new genetic reagents to study cornea disease.

Abstract Uveal melanoma is the most common ocular tumor in adults. It harbors genetic mutations very different from those seen in cutaneous melanoma. Liver metastasis is common in uveal melanoma and contributes to the very poor prognosis with average survival of several months. Currently there is no treatment for metastatic uveal melanoma. Activating mutations in GNAQ and GNA11 are the most important cancer drivers, as approximately 80% of uveal melanomas have mutations in either GNAQ or GNA11. Our recent studies have shown that the mutant GNAQ/11 potently activates the YAP oncoprotein, which is a key component of the Hippo tumor suppressor pathway. Moreover, the elevated YAP activity is essential for tumor growth of uveal melanoma cells containing an activating mutation in GNAQ/11. Besides GNAQ/11, mutations in BAP1, SF3B1, and EIF1AX are also frequently observed in uveal melanoma in a mutually exclusive manner. However, a mechanistic understanding of these genes in uveal melanoma and their functional interactions are largely unknown. The major goals of this project are to determine the contributions of the genes commonly mutated in uveal melanoma and their functional interaction in promoting tumorigenesis of uveal melanoma. We also aim to characterize and validate potential therapeutic targets and tools for treatment of uveal melanoma.