Molecular mechanisms underlying the development of mammalian retina and of genetic defects leading to retinal diseases broadly constitute the research interests of my laboratory. The major projects are: (1) Transcriptional Regulatory Networks underlying Photoreceptor Differentiation. (2) Ciliary Transport, Microtubule Organization and Retinal Dystrophies. (3) Genetic Susceptibility to Age-related Macular Degeneration. (4) Genetics of Retinal Degeneration and Treatment Strategies.

Regulation of quantitatively precise expression of genes in the right cell type and at the right time is mediated by the combinatorial and synergistic (or antagonistic) action of a limited number of transcription factors. Soon after joining the faculty at UM, my lab discovered the NRL (neural retina leucine zipper) gene from a human retinal cDNA library after subtraction cloning. NRL is a key basic motif-leucine zipper (bZIP) transcription factor, which has now been established as a prime mediator of gene regulation in both developing and mature rod photoreceptors. Extensive studies by our laboratory showed that NRL interacts with the cone-rod homeobox protein CRX and synergistically regulates the expression of rhodopsin and other rod-specific genes. Our research demonstrated that mutations in the human NRL and CRX genes are associated with retinal degenerative diseases. A major exciting finding came from gene knockout studies. The deletion of Nrl in mice (Nrl-/-) resulted in the complete lack of rod function and rod-specific gene expression; instead, there was enhanced S-cone function indicating a phenotypic transformation. This has provided an excellent model to study photoreceptor differentiation. It should be noted that cones provide day-light vision, color perception and high visual acuity yet they constitute only a small fraction (3-5%) of photoreceptors in most mammals (including mice and humans). The availability of the Nrl-knockout mice has now opened new avenue for exploring cone function. These mice are now being used by more than 30 different laboratories all over the world. Using the Nrl-promoter to drive enhanced green fluorescent protein (EGFP) in transgenic mice, we have now accomplished another major feat, i.e., they have now tagged rod precursors at birth with GFP (published recently in PNAS and on the cover). This has allowed us to study the differentiation of photoreceptor regulatory pathways by producing gene profiles of purified rods from normal and mutant mouse retinas. Microarray profiling, combined with chromatin immunoprecipitation and bioinformatic analysis, has led to the identification of numerous direct targets of NRL. One such target of NRL is an orphan nuclear receptor NR2E3, mutations in which cause enhanced S-cone syndrome and related diseases in humans. Comprehensive investigations of NRL, NR2E3 and CRX, their interacting proteins (identified by yeast two-hybrid analysis, co-immunoprecipitation and mass spectrometry), and their transcriptional targets are in progress and are expected to yield the complete regulatory pathway underlying the differentiation of photoreceptors. Since mutations in several of the regulatory proteins and their downstream targets result in retinal diseases, these studies may allow us to experimentally manipulate the expression of specific target gene(s) to correct a disease phenotype. The GFP-tagged rod precursors are also being used for transplantation to degenerative retinas to restore photoreceptor function. We are also exploring how mutations in retinal transcription factors lead to developmental defects or degeneration.

X-linked retinitis pigmentosa (XLRP) is a relatively severe progressive degenerative disease affecting photoreceptors. We have been involved in research on XLRP since 1988. We have a large (perhaps the largest in the world) collection of families and patients with this disease and mapped/identified genetic defects in almost 150 families. Mutations in RPGR and RP2 account for 80-90% of XLRP. These two genes are ubiquitously expressed; however mutations in these primarily result in photoreceptor cell death. Their studies have two distinct parts: the first focuses on delineating the function of RPGR and RP2 proteins using biochemical, genetic, cell and molecular biology techniques and by developing mouse models with altered Rpgr and Rp2 function; and the second is designed to identify the molecular lesion in XLRP patients, who do not show mutations in the RPGR and RP2 genes. Exciting recent studies suggest novel roles for RPGR and RP2 in microtubule dynamics and/or intracellular trafficking. In collaborative investigations, our lab has identified the interaction and co-localization of RPGR with the newly cloned NPHP5 protein, which is mutated in Senior Loken Syndrome, thereby suggesting the possible mechanism of retinal-renal disease. More recently, we have identified the NPHP6/CEP290 gene responsible for Jobert Syndrome. An in-frame deletion in CEP290/NPHP6 affects its interaction with RPGR and leads to retinal degeneration. CEP290 mutations have recently been identified as a major cause of Leber congenital amaurosis, a congenital blinding disease. Using yeast two-hybrid strategy, co-immunoprecipitation and mass-spectrometry, our lab has discovered several novel interactors of RPGR and RP2. We are also generating mouse models of XLRP by creating conditional null mutations of mouse Rpgr and Rp2 in retinal photoreceptors or RPE using the Cre-lox strategy (complete knockout has not been possible as yet). The long-term objectives of these studies are to design gene- or pathway-based treatment strategies.

Age-related macular degeneration (AMD) is a multi-factorial disease encompassing a broad spectrum of often progressive clinical phenotypes that is a major cause of visual impairment in the elderly population of the Western countries. Exciting findings, from several groups including that of Dr. Swaroop, have opened new avenues for genetic investigations since significant evidence now exists in support of multiple chromosomal regions that harbor AMD susceptibility loci. The primary goals of this project are to identify genetic variations and/or haplotypes that are associated with susceptibility to AMD. To identify the relevant genetic variations, Swaroop lab is utilizing three complimentary approaches: (1) genome-wide linkage analysis using sib-pairs (and relative-pairs) with primarily late-stage disease; (2) haplotype mapping of selected chromosomal regions that harbor susceptible loci; and (3) association studies using candidate genes. Their emphasis will primarily be on candidate genes from specific biological pathways, with an ultimate goal of identifying molecular targets for therapeutic interventions and drug discovery. Our group has collected a large well-characterized population of AMD patients, family members, and controls. Our genetic studies have identified several AMD susceptibility loci, including sequence variants in CFH, TLR4 and other genes that exhibit significant association with the disease. Our studies show that a number of noncoding variants in and around CFH influence the susceptibility to AMD. More recently, we are working of several novel strongly associated gene variants and their functional relevance to AMD pathology.

Our lab has been developing extensive gene profiles of the mouse and human retina using custom slide microarrays of eye-expressed genes (generated by his group) and Affymetrix GeneChips. The primary goals are to identify signaling pathways that contribute to retinal aging and/or disease pathogenesis. Towards this goal, they have ongoing and highly-productive collaborations with colleagues in Biostatistics, Bioinformatics and Electrical Engineering and Computer Science departments. We have identified a number of new disease/mutation genes, and functional studies are in progress. Novel strategies, including transplantation of purified rod precursors into degenerating retina, are being evaluated with a goal to treat retinal disorders.

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