SIGNALING MECHANISMS THAT CONTROL GAP JUNCTIONAL COUPLING BETWEEN RETINAL NEURONS

Gap junctions between neurons form the structural substrate for electrical synapses. Connexin 36 (Cx36, and its non-mammalian ortholog connexin 35) is the major neuronal gap junction protein in the central nervous system (CNS), and contributes to several important neuronal functions including neuronal synchronization, signal averaging, network oscillations, and motor learning. Connexin 36 is strongly expressed in the retina, where it is an obligatory component of the high-sensitivity rod photoreceptor pathway. A fundamental requirement of the retina is to adapt to broadly varying inputs in order to maintain a dynamic range of signaling output. Modulation of the strength of electrical coupling between networks of retinal neurons, including the Cx36-coupled AII amacrine cell in the primary rod circuit, is a hallmark of retinal luminance adaptation. However, very little is known about the mechanisms regulating dynamic modulation of Cx36-mediated coupling. The primary goal of this work was to understand how cellular signaling mechanisms regulate coupling through Cx36 gap junctions. We began by developing and characterizing phospho-specific antibodies against key regulatory phosphorylation sites on Cx36. Using these tools we showed that phosphorylation of Cx35 in fish models varies with light adaptation state, and is modulated by acute changes in background illumination. We next turned our focus to the well-studied and readily identifiable AII amacrine cell in mammalian retina. Using this model we showed that increased phosphorylation of Cx36 is directly related to increased coupling through these gap junctions, and that the dopamine-stimulated uncoupling of the AII network is mediated by dephosphorylation of Cx36 via protein kinase A-stimulated protein phosphatase 2A activity. We then showed that increased phosphorylation of Cx36 on the AII amacrine network is driven by depolarization of presynaptic ON-type bipolar cells as well as background light increments. This increase in phosphorylation is mediated by activation of extrasynaptic NMDA receptors associated with Cx36 gap junctions on AII amacrine cells and by Ca2+-calmodulin-dependent protein kinase II activation. Finally, these studies indicated that coupling is regulated locally at individual gap junction plaques. This work provides a framework for future study of regulation of Cx36-mediated coupling, in which increased phosphorylation of Cx36 indicates increased neuronal coupling

The Role of Fibrillin-1 in Eye Development and Disease

The ciliary zonule of the human eye consists of a circumferential array of fibers that connect the ocular lens to the nonpigmented ciliary epithelium (NPCE) located at the inner wall of the eye. Zonular fibers consist of bundles of beaded filaments called microfibrils. Microfibrils are major structural elements of the extracellular matrix and are present in pure form in the ciliary zonule. Microfibrils are composed principally of fibrillin-1 (FBN-1); a large extracellular matrix glycoprotein. In humans, mutations in FBN1 underlie Marfan syndrome; a pleiotropic connective tissue disorder that profoundly affects the eye. Ocular manifestations include ectopia lentis (dislocated lenses), cataracts, glaucoma and axial myopia. The ocular phenotypes in Marfan syndrome suggest an important role for FBN-1 in eye development. In this report, I used mice as a model system to test the role of FBN1 in eye development and disease with an emphasis on understanding the role of FBN-1 in the synthesis of the ciliary zonule. Our lab has shown that the organization and composition of the mouse ciliary zonule is similar to humans. A recent proteomic study identified FBN-1 as the main component of the ciliary zonule, accounting for 60% to 70% of total zonule protein. In order to understand the role of FBN-1 in eye development, I had to first identify the cells in the eye responsible for expressing FBN-1 and other core components of the zonule (FBN-2, MFAP2, LTBP2 and ADAMTSL4). I used in situ hybridization on wild type mouse eye tissue to show that some zonule components (Fbn1, Fbn2, Ltbp2, Mfap2) are expressed by cells of the NPCE while others (Adamtsl4, Mfap2) are expressed by the lens. This suggests that the ciliary zonule is synthesized by both tissues. Immunofluorescence experiments on adult wild type mouse eyes using antibodies against core zonule proteins showed that the ciliary zonule is heterogeneous in composition and nonuniform along the length of fibers suggesting that the spatial distribution of proteins in the ciliary zonule could reflect the temporal expression of zonule components during eye development. The identification of NPCE cells as the likely source of FBN-1 in the zonule allowed me to test the contribution of Fbn1 directly. I used a conditional knockout approach to delete Fbn1 in the nasal and temporal regions of the mouse NPCE. Three-dimensional reconstructions of conditional Fbn1 knockout mouse eyes (Fbn1-NPCE) demonstrated that the zonule was produced in the nominal absence of its most abundant component. However, the Fbn1-NPCE mice developed ectopia lentis; the major ocular manifestation and diagnostic criterion for MFS in humans. Given the clinical importance of the disease, I decided to investigate the structural and biomechanical changes leading to ectopia lentis in the Fbn1-NPCE mouse model. Ultrastructural studies on FBN-1 deficient mouse eyes using scanning electron microscopy revealed that Fbn1-NPCE mice had significant changes in zonular fiber density and thickness. Biomechanical testing on wild type and Fbn1-NPCE mouse zonules showed that FBN-1 deficient zonular fibers were significantly weaker (~50%) than their wild type counterparts. Conditional deletion of Fbn1 in the mouse lens (Fbn1-lens) had no discernible effect on the structure or function of the ciliary zonule, suggesting that FBN-1 protein synthesized by the lens is not required for the stability of the ciliary zonule. Together, these data show that the ectopia lentis phenotype in Fbn1-NPCE mice is secondary to changes in ciliary zonule ultrastructure and biomechanical properties and that these effects may give some insights to the mechanism of ectopia lentis in Marfan syndrome. In addition to ectopia lentis, Fbn1-NPCE mice express other ocular phenotypes commonly associated with Marfan syndrome including cataracts, increased axial length and, in rare cases, glaucoma. The data presented in this report provide insights into the role of FBN-1 in eye development and the biology of the ciliary zonule. In addition, the development of the Fbn1-NPCE mouse model will serve as a platform for understanding the role of FBN-1 in ocular disease associated with Marfan and related syndromes

Long-term genetic modification of the ocular outflow tract and the retinal pigment epithelium with feline immunodeficiency viral vectors

The central goal to engineer feline immunodeficiency viral (FIV) vectors into a more modular platform for long-term ocular gene therapy was accomplished collaboratively by separating envelope, packaging and transfer function, the latter of which I optimized. FIV mediated gene transfer to the aqueous humor outflow tract and to the retina was established and long-term vector function and biocompatibility were confirmed. Transduction of the outflow tract was highly efficient and resulted in genetic modification of nearly the entire trabecular meshwork. For this purpose, I developed methods for administration and sensitive monitoring of FIV vectors in the anterior chamber and a novel subretinal injection technique. Bicistronic FIV vectors generated high levels of two different transgenes eGFP and β-galactosidase, which allowed live in vivo tracking and sensitive yet specific cell labeling in tissue specimen, respectively. An integrase mutant control vector that differed in only one amino acid resulted in no significant transduction while preserving all other biochemical properties. I developed a novel protocol for scaled-up production FIV vectors using cell factories and large-volume concentration that proved useful in the animal studies of this thesis. Especially the results achieved in the larger animal model, the domestic cat, validated the protocol for large-scale production. Transient transfection of 10 times lessDNA into 293T cells within high surface area cell factories and high volume, fixed-angle ultracentrifugation resulted in high titer vectors

Choroidal Vessel Wall: Hypercholesterolaemia-Induced Dysfunction and Potential Role of Statins

© 2012 Ramírez et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Unidad Docente de Inmunología, Oftalmología y ORLFac. de Óptica y OptometríaTRUEMinisterio de Ciencia e Innovación de EspañaFundación Mutua MadrileñaUniversidad Complutense de Madridpu

The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs) mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation

Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis

It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro

The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs) mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation