Debora B. Farber - US grants

The objective is to investigate the metabolism of cyclic nucleotides in normal and diseased visual cells with a special emphasis in the less biochemically characterized cones. In addition, we wish to investigate the mechanism by which elevated cyclic nucleotide levels affect the viability of photoreceptor cells in some retinal degenerations. Our work has suggested that in contrast to the function that cGMP plays in rods, cAMP may be more important than cGMP in cone-dominant retinas since cAMP concentration is higher, cAMP levels are decreased by light, and elevated levels of cAMP seem to be more toxic and cause the specific degeneration of cones. However, to establish that cones and rods utilize different cyclic nucleotides as intracellular regulators it is necessary to study isolated cone photoreceptors because the rest of the retina interferes with its own cAMP metabolism. Using isolated cone cells we will study their cyclic nucleotide content, activities, kinetics and regulation of cyclic nucleotide synthetic and degradative enzymes, and investigate if cyclic nucleotide action is modulated by the phosphorylation of specific proteins. We will determine whether light reduces cyclic nucleotide levels and whether activities of the metabolic enzymes are coupled to bleaching of the visual pigment. The same enzyme systems will also be studied in developing cone-like photoreceptors grown in culture, obtained from dissociated retinas of chick embryos. In addition, we will continue to collect baseline data on cyclic nucleotide metabolism in normal human retina to be used for comparison with results from retinas affected with Retinitis Pigmentosa, macular degeneration or other retinal diseases, whenever these retinas become available. To gain insight into how elevated cyclic nucleotide levels participate in visual cell degeneration, we will study first degeneration of rods in the rd mouse, since we have several probes on hand and clues about what components need to be analyzed. In the future, when more is known about cones and probes are developed, we will focus our attention on cone degeneration. We will use antibodies and molecular biology methods to measure concentration and synthesis of cGMP-phosphodiesterase (PDE), the activity of which is abnormal in rd retina, and mRNAs that code for G-protein and rhodopsin, which are needed for PDE activation. We will also clone cDNAs for PDE and rhodopsin from control and rd retinas. Analyses of the cDNA sequences may reveal anomalies associated with the cause of the rd mouse disease and further our understanding of photoreceptor degeneration.

DESCRIPTION (Adapted from the applicant's abstract): The long-term objectives of this application are to elucidate the molecular events that control transcription and translation of the genes encoding rod- and cone-specific cyclic GMP phosphodiesterases (PDEs), enzymes that are fundamental for integrity and normal function of photoreceptors, and to identify the genes encoding regulatory proteins essential for expression of PDEs. Specifically, the investigators will focus on the isolation and characterization of protein(s) (YP) interacting with a cis-element (Y element) adjacent to the transcription start site of the rod beta-PDE gene (mutations in this element abolish transcription) and RNA-binding proteins (UBPs) which may regulate translational efficiency of rod and cone PDE subunits. The genes encoding YP and UBP will constitute new candidates to screen for mutations in the DNA of patients affected with different types of inherited retinal degenerations. They will characterize the Y element and also examine if a Y-like sequence in the cone alpha-PDE gene plays a similar role in transcription of this gene. Deletion and substitution mutational analyses using transfections and gel mobility shift assays will be employed. To obtain YP, the one-hybrid system will be used to clone the YP cDNA followed by expression of YP. Alternatively, they will use classical column and affinity chromatography methods. Recombinant YP will be characterized and tested for its ability to activate beta-PDE transcription. To study the role of rod and cone PDE mRNA structures in translational regulation, constructs containing the 5'- and/or 3'-untranslated regions or both will be analyzed for their effect on protein synthesis using in vitro translation assays or by expressing them in the appropriate cell lines. UBP will be obtained using affinity chromatography and its cDNA will be cloned and expressed. They will then clone, determine the structure, and map them o their corresponding human chromosomes the YP and UBP genes. The DNA of patients with different retinal degenerations will be screened for potential disease-causing mutations in the YP and UBP genes using haplotype/exon screening and single strand conformational polymorphism electrophoresis (SSCP). Overall, these studies are designed to increase our knowledge of the etiology and possibly the pathogenesis of certain hereditary retinal degenerations and open new avenues for future therapeutic modalities.

DESCRIPTION (Adapted from the applicant's abstract): The long-term objectives of this application are to elucidate the molecular events that control transcription and translation of the genes encoding rod- and cone-specific cyclic GMP phosphodiesterases (PDEs), enzymes that are fundamental for integrity and normal function of photoreceptors, and to identify the genes encoding regulatory proteins essential for expression of PDEs. Specifically, the investigators will focus on the isolation and characterization of protein(s) (YP) interacting with a cis-element (Y element) adjacent to the transcription start site of the rod beta-PDE gene (mutations in this element abolish transcription) and RNA-binding proteins (UBPs) which may regulate translational efficiency of rod and cone PDE subunits. The genes encoding YP and UBP will constitute new candidates to screen for mutations in the DNA of patients affected with different types of inherited retinal degenerations. They will characterize the Y element and also examine if a Y-like sequence in the cone alpha-PDE gene plays a similar role in transcription of this gene. Deletion and substitution mutational analyses using transfections and gel mobility shift assays will be employed. To obtain YP, the one-hybrid system will be used to clone the YP cDNA followed by expression of YP. Alternatively, they will use classical column and affinity chromatography methods. Recombinant YP will be characterized and tested for its ability to activate beta-PDE transcription. To study the role of rod and cone PDE mRNA structures in translational regulation, constructs containing the 5'- and/or 3'-untranslated regions or both will be analyzed for their effect on protein synthesis using in vitro translation assays or by expressing them in the appropriate cell lines. UBP will be obtained using affinity chromatography and its cDNA will be cloned and expressed. They will then clone, determine the structure, and map them o their corresponding human chromosomes the YP and UBP genes. The DNA of patients with different retinal degenerations will be screened for potential disease-causing mutations in the YP and UBP genes using haplotype/exon screening and single strand conformational polymorphism electrophoresis (SSCP). Overall, these studies are designed to increase our knowledge of the etiology and possibly the pathogenesis of certain hereditary retinal degenerations and open new avenues for future therapeutic modalities.

DESCRIPTION: (Applicant's Abstract) Individuals with ocular albinism (OA) lack stereoscopic vision due to a reduction of the ipsilateral component of the optic tract and have deficient melanin levels in the retinal pigment epithelium (RPE). The gene that causes the X-linked form of this disease, 0A1, has been identified and characterized. It encodes a G-protein coupled-receptor of unknown function that is localized on the membrane of melanosomes. Melanogenesis occurs in these organelles and tyrosinase is the key enzyme involved in this process. Melanosornes are present in the melanocytes, of the skin and in the RPE. The goal of this proposal is to investigate the molecular mechanisms of axon guidance that lead to the formation of abnormal synaptic connections in the brain of individuals affected with OA. The mouse albino mutant, that carries a point mutation in tyrosinase leading to decreased numbers of uncrossed retinal axons, offers a genetic model to address why the deficiency in melanin results in the abnormality at the optic chiasm seen in OA. We propose to use a genetic approach to identify the cues provided by tyrosinase, RPE cells and Oal that direct retinal axon divergence at the chiasm, and the mechanisms underlying specification of retinal ganglion cells to respond to these cues. Initially, we will genetically engineer mice having Cre-recombinase. These animals will allow us to control the timing of expression of specific genes. The Cre-mice will be crossed with transgenic albino mice expressing tyrosinase or diphteria toxin, and with transgenic mice carrying a conditional allele of 0al. The inducible restoration of melanine by tyrosinase will allow us to determine whether pigmentation has a role in axonal pathfinding. The inducible expression of diphteria toxin to ablate the RPE will indicate whether these cells influence both the differentiation of ganglion cells and their axonal pathfinding. The introduction of an "on/off" switch to flip Oal coding sequences will allow us to determine whether the stages of axonal crossing can be reversed by the re-expression of the wild type gene. The information obtained with these studies will. increase our understanding of the pathology of ocular albinism and will help us . to unravel molecular mechanisms of pathfinding in the optic chiasm, a "choice point" where growth cones navigate to the same or opposite side of the brain. Finding how pigmentation defects in the RPE cause abnormalities in axonal guidance and in retinal development may provide insights applicable to future therapy.

DESCRIPTION (provided by applicant): Genetic mutations that alter ocular pigmentation produce abnormalities within the developing retina and visual pathways that cause permanent visual impairment. While much is known about the neural phenotype associated with ocular albinism (OA) and related hypopigmentation conditions affecting the retinal pigment epithelium (RPE), how these changes within the RPE affect the nervous system remain an enigma. This research program will directly address these issues, seeking an integrated understanding of the relationship between tyrosinase, melanin synthesis, OA1 signaling, G-protein activation and the downstream effectors that ultimately modulate gene expression in the neural retina. Novel inducible site-specific recombination strategies for generating transgenic mice will be used that permit tissue-specific expression of desired genes at different times during development and control of transgene dosage. Three different Gi protein knockout mice will also be examined to define the Gi protein through which OA1 normally functions, and constitutively active Gi-expressing transgenic mice will be generated and then crossed to Oa1-knockout mice to see whether the Oa1-knockout phenotype can be rescued. The primary neural abnormality associated with ocular hypopigmentation is a defect in axonal navigation at the optic chiasm during development, manifested as a misrouting of optic axons from the temporal retina into the opposite side of the brain. The decussation patterns associated with the retinofugal pathways in these various transgenic and knockout mice will be defined using anterograde and retrograde tract-tracing techniques, while various features associated with their RPE will be quantified, including the degree of tyrosinase expression, total melanin content and melanosomal morphology. Having identified the critical stages during development when an RPE-derived signal affects the neural retina altering decussation patterns at the optic chiasm, a subtractive hybridization strategy combined with microarray analysis will be conducted to identify candidate genes involved in this signaling. Differences in gene expression within the neural retina and in RPE cells will be examined in Oa1-knockout and Gi-knockout mice relative to wild-type control mice, and then compared with patterns of differential gene expression derived from albino mice in which a tyrosinase transgene is activated or remains inactive. Using this combination of approaches drawing on the fields of developmental biology, molecular genetics and neuroanatomy, this research program will identify the critical signaling events initiated within the RPE and ultimately manifested at the optic chiasm. Our studies should lead to the development of new approaches for devising therapeutic strategies based on gene therapy or pharmacological manipulations of Gi signaling in order to prevent the visual impairments in ocular albinism and other hypopigmentation mutations.

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to characterize genes involved in human retinal degenerations and to find possible ways to halt or eventually cure these blinding diseases. Specifically, we will study two proteins present in cones, 15A15 and retinoschisin, encoded by genes isolated previously in our laboratory, that may be interrelated in their function. We have found that the 15A15 protein binds to several DNA fragments containing the core consensus sequences for hormone response elements and that it has several features characteristic of coactivators/ co-repressors of nuclear hormone receptors (NHRs). In our first Specific Aim we will: corroborate that 15A15 plays this role studying its protein-protein interactions with GST pull-down assays;investigate if 15A15 interactions with NHRs are hormone-dependent and enhance the transactivation of endogenous NHRs;map the region of 15A15 necessary for these interactions;determine if 15A15 binds directly to response elements present in the promoter of retinoschisin and other genes expressed in cones, regulating their transcription, and if mutations in 15A15 modules abolish transcriptional enhancement of NHRs. Chromatin immunoprecipitation assays will show if 15A15 exists as part of a complex with other factors, in vivo. We will also screen the DNA of patients with retinal degenerations affecting cones for mutations in 15A15 that may result in disease. In our second Specific Aim, we will continue studying retinoschisin, the secreted protein involved in cell-cell interactions that when mutated causes X-linked juvenile retinoschisis (XLRS). We have found that cGMP, Ca2+ and possibly G-proteins may participate in regulation of secretion of retinoschisin from the photoreceptor inner segments. On the basis of these data, we will carry out a systematic study on the mechanisms that control this process, investigating the involvement of membrane and soluble guanylate cyclases (GC), the effects of GC-activating proteins, nitric oxide and carbon monoxide;the participation of rod, cone and Ca2+/calmodulin-dependent PDEs, and the role of guanine deaminase, which converts cGMP into cXMP. We will also determine whether L-type voltage gated Ca2+ channels and intracellular Ca2+ stores, as well as Gbeta-gamma and mGluR8 influence the secretion of retinoschisin. For each photoreceptor component studied, we will start with a pharmacological approach to establish its involvement, followed by its removal from the system with the use of shRNAs to corroborate its effect. Overall, our research will increase the understanding of the interaction and function of 15A15 and retinoschisin, two important proteins in cones. By learning about the biochemical pathways involved in the regulation of retinoschisin secretion in normal physiological conditions, that may be important for potential pharmacological treatment of XLRS, we hope to open new avenues to explore possible ways to rescue vision for diseases for which there are no current cures.

[unreadable] DESCRIPTION (provided by applicant): The retina in lower vertebrates shows a remarkable regenerative ability that is lost in mammals. Multipotent progenitor cells, that are capable of differentiating into a variety of retinal cell types, have been isolated from the ciliary margin and retina of mammalian eyes. Despite their presence, these progenitor cells are normally quiescent and unable to regenerate damaged retina. This proposal will investigate the ability of microvesicles, released from mouse embryonic stem cells, to reactivate mouse retinal progenitor cells. The long term goal of this project is to discover novel ways of reactivating quiescent progenitor cell populations in the human eye, so that regeneration of damaged retina may be possible. [unreadable] [unreadable] Microvesicles are plasma-membrane particles that are released into the extracellular environment. Very recently, microvesicles have been reported from embryonic stem cells cultured in vitro. Our preliminary results show that these embryonic stem cell microvesicles contain RNA and protein. Most interestingly, they contain a specific class of RNA molecules called microRNAs, which are potent regulators of translation. Microvesicles may serve a role in intercellular communication in one of several manners. They may transfer microRNAs, mRNAs, or proteins to cells. Alternatively they can signal cells through surface proteins found on microvesicles. [unreadable] [unreadable] Our first aim is to characterize the RNA and protein contents of mouse embryonic stem cell microvesicles to look for candidates that might alter stem cell programming. We will use microarray analysis and qRT-PCR for mRNA and microRNA profiling, and mass spectrometry, Western blot analysis, and immunocytochemistry for protein profiling. We will also explore in Aim I the ability of these microvesicles to directly transfer RNA or protein to cells in vitro. Our second aim is to determine if these microvesicles can activate the quiescent stem cell population found in the ciliary margin and retina of mouse eyes. We will inject microvesicles into the aqueous, vitreous, and subretinal space of mice and look for increased proliferation of stem cells with BrDU- labeling. Our final aim is to look for endogenous microvesicles in the aqueous and vitreous and also to characterize them using microarray profiling, qRT-PCR, mass spectrometry, Western blot analysis, immunocytochemistry, and electron microscopy. The information generated in this proposal will not only lead to an increased understanding of the role that extrinsic mRNA, microRNAs, and proteins play in determining stem cell fate, but may also identify microvesicles as novel endogenous signaling factors in the eye, and possibly contributing to the stem cell niche. [unreadable] [unreadable]