Ultrastructural localization of retinal guanylate cyclase in human and monkey retinas
ABSTRACT Immuno-imaging with confocal and electron microscopy revealed the localization of retinal guanylate cyclase (RetGC) in human and monkey retinas. Using an antibody against a peptide derived from human RetGC, RetGC was found predominantly in the photoreceptor layer in these retinas, although a small amount of RetGC was detected in various other retinal cells. In particular, the cone outer segments were more densely labeled with the antibody than the rod outer segments. The RetGC in outer segments was localized exclusively in the membrane-rich domains, and appeared to be associated with the marginal region of the disk membrane and/or the plasma membrane. The connecting cilium and its cytoplasmic extension never showed immunoreactivity with the antibody. The localization of RetGC in photoreceptor cells is discussed from the viewpoint of mechanisms for the recovery of photoreceptors to the dark level.
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ABSTRACT: Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated AAV-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually- guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic WT mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.Human gene therapy 12/2012; DOI:10.1089/hum.2012.193 · 3.62 Impact Factor
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ABSTRACT: Analysis of the other segments of rod and cone photoreceptors in vertebrates has provided a rich molecular understanding of how light absorbed by a visual pigment can result in changes in membrane polarity that regulate neurotransmitter release. These events are carried out by a large group of phototransduction proteins that are enriched in the outer segment. However, the mechanisms by which phototransduction proteins are sequestered in the outer segment are not well defined. Insight into those mechanisms has recently emerged from the findings that outer segments arise from the plasma membrane of a sensory cilium, and that intraflagellar transport (IFT), which is necessary for assembly of many types of cilia and flagella, plays a crucial role. Here we review the general features of outer segment assembly that may be common to most sensory cilia as well those that may be unique to the outer segment. Those features illustrate how further analysis of photoreceptor IFT may provide insight into both IFT cargo and the role of alternative IFT kinesins.Developmental Dynamics 08/2008; 237(8):1982-92. DOI:10.1002/dvdy.21554 · 2.67 Impact Factor
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ABSTRACT: Rod and cone cells of the mammalian retina harbor two types of a membrane bound guanylate cyclase (GC), rod outer segment guanylate cyclase type 1 (ROS-GC1) and ROS-GC2. Both enzymes are regulated by small Ca(2+)-binding proteins named GC-activating proteins that operate as Ca2+ sensors and enable cyclases to respond to changes of intracellular Ca2+after illumination. We determined the expression level of ROS-GC2 in bovine ROS preparations and compared it with the level of ROS-GC1 in ROSs. The molar ratio of a ROS-GC2 dimer to rhodopsin was 1 : 13 200. The amount of ROS-GC1 was 25-fold higher than the amount of ROS-GC2. Heterologously expressed ROS-GC2 was differentially activated by GC-activating protein 1 and 2 at low free Ca2+ concentrations. Mutants of GC-activating protein 2 modulated ROS-GC2 in a manner different from their action on ROS-GC1 indicating that the Ca2+ sensitivity of the Ca2+ sensor is controlled by the mode of target-sensor interaction.Journal of Neurochemistry 12/2007; 103(4):1439-46. DOI:10.1111/j.1471-4159.2007.04923.x · 4.24 Impact Factor