Rhodopsin Expression Level Affects Rod Outer Segment Morphology and Photoresponse Kinetics

Background: The retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly. Methodology/Principal Findings: The structure and function of rods from hemizygous rhodopsin knockout (R+/−) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/− rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics. Conclusions/Significance: Rhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/− rods to light

Le FORUM, Vol. 40 No. 1

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F.A.R.O.G. FORUM, Vol. 5 No. 7

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Le Forum, Vol. 44 #4

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Evidence for Baseline Retinal Pigment Epithelium Pathology in the Trp1-Cre Mouse

The increasing popularity of the Cre/loxP recombination system has led to the generation of numerous transgenic mouse lines in which Cre recombinase is expressed under the control of organ- or cell-specific promoters. Alterations in retinal pigment epithelium (RPE), a multifunctional cell monolayer that separates the retinal photoreceptors from the choroid, are prevalent in the pathogenesis of a number of ocular disorders, including age-related macular degeneration. To date, six transgenic mouse lines have been developed that target Cre to the RPE under the control of various gene promoters. However, multiple lines of evidence indicate that high levels of Cre expression can be toxic to mammalian cells. In this study, we report that in the Trp1-Cre mouse, a commonly used transgenic Cre strain for RPE gene function studies, Cre recombinase expression alone leads to RPE dysfunction and concomitant disorganization of RPE layer morphology, large areas of RPE atrophy, retinal photoreceptor dysfunction, and microglial cell activation in the affected areas. The phenotype described herein is similar to previously published reports of conditional gene knockouts that used the Trp1-Cre mouse, suggesting that Cre toxicity alone could account for some of the reported phenotypes and highlighting the importance of the inclusion of Cre-expressing mice as controls in conditional gene targeting studies

Increased Choroidal Neovascularization following Laser Induction in Mice Lacking Lysyl Oxidase-like 1

PURPOSE. Age-related degradation of the elastic lamina in Bruch's membrane may have a permissive effect on the growth of choroidal neovascularization (CNV). This study investigated the influence of defective elastic fiber maintenance in the development of laser-induced CNV. METHODS. A mouse lacking lysyl oxidase-like (LOXL)-1, an enzyme essential for elastin polymerization, was studied. The morphologic characteristics of the elastic lamina within Bruch's membrane were examined in mutant and wild-type (WT) eyes. Laser-induced CNV was evaluated by fluorescein angiography and choroidal flat mounts. Immunohistochemistry for elastin was performed on the CNV lesions, and vascular endothelial growth factor (VEGF) levels were determined by ELISA. Soluble elastin and matrix metalloproteinase (MMPs) levels were also analyzed by immunoblotting. RESULTS. The elastic lamina of Bruch's membrane in the LOXL1-deficient mice was fragmented and less continuous than in the WT controls. The mutant mice showed increased levels of soluble elastin peptides and reduced elastin polymer deposition in neovascular membranes. Significantly larger CNV with greater leakage on fluorescein angiography developed in mutant mice. VEGF levels in the RPE/choroid were higher in the knockout mice on days 7 and 14 after laser (P Ͻ 0.05). MT1-MMP (MMP14) was also elevated after laser in the LOXL1 mutant eyes compared to the WT controls. CONCLUSIONS. These results show that a systemic defect in elastic fiber deposition affects Bruch's membrane integrity and leads to more aggressive CNV growth. The latter may be partially mediated by abnormal signaling from the accumulation of soluble elastin peptides. (Invest Ophthalmol Vis Sci. 2008;49:2599 -2605) DOI:10.1167/iovs.07-1508 C horoidal neovascularization (CNV) is the predominant cause of severe visual loss in age-related macular degeneration (AMD) and other macular diseases. Bruch's membrane integrity is known to be compromised in many conditions characterized by choroidal neovascularization, such as traumatic choroidal rupture, angioid streaks, and myopia. Additionally, laser rupture of Bruch's membrane is a well-established means of inducing experimental choroidal neovascularization. Such evidence indicates that Bruch's membrane serves as an important barrier to CNV formation, but the structural characteristics important in this barrier function are still under investigation. Bruch's membrane lies between the neural retina and the choriocapillaris. There are five layers to this structure: the basement membrane of the retinal pigment epithelium, an inner collagenous layer, the central elastic lamina composed of elastic fibers, the outer collagenous layer, and the basement membrane of the choriocapillaris endothelium. Numerous morphologic changes in Bruch's membrane have been described with aging, particularly the development of drusen and basal laminar deposits, but also calcification, changes in thickness, and decreases in hydraulic conductivity. 1 Loss of elastic fibers is a hallmark of connective tissue aging, and the central elastic lamina of Bruch's membrane has long been postulated to have a barrier function against CNV. 2,3 Recently, Chong et al. 5 Elastic fibers are amorphous polymers composed of the protein elastin, known as tropoelastin in its monomeric form. Oxidative deamination of lysine residues, an initial step of elastin polymerization, requires lysyl oxidases. 6 Lysyl oxidaselike (LOXL) protein 1 has been shown to guide the spatially defined deposition of elastin and is essential for the maintenance of elastic fibers. 7 LOXL1-deficient mice develop multiple systemic defects including pelvic organ prolapse, emphysematous changes in the lungs, and vascular abnormalities attributable to a failure in elastic fiber maintenance

A step forward for understanding the morbidity burden in Guinea: a national descriptive study

<p>Abstract</p> <p>Background</p> <p>Little evidence on the burden of disease has been reported about Guinea. This study was conducted to demonstrate the morbidity burden in Guinea and provide basic evidence for setting health priorities.</p> <p>Methods</p> <p>A retrospective descriptive study was designed to present the morbidity burden of Guinea. Morbidity data were extracted from the National Health Statistics Report of Guinea of 2008. The data are collected based on a pyramid of facilities which includes two national hospitals (teaching hospitals), seven regional hospitals, 26 prefectural hospitals, 8 communal medical centers, 390 health centers, and 628 health posts. Morbidity rates were calculated to measure the burden of non-fatal diseases. The contributions of the 10 leading diseases were presented by sex and age group.</p> <p>Results</p> <p>In 2008, a total of 3,936,599 cases occurred. The morbidity rate for males was higher than for females, 461 versus 332 per 1,000 population. Malaria, respiratory infections, diarrheal diseases, helminthiases, and malnutrition ranked in the first 5 places and accounted for 74% of the total burden, respectively having a rate of 148, 64, 33, 32, and 14 per 1,000 population. The elderly aged 65+ had the highest morbidity rate (611 per 1,000 population) followed by working-age population (458 per 1,000 population) and children (396 per 1,000 population) while the working-age population aged 25-64 contributed the largest part (39%) to total cases. The sex- and age-specific spectrum of morbidity burden showed a similar profile except for small variations.</p> <p>Conclusion</p> <p>Guinea has its unique morbidity burden. The ten leading causes of morbidity burden, especially for malaria, respiratory infections, diarrheal diseases, helminthiases, and malnutrition, need to be prioritized in Guinea.</p