Segment Membranes Research Articles

A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa

The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) × rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca(2+))-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca(2+)-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.

The ins and outs of cholesterol in the vertebrate retina

The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterified form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that significantly compromises the structure and function of the retina. Although the relative contributions of de novo synthesis versus extraretinal uptake are not yet known, herein we review what is known about these processes and the dynamics of cholesterol in the vertebrate retina and indicate some future avenues of research in this area.

The Retinal Pigment Epithelium in Health and Disease

Retinal pigment epithelial cells (RPE) constitute a simple layer of cuboidal cells that are strategically situated behind the photoreceptor (PR) cells. The inconspicuousness of this monolayer contrasts sharply with its importance [1]. The relationship between the RPE and PR cells is crucial to sight; this is evident from basic and clinical studies demonstrating that primary dysfunctioning of the RPE can result in visual cell death and blindness. RPE cells carry out many functions including the conversion and storage of retinoid, the phagocytosis of shed PR outer segment membrane, the absorption of scattered light, ion and fluid transport and RPE-PR apposition. The magnitude of the demands imposed on this single layer of cells in order to execute these tasks, will become apparent to the reader of this review as will the number of clinical disorders that take origin from these cells.

Novel Ionophores for Barium‐Selective Electrodes: Synthesis and Analytical Characterization

AbstractA novel way of synthesis is developed for the Ba2+ selective neutral Ionophore 2a: 2,2′‐[1,2‐phenylenebis(oxyethane‐2,1‐diyloxy)]bis(N‐benzyl‐N‐phenylacetamide) and its methyl (2b), buthyl (2c), and hexyl (2d) derivatives. Ba2+ selective electrodes based on Ionophores 2a–d are compared with those with commonly used Ionophore 1: N,N,N′,N′‐tetracyclohexyl‐oxybis(o‐phenyleneoxy) diacetamide. It is shown that Ionophores 2a–d, particularly 2b, are superior for measurements of Ba2+ in the presence of Ca2+, and in acidic solutions. Segmented sandwich membrane studies suggest formation of complexes IL22+ for Ba2+, Ca2+ and Mg2+ ions with Ionophore 2b, while H+ ions apparently form complexes H2L2+. The values of the complex formation constants are consistent with the selectivity coefficients.

Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the beta1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type beta1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the beta1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with beta1(C121W) subunits. We therefore conclude that Na+ channel beta1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.

Functional and behavioral restoration of vision by gene therapy in the guanylate cyclase-1 (GC1) knockout mouse.

BackgroundRecessive mutations in guanylate cyclase-1 (Gucy2d) are associated with severe, early onset Leber congenital amaurosis-1(LCA1). Gucy2d encodes guanylate cyclase (GC1) is expressed in photoreceptor outer segment membranes and produces cGMP in these cells. LCA1 patients present in infancy with severely impaired vision and extinguished electroretinogram (ERG) but retain some photoreceptors in both their macular and peripheral retina for years. Like LCA1 patients, loss of cone function in the GC1 knockout (GC1KO) mouse precedes cone degeneration. The purpose of this study was to test whether delivery of functional GC1 to cone cells of the postnatal GC1KO mouse could restore function to these cells.Methodology/Principal FindingsSerotype 5 AAV vectors containing either a photoreceptor-specific, rhodopsin kinase (hGRK1) or ubiquitous (smCBA) promoter driving expression of wild type murine GC1 were subretinally delivered to one eye of P14 GC1KO mice. Visual function (ERG) was analyzed in treated and untreated eyes until 3 months post injection. AAV-treated, isogenic wild type and uninjected control mice were evaluated for restoration of visual behavior using optomotor testing. At 3 months post injection, all animals were sacrificed, and their treated and untreated retinas assayed for expression of GC1 and localization of cone arrestin. Cone-mediated function was restored to treated eyes of GC1KO mice (ERG amplitudes were ∼45% of normal). Treatment effect was stable for at least 3 months. Robust improvements in cone-mediated visual behavior were also observed, with responses of treated mice being similar or identical to that of wild type mice. AAV-vectored GC1 expression was found in photoreceptors and cone cells were preserved in treated retinas.Conclusions/SignificanceThis is the first demonstration of gene-based restoration of both visual function/vision-elicited behavior and cone preservation in a mammalian model of GC1 deficiency. Importantly, results were obtained using a well characterized, clinically relevant AAV vector. These results lay the ground work for the development of an AAV-based gene therapy vector for the treatment of LCA1.

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

In a previous study, we used differential proteomics to identify retinal proteins whose steady-state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady-state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina-retinal recycling and generation of retinoic acid-we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11-cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Do vertebrate rhodopsins contain an allosteric binding site(s) for retinoids?

We found by microspectrophotometry that β‐ionone partitioned into the outer segment membranes of salamander green‐sensitive (GS) and blue‐sensitive (BS) rods with greater uptake into BS rods. Electrophysiology showed that BS rod rhodopsin, but not that of GS rods, was activated and bleached by β‐ionone. But biochemical assays using rhodopsins formed from recombinant salamander GS rod and BS rod opsins and 11‐cis retinal (A1) showed little activation by β‐ionone. Since salamander photoreceptors normally contain both A1 and 3‐dehydro 11‐cis retinal (A2)‐based visual pigments, we hypothesized that only the latter activated pharmacologically. We recorded BS rods whose native chromophore had been partially replaced by A1. Lowering the amount of A2 rhodopsin decreased pharmacological activation. Because β‐ionone does not occur naturally in the retina, we also examined the effects of vitamin A on rod activity. In contrast to β‐ionone, vitamin A activated rhodopsin in both GS and BS rods, and also sensitized rods to UV light, presumably by resonance energy transfer. Thus the different effects of retinoids on rods argue for the existence of allosteric binding sites on rhodopsins.Research Support: NEI EY011358 and Lions of Massachusetts

Light-Induced Tyrosine Phosphorylation of Rod Outer Segment Membrane Proteins Regulate the Translocation, Membrane Binding and Activation of Type II α Phosphatidylinositol-5-Phosphate 4-Kinase

Type II phosphatidylinositol 5-phosphate 4-kinase (PIPKIIα) catalyzes the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)), an essential lipid second messenger that may be involved in the regulation of phototransduction, neuroprotection, and morphogenesis in the vertebrate retina. Here we report that in rodent and transgenic frogs, the light-mediated activity and membrane binding of PIPKIIα in rod outer segments (ROS) is dependent on tyrosine phosphorylation of ROS proteins. The greater type II α PIP kinase activity in the light-adapted ROS membrane results from light-driven translocation of PIPKIIα from the rod inner segment to ROS, and subsequent binding to the ROS membrane, thus improving access of the kinase to its lipid substrates. These results indicate a novel mechanism of light regulation of the PIPKIIα activity in photoreceptors, and suggest that the greater PIPKIIα activity in light-adapted animals and the resultant accumulation of PI-4,5-P(2) within the ROS membrane may be important for the function of photoreceptor cells.

Phospholipid meets all-trans-retinal: the making of RPE bisretinoids

The lipid phase of the photoreceptor outer segment membrane is essential to the photon capturing and signaling functions of rhodopsin. Rearrangement of phospholipids in the bilayer accompanies the formation of the active intermediates of rhodopsin following photon absorption. Furthermore, evidence for the formation of a condensation product between the photolyzed chromophore all-trans-retinal and phosphatidylethanolamine indicates that phospholipid may also participate in the movement of the retinoid in the membrane. The downside of these interactions is the formation of bisretinoid-phosphatidylethanolamine compounds that accumulate in retinal pigment epithelial cells with age and that are particularly abundant in some retinal disorders. The propensity of these compounds to negatively impact on the cells has been linked to the pathogenesis of some retinal disorders including juvenile onset recessive Stargardt disease and age-related macular degeneration.

Biochemical Analysis of Phenotypic Diversity Associated with Mutations in Codon 244 of the Retinal Degeneration Slow Gene

Mutations in the protein product of the retinal degeneration slow (RDS) gene cause both rod-dominant retinitis pigmentosa and different forms of cone-dominant macular dystrophies. In particular, mutations in codon 244 can cause either of these types of disease. In this study, we examine the biochemical effects of N244H and N244K in an effort to understand the mechanism underlying rod- and cone-dominant defects, respectively. COS-1 cells were cotransfected with either wild-type (WT) RDS or RDS containing an N244H or N244K mutation along with its binding partner, ROM-1 (rod outer segment membrane protein 1). Cell extracts were analyzed for mutant protein stability by Western blot, and localization was examined by immunocytochemistry. Interactions between transfected proteins were assessed by reciprocal co-immunoprecipitation, and nonreducing velocity sedimentation was used to identify the pattern of RDS complex assembly. Interactions were confirmed using GST fusion constructs of WT and mutant RDS in GST pull-down assays from WT mouse retinal extract. In COS-1 cells, recombinant N244H RDS had a weakened ability to assemble into higher-order complexes but retained the ability to co-immunoprecipitate with ROM-1 as well as localize properly throughout the cells. In contrast, recombinant N244K protein did not associate with ROM-1, showed signs of protein aggregation, and colocalized with an ER marker. These experiments support the hypothesis that RDS mutations that interrupt higher-order oligomer formation but still interact with ROM-1 and fold properly in membranes may cause dominant, gain-of-function disease phenotypes while mutations that cause RDS misfolding (and thus incorrect trafficking and assembly) may be associated with a loss-of-function haploinsufficiency phenotype.

Orange Peeling Technologies

Consumer demand for ready-to-eat products has stimulated the development of peeling techniques to prepare fresh-cut fruit and vegetables. The total peeling of oranges, that is, the removal of flavedo and albedo is difficult due to the adherence of the albedo to the segment membrane. Therefore, treatments which make peeling easier have been studied. This review comprehends patents and publications about orange peeling.

In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

To determine the identity of the XAP-1 antigen. The XAP-1 antibody has been used by many investigators and is recognized as an index of photoreceptor outer segment maturity, yet its antigen remains unknown. Previous studies documented that the XAP-1 antigen is a photoreceptor membrane-associated protein. To enrich for this protein, the authors prepared outer segment preparations from mouse retinas. Crude membrane and cytoplasmic fractions from this preparation were then generated using ultracentrifugation. Proteins were solubilized using n-dodecyl beta-D-maltoside and separated using SDS-PAGE. Aliquots of the crude membrane fraction were run on multiple lanes of a single gel, one lane of which was transferred to PVDF membrane and probed with the XAP-1 antibody. The remaining lanes were silver-stained. Very careful alignment of the Western blot with the silver-stained lanes indicated the presence of a single lightly stained band at the same position as the immunopositive band. nanoLC-ESI-MS/MS analysis was performed on the pooled protein bands. On determining the protein identity, confirmatory Western blot analysis and immunohistochemistry studies were performed. Western blot analysis performed using the XAP-1 antibody indicated a single immunoreactive band at approximately 74 kDa in lysates from both total outer segment and crude membrane preparations. No immunoreactive band was present in the cytoplasmic lysate. MS analysis of pooled silver stained bands determined that the XAP-1 antigen is Grp78. Western blot analysis and immunohistochemistry both support this identification. Present evidence indicates that the XAP-1 antigen is Grp78, a protein that has been previously documented in the interphotoreceptor matrix surrounding cones.

Comparative proteomics of a lycopene‐accumulating mutant reveals the important role of oxidative stress on carotenogenesis in sweet orange (Citrus sinensis [L.] osbeck)

A spontaneous sweet orange (Citrus sinenesis [L.] Osbeck) mutant 'Hong Anliu' is of high value due to lycopene accumulation in the pulp. In this study, we analyzed the proteomic alterations in the pulp of 'Hong Anliu' versus its wild type (WT) at four maturing stages by using 2-DE combined with MALDI-TOF-TOF MS. Among the 74 differentially expressed proteins identified, the majority are predicted to be involved in stress response, carbohydrate/energy metabolism and regulation, or protein fate, modification and degradation. Particularly, expression levels of six anti-oxidative enzymes were altered by the mutation; and assays of their respective enzymatic activities indicated an enhanced level of oxidative stress in 'Hong Anliu', implying a regulatory role of oxidative stress on carotenogenesis. This conclusion was further confirmed by our observation that treatment of fruit pulps with tert-butylhydroperoxide (a ROS progenitor) induced lycopene accumulation in 'Hong Anliu' only. Gene expression showed that genes predicted to function upstream of lycopene biosynthesis were generally upregulated in juice sacs, but downregulated in segment membranes in both 'Hong Anliu' and its WT. The result suggests an important role of post-transcriptional regulation on carotenogenesis since lycopene was induced in 'Hong Anliu' but not WT. The result also implies that carotenogenesis in juice sacs and segment membranes of citrus fruits may be regulated by different mechanisms.

Dual roles of polyunsaturated fatty acids in retinal physiology and pathophysiology associated with retinal degeneration

Retinas are highly enriched with long-chain polyunsaturated fatty acids (PUFAs). The reduction of docosahexenoic acid in the photoreceptor outer segment membrane leads to a reduction in the electroretinographic response, suggesting crucial roles for PUFAs in normal retinal physiology. Epidemiological studies suggest a correlation between environmental light exposure and the development/progression of human retinal degenerations such as age-related macular degeneration and retinitis pigmentosa. The double bonds in PUFAs could be target substrates to propagate photooxidative stress in photoreceptors. Light exposure to animals results in selective losses of photoreceptor and retinal pigment epithelial cells and post-translational modifications of retinal proteins by 4-hydroxynonenal and 4-hydroxyhexenal. These end products of nonezymatic oxidation of n-6 and n-3 PUFAs, respectively, are likely to be involved in the light-induced retinal degeneration. Low levels of 4-HNE generated by low levels of environmental light exposure upregulate endogenous redox molecules such as thioreodoxin and thioreodoxin reductase via the nuclearfactor E2-related factor 2/antioxidant-responsive element pathway in the retina, and confer retinal neuroprotection. This paper highlights the dual roles of retinal PUFAs in cellular physiology and pathology.

La Cochrane Library : une base de données et de revues systématiques indispensable à connaître (www.cochrane.org/)

Photoreceptors are polarized neurons, with very specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment, the site of photon capture that initiates vision, an inner segment that houses the biosynthetic machinery and a synaptic terminal for signal transmission to downstream neurons. Outer segments and inner segments are connected by a connecting cilium (CC), the equivalent of a transition zone (TZ) of primary cilia. The connecting cilium is part of the basal body/axoneme backbone that stabilizes the outer segment. This report will update the reader on late developments in photoreceptor ciliogenesis and transition zone formation, specifically in mouse photoreceptors, focusing on early events in photoreceptor ciliogenesis. The connecting cilium, an elongated and narrow structure through which all outer segment proteins and membrane components must traffic, functions as a gate that controls access to the outer segment. Here we will review genes and their protein products essential for basal body maturation and for CC/TZ genesis, sorted by phenotype. Emphasis is given to naturally occurring mouse mutants and gene knockouts that interfere with CC/TZ formation and ciliogenesis.

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

P(4)-ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P(4)-ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P(4)-ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

Orange Peeling Technologies

Consumer demand for ready-to-eat products has stimulated the development of peeling techniques to prepare fresh-cut fruit and vegetables. The total peeling of oranges, that is, the removal of flavedo and albedo is difficult due to the adherence of the albedo to the segment membrane. Therefore, treatments which make peeling easier have been studied. This review comprehends patents and publications about orange peeling. Keywords: Orange, peeling, enzyme, vacuum, pressure, hydrothermal treatment