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Timothy S Kern - One of the best experts on this subject based on the ideXlab platform.
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Photoreceptor Cells produce inflammatory products that contribute to retinal vascular permeability in a mouse model of diabetes
Diabetologia, 2017Co-Authors: Deoye Tonade, Timothy S Kern, Krzysztof Palczewski, Haitao LiuAbstract:Recent studies suggest that Photoreceptor Cells produce mediators or products that contribute to retinal capillary damage in diabetes. The purpose of this study was to determine if Photoreceptor Cells release soluble factors that contribute to retinal vascular permeability in diabetes. To assess retinal vascular leakage, a streptozotocin-induced mouse model of diabetes, with hyperglycaemia for 8 months, and age-matched control mice, were injected with FITC-BSA. Fluorescence microscopy was used to detect leakage of FITC-BSA from the retinal vasculature into the neural retina. Ex vivo and in vitro experiments were performed to determine if Photoreceptor Cells released products that directly increased retinal endothelial cell permeability or cell death. Effects of products released by Photoreceptors on tight junction and cell adhesion proteins were assessed by quantitative reverse transcription PCR (qRT-PCR). Inflammatory products released by Photoreceptors into media were measured using protein arrays. Eight months duration of diabetes increased retinal vascular permeability in wild-type mice, but this defect was inhibited in opsin-deficient diabetic mice in which Photoreceptor Cells had degenerated earlier. Photoreceptor Cells from diabetic wild-type mice released inflammatory products (e.g. IL-1α, IL-1β, IL-6, IL-12, chemokine C-X-C motif ligand 1 [CXCL1], monocyte chemoattractant protein 1 [MCP-1], CXCL12a, I-309, chemokine ligand 25 [CCL25] and TNF-α), which directly contributed to increased retinal endothelial cell permeability, at least in part via changes in claudin (tight junction) mRNA. Products released from Photoreceptor Cells from diabetic mice or under diabetes-like conditions did not directly kill retinal endothelial Cells in vitro. Photoreceptor Cells can produce inflammatory products that contribute to retinal vascular permeability in mouse models of diabetes.
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Photoreceptor Cells Influence Retinal Vascular Degeneration in Mouse Models of Retinal Degeneration and Diabetes.
Investigative ophthalmology & visual science, 2016Co-Authors: Haitao Liu, Krzysztof Palczewski, Deoye Tonade, Jie Tang, Aicha Saadane, Ivy S. Samuels, Alex Veenstra, Timothy S KernAbstract:PURPOSE Loss of Photoreceptor Cells is associated with retinal vascular degeneration in retinitis pigmentosa, whereas the presence of Photoreceptor Cells is implicated in vascular degeneration in diabetic retinopathy. To investigate how both the absence and presence of Photoreceptors could damage the retinal vasculature, we compared two mouse models of Photoreceptor degeneration (opsin-/- and RhoP23H/P23H ) and control C57Bl/5J mice, each with and without diabetes. METHODS Retinal thickness, superoxide, expression of inflammatory proteins, ERG and optokinetic responses, leukocyte cytotoxicity, and capillary degeneration were evaluated at 1 to 10 months of age using published methods. RESULTS Retinal Photoreceptor Cells degenerated completely in the opsin mutants by 2 to 4 months of age, and visual function subsided correspondingly. Retinal capillary degeneration was substantial while Photoreceptors were still present, but slowed after the Photoreceptors degenerated. Diabetes did not further exacerbate capillary degeneration in these models of Photoreceptor degeneration, but did cause capillary degeneration in wild-type animals. Photoreceptor Cells, however, did not degenerate in wild-type diabetic mice, presumably because the stress responses in these Cells were less than in the opsin mutants. Retinal superoxide and leukocyte damage to retinal endothelium contributed to the degeneration of retinal capillaries in diabetes, and leukocyte-mediated damage was increased in both opsin mutants during Photoreceptor cell degeneration. CONCLUSIONS Photoreceptor Cells affect the integrity of the retinal microvasculature. Deterioration of retinal capillaries in opsin mutants was appreciable while Photoreceptor Cells were present and stressed, but was less after Photoreceptors degenerated. This finding proves relevant to diabetes, where persistent stress in Photoreceptors likewise contributes to capillary degeneration.
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Photoreceptor Cells Produce Inflammatory Mediators That Contribute to Endothelial Cell Death in Diabetes.
Investigative ophthalmology & visual science, 2016Co-Authors: Deoye Tonade, Haitao Liu, Timothy S KernAbstract:PURPOSE Recent studies suggest that Photoreceptor Cells regulate local inflammation in the retina in diabetes. The purpose of this study was to determine if Photoreceptor Cells themselves produce inflammatory proteins in diabetes and if soluble factors released by Photoreceptors in elevated glucose induce inflammatory changes in nearby Cells. METHODS Laser capture microdissection was used to isolate the outer retina (Photoreceptors) from the inner retina in nondiabetic and diabetic mice. Diabetes-induced changes in the expression of inflammatory targets were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. Cell culture experiments were carried out to determine if Photoreceptors in vitro and ex vivo release soluble mediators that can stimulate nearby Cells. Photoreceptor contribution to leukocyte-mediated endothelial cell death was tested using coculture models. RESULTS Messenger ribonucleic acid and protein expression levels for inflammatory proteins intercellular adhesion molecule 1 (ICAM1), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) were increased in Photoreceptors Cells in diabetes. In vitro and ex vivo studies show that Photoreceptor Cells in elevated glucose release mediators that can induce tumor necrosis factor-α in leukocytes and endothelial Cells, but not in glia. The soluble mediators released by Photoreceptor Cells in elevated glucose are regulated by transforming growth factor β-activated kinase 1 and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) signaling. In contrast to enhanced leukocyte-mediated killing of endothelial Cells by leukocytes from wild-type diabetic mice, leukocytes from diabetic mice lacking Photoreceptor Cells (opsin-/-) did not kill endothelial Cells. CONCLUSIONS These data indicate that Photoreceptor Cells are a source of inflammatory proteins in diabetes, and their release of soluble mediators can contribute to the death of retinal capillaries in diabetes.
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Photoreceptor Cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina
Proceedings of the National Academy of Sciences, 2013Co-Authors: Yunpeng Du, Alma Veenstra, Timothy S KernAbstract:Accumulating evidence suggests that Photoreceptor Cells play a previously unappreciated role in the development of early stages of diabetic retinopathy, but the mechanism by which this occurs is not clear. Inhibition of oxidative stress is known to inhibit the vascular lesions of early diabetic retinopathy, and we investigated whether the diabetes-induced oxidative stress in the retina emanates from Photoreceptors. Superoxide generation was assessed in retinas of male C57BL/6J mice made diabetic for 2 mo (4 mo of age when killed) using histochemical (dichlorofluorescein and dihydroethidine) and bioluminescence (lucigenin) methods. Photoreceptors were eliminated in vivo by genetic (opsin(-/-)) and chemical (iodoacetic acid) techniques. Immunoblots were used to measure expression of intercellular adhesion molecule 1 and the inducible form of nitric oxide synthase. Diabetes increased the generation of superoxide by diabetic mouse retina more at night than during the day. Photoreceptors were the major source of reactive oxygen species in the retina, and their deletion (either genetically in opsin(-/-) mice or acutely with iodoacetic acid) inhibited the expected diabetes-induced increase in superoxide and inflammatory proteins in the remaining retina. Both mitochondria and NADPH oxidase contributed to the observed retinal superoxide generation, which could be inhibited in vivo with either methylene blue or apocynin. Photoreceptors are the major source of superoxide generated by retinas of diabetic mice. Pharmaceuticals targeting Photoreceptor oxidative stress could offer a unique therapy for diabetic retinopathy.
Uwe Wolfrum - One of the best experts on this subject based on the ideXlab platform.
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light dependent phosphorylation of bardet biedl syndrome 5 in Photoreceptor Cells modulates its interaction with arrestin1
Cellular and Molecular Life Sciences, 2013Co-Authors: Tyler S Smith, Elisabeth Sehn, Uwe Wolfrum, Benjamin Spitzbarth, Donald R Dugger, Gabi Sternschneider, Susan Bolch, Hugh J Mcdowell, Jeremiah D Tipton, Clay W SmithAbstract:Arrestins are dynamic proteins that move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate Photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the light conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod Photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that Bardet–Biedl syndrome 5 (BBS5) is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in Photoreceptor inner segments, and synaptic regions. Our principal findings in this study are threefold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in Photoreceptor Cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the Photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.
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Intraflagellar transport proteins in ciliogenesis of Photoreceptor Cells.
Biology of the cell, 2011Co-Authors: Tina Sedmak, Uwe WolfrumAbstract:Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian Photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse Photoreceptor Cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron-dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light-sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of Photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in Photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella.
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immunoelectron microscopy of vesicle transport to the primary cilium of Photoreceptor Cells
Methods in Cell Biology, 2009Co-Authors: Tina Sedmak, Elisabeth Sehn, Uwe WolfrumAbstract:Cilia are organelles of high structural complexity. Since the biosynthetic machinery is absent from cilia all their molecular components must be synthesized in organelles of the cytoplasm and subsequently transported to the cilium. Ciliary cargos are thought to be translocated in the membrane of transport vesicles or association with these vesicles to the base of the cilium where the vesicles fuse with the periciliary target membrane for further delivery of their cargo into the ciliary compartment by the intraflagellar transport (IFT). Here we describe a modified preembedding labeling method as an alternative technique to conventional postembedding methods eligible for analyses of ciliary cargo vesicles and the distribution of ciliary molecules in subciliary compartments for immunoelectron microscopy. The preembedding labeling method preserves the antigenicity of ciliary antigens and its application reveals differential localization of individual IFT proteins in vertebrate Photoreceptor cilia. Since membrane vesicles are conserved, the preembedding protocol additionally allows the identification of ciliary cargo vesicles by immunolabeling of individual IFT proteins and ciliary targeting molecules in ciliary Photoreceptor Cells. These results do not only confirm the central function of IFT molecules in ciliary transport, but further strengthen their role in transport processes in the cytoplasm. Furthermore, evidence for different alternative transport routes of cargo vesicles directed to different target membranes is gathered.
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centrins in retinal Photoreceptor Cells regulators in the connecting cilium
Progress in Retinal and Eye Research, 2008Co-Authors: Philipp Trojan, Andreas Giessl, Alexander Pulvermuller, Huiwoog Choe, Norbert Krauss, Uwe WolfrumAbstract:Changes in the intracellular Ca2+ concentration regulate the visual signal transduction cascade directly or more often indirectly through Ca2+-binding proteins. Here we focus on centrins, which are members of a highly conserved subgroup of the EF-hand superfamily of Ca2+-binding proteins in Photoreceptor Cells of the vertebrate retina. Centrins are commonly associated with centrosome-related structures. In mammalian retinal Photoreceptor Cells, four centrin isoforms are expressed as prominent components in the connecting cilium linking the light-sensitive outer segment compartment with the metabolically active inner segment compartment. Our data indicate that Ca2+-activated centrin isoforms assemble into protein complexes with the visual heterotrimeric G-protein transducin. This interaction of centrins with transducin is mediated by binding to the betagamma-dimer of the heterotrimeric G-protein. More recent findings show that these interactions of centrins with transducin are reciprocally regulated via site-specific phosphorylations mediated by the protein kinase CK2. The assembly of centrin/G-protein complexes is a novel aspect of translocation regulation of signalling proteins in sensory Cells, and represents a potential link between molecular trafficking and signal transduction in general.
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differential expression and interaction with the visual g protein transducin of centrin isoforms in mammalian Photoreceptor Cells
Journal of Biological Chemistry, 2004Co-Authors: Andreas Giessl, Alexander Pulvermuller, Philipp Trojan, Jung Hee Park, Huiwoog Choe, Oliver P Ernst, Klaus Peter Hofmann, Uwe WolfrumAbstract:Abstract Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of Photoreceptor Cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of Photoreceptor Cells. Ca2+-triggered assembly of a centrin-transducin complex in the connecting cilium of Photoreceptor Cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtβγ heterodimers. High affinity binding to Gtβγ and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the Photoreceptor cilium. Binding of Cen2 and Cen4 to Gβγ of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions.
Robert S Molday - One of the best experts on this subject based on the ideXlab platform.
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atp binding cassette transporter abca4 and chemical isomerization protect Photoreceptor Cells from the toxic accumulation of excess 11 cis retinal
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Faraz Quazi, Robert S MoldayAbstract:The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone Photoreceptor Cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from Photoreceptors following photoexcitation. How rod and cone Cells prevent the accumulation of 11-cis-retinal in Photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in Photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration.
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rd3 the protein associated with leber congenital amaurosis type 12 is required for guanylate cyclase trafficking in Photoreceptor Cells
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Seifollah Azadi, Laurie L Molday, Robert S MoldayAbstract:Guanylate cyclases, GC1 and GC2, are localized in the light-sensitive outer segment compartment of Photoreceptor Cells, where they play a crucial role in phototransduction by catalyzing the synthesis of cGMP, the second messenger of phototransduction, and regulating intracellular Ca2+ levels in combination with the cGMP-gated channel. Mutations in GC1 are known to cause Leber congenital amaurosis type 1 (LCA1), a childhood disease associated with severe vision loss. Although the enzymatic and regulatory properties of guanylate cyclases have been studied extensively, the molecular determinants responsible for their trafficking in Photoreceptors remain unknown. Here we show that RD3, a protein of unknown function encoded by a gene associated with Photoreceptor degeneration in humans with Leber congenital amaurosis type 12 (LCA12), the rd3 mouse, and rcd2 collie, colocalizes and interacts with GC1 and GC2 in rod and cone Photoreceptor Cells of normal mice. GC1 and GC2 are undetectable in Photoreceptors of the rd3 mouse deficient in RD3 by immunofluorescence microscopy. Cell expression studies show that RD3 mediates the export of GC1 from the endoplasmic reticulum to endosomal vesicles, and that the C terminus of GC1 is required for RD3 binding. Our results indicate that Photoreceptor degeneration in the rd3 mouse, rcd2 dog, and LCA12 patients is caused by impaired RD3-mediated guanylate cyclase expression and trafficking. The resulting deficiency in cGMP synthesis and the constitutive closure of cGMP-gated channels might cause a reduction in intracellular Ca2+ to a level below that required for long-term Photoreceptor cell survival.
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interaction and localization of the retinitis pigmentosa protein rp2 and nsf in retinal Photoreceptor Cells
Biochemistry, 2010Co-Authors: Juha M Holopainen, Laurie L Molday, Christiana L Cheng, Gurp Johal, Jonathan A Coleman, Frank M Dyka, Robert S MoldayAbstract:Retinitis pigmentosa (RP) is a leading cause of inherited blindness with an incidence of 1 in 3500 individuals worldwide. It is a heterogeneous group of retinal degenerative diseases characterized by a reduction in visual field, night blindness and progressive loss of central vision often leading to complete blindness (1–3). RP can be inherited as an autosomal dominant, autosomal recessive or X-linked trait. To date, over 48 different genes have been implicated in the various forms of RP (http://www.sph.uth.tmc.edu/Retnet/) with most genes encoding proteins that are expressed in Photoreceptor or retinal pigment epithelial (RPE) Cells and are critical for Photoreceptor cell structure, function and survival. X-linked RP (XLRP) accounts for approximately 10–20% of RP cases. It is a particularly severe form of the disease, typically resulting in significant vision loss in the first decade and progressing to total blindness by the third or fourth decade of life (3–6). Approximately, 10–15% of the XLRP cases are caused by mutations in the RP2 gene. These include missense, splice-site, nonsense and frame shift mutations (7–11). The RP2 gene encodes a ubiquitously expressed protein of 350 amino acids known as retinitis pigmentosa 2 protein or RP2 (8, 12, 13). Using polyclonal antibodies, Grayson et al. (14) first reported that RP2 is distributed throughout the human retina with immunoreactivity in Photoreceptors extending from the tips of the outer segments to the synaptic terminals. RP2 is both myristoylated and palmitoylated at the N-terminus. This dual post-translational acylation is believed to target the protein to the plasma membrane of Cells (12, 14). The N-terminal region of RP2 consisting of 151 amino acids (amino acid 42–192) shares a 30% sequence identity and partial functional conservation with the tubulin-specific chaperone protein (TBCC) (8, 15). In the presence of tubulin-specific co-factor D (TBCD), RP2 can substitute for TBCC by stimulating the GTPase activity of native tubulin (15). However, RP2 cannot replace TBCC in promoting the assembly of newly folded tubulin into heterodimers. The C-terminal region exhibits sequence and structural homology to nucleoside diphosphate (NDP) kinase, but the function of this domain remains to be determined. A high resolution structure of RP2 has been determined by X-ray crystallography (16). The N-terminal 228 amino acids fold into a β helix domain while the C-terminal domain (amino acid 229–350) forms a ferrredoxin-like α/β structure. RP2 has been shown to bind to GTP bound form of ADP ribosylation factor-like 3 (Arl3), a member of the Arl subfamily of Ras-related GTP-binding proteins (15, 16). The high resolution structure of RP2 as a complex with Arl3-GppNHp and Arl3-GDP-AlF4 has been determined (16). The β-helix domain and a short upstream unstructured segment within the N-terminal region of RP2 serves as a high affinity binding site for Arl3 containing a bound GTP analogue (15–17). Myristoylation of RP2 weakens its interaction with Arl3 (15). Recently, Veltel et al. (17) have shown that RP2 is an efficient GTPase activating protein (GAP) for Arl3. The binding of RP2 to GTP-Arl3 resulted in a 90,000-fold stimulation of the intrinsic GTPase activity of Arl3. Although structural studies of RP2 and its interaction with Arl3 have provided insight into the role of RP2 as a GAP protein for Arl3, the interaction of RP2 with Arl3 and other proteins in Photoreceptor Cells has not been investigated at a molecular level. We have generated a monoclonal antibody to RP2 and used this reagent to examine the distribution of RP2 in rodent and human Photoreceptors and identify proteins that interact with RP2 in the retina. Here, we show that RP2 co-localizes and directly binds N-ethylmaleimide sensitive factor (NSF) in Photoreceptor Cells and cultured cell lines. Our data suggests that RP2 may have multiple functions in Cells related to the trafficking of NSF to the cilium and synaptic region of Photoreceptors in addition to its role as a GAP protein for Arl3.
Michael P. Iuvone - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional screening of key enzymes of energy metabolism to be under daily and circadian regulation in retina and Photoreceptor Cells.
2016Co-Authors: Patrick Vancura, Tanja Wolloscheck, Kenkichi Baba, Gianluca Tosini, Michael P. Iuvone, Rainer SpessertAbstract:Transcript levels of key enzymes of glycolysis (Hk1), gluconeogenesis (Pck2 and G6p), glycogen synthesis (Gys1 and Gsk3) and fatty acid oxidation (Cpt-1a, Cpt-1b, Cpt-1c, Cpt-2, Acadm, Acads, Acadl, Acadvl, Hadha, Hadhb) are recorded in preparations of the whole retina under light/dark (LD) 12:12 (black lines), in preparations of the whole retina under constant darkness (DD) (blue lines) and in microdissected Photoreceptor Cells (PRCs) under light/dark (LD) 12:12 (red lines) using qPCR. The mRNA levels are plotted as a function of Zeitgeber time (ZT) and the lines represent the periodic sinusoidal functions determined by cosinor analysis (solid and broken line for p < 0.05 and p > 0.05 in cosinor analysis). Data represent a percentage of the average value of the transcript amount during the 24-h period. Statistical analysis of transcriptional profiling is provided in Table 2. The value of ZT0 was plotted twice at both ZT0 and ZT24. The solid bars indicate the dark period. Each value represents mean ± SEM (n = 4; each n represents two animals / four retinae). Note that the FAO genes Cpt-1α and Acadm as well as the reference gene Dbp exhibit significant fluctuations in all applied settings.
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essential roles of dopamine d4 receptors and the type 1 adenylyl cyclase in photic control of cyclic amp in Photoreceptor Cells
Journal of Neurochemistry, 2009Co-Authors: Chad R Jackson, Shyam S Chaurasia, Hong Zhou, Rashidul Haque, Daniel R Storm, Michael P. IuvoneAbstract:Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in Photoreceptor Cells. Dopamine D4 receptor (D4R) activation promotes light adaptation and suppresses the light-sensitive pool of cyclic AMP in Photoreceptor Cells. The key signaling pathways involved in regulating cyclic AMP in Photoreceptor Cells have not been identified. In the present study, we show that the light- and D4R-signaling pathways converge on the type 1 Ca2+/calmodulin-stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in Photoreceptor Cells. In addition, we present evidence that D4R activation tonically regulates the expression of AC1 in Photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca2+/calmodulin-stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D4R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D4R, cyclic AMP levels in the dark-adapted retina are significantly lower compared to wild-type retina and are unresponsive to light. These changes in Drd4−/− mice were accompanied by significantly lower Adcy1 mRNA levels in Photoreceptor Cells and lower Ca2+/calmodulin-stimulated adenylyl cyclase activity in retinal membranes compared with wild-type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild-type mice treated chronically with a D4R antagonist, L-745870. Thus, activation of D4R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross-talk between dopamine and photic signaling pathways regulating cyclic AMP in Photoreceptor Cells.
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dopamine modulates diurnal and circadian rhythms of protein phosphorylation in Photoreceptor Cells of mouse retina
European Journal of Neuroscience, 2008Co-Authors: Nikita Pozdeyev, Gianluca Tosini, Li Li, Stanislav Rozov, Michael P. IuvoneAbstract:Many aspects of Photoreceptor metabolism are regulated as diurnal or circadian rhythms. The nature of the signals that drive rhythms in mouse Photoreceptors is unknown. Dopamine amacrine Cells in mouse retina express core circadian clock genes, leading us to test the hypothesis that dopamine regulates rhythms of protein phosphorylation in Photoreceptor Cells. To this end, we investigated the phosphorylation of phosducin, an abundant Photoreceptor-specific phosphoprotein. In mice exposed to a daily light-dark cycle, robust daily rhythms of phosducin phosphorylation and retinal dopamine metabolism were observed. Phospho-phosducin levels were low during the daytime and high at night, and correlated negatively with levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid. The effect of light on phospho-phosducin levels was mimicked by pharmacological activation of dopamine D4 receptors. The amplitude of the diurnal rhythm of phospho-phosducin was reduced by more than 50% in D4 receptor knockout mice, due to higher daytime levels of phospho-phosducin. In addition, the daytime level of phospho-phosducin was significantly elevated by L-745,870, a dopamine D4 receptor antagonist. These data indicate that dopamine and other light-dependent processes cooperatively regulate the diurnal rhythm of phosducin phosphorylation. Under conditions of constant darkness, a circadian rhythm of phosducin phosphorylation was observed, which correlated negatively with a circadian rhythm of 3,4-dihydroxyphenylacetic acid level. The circadian fluctuation of phospho-phosducin was completely abolished by constant infusion of L-745,870, indicating that the rhythm of phospho-phosducin level is driven by dopamine. Thus, dopamine release in response to light and circadian clocks drives daily rhythms of protein phosphorylation in Photoreceptor Cells.
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photic regulation of arylalkylamine n acetyltransferase binding to 14 3 3 proteins in retinal Photoreceptor Cells
Journal of Neuroscience Nursing, 2006Co-Authors: Nikita Pozdeyev, Shyam S Chaurasia, Rashidul Haque, Carla Taylor, Amy Visser, Aamera Thazyeen, Joan L Weller, David C Klein, Michael P. IuvoneAbstract:14-3-3 proteins are a ubiquitous, highly conserved family of chaperone proteins involved in signal transduction, regulation of cell cycle, intracellular trafficking/targeting, cytoskeletal structure, and transcription. Although 14-3-3 proteins are among the most abundant proteins in the CNS, very little is known about their functional roles in the vertebrate retina. In the present study, we demonstrated that Photoreceptors express 14-3-3 protein(s) and identified a 14-3-3 binding partner in Photoreceptor Cells, the melatonin-synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Importantly, our data demonstrate that the binding of 14-3-3 to AANAT is regulated by light, with dramatic functional consequences. During the night in darkness, retinal AANAT is phosphorylated and forms a complex with 14-3-3 proteins with an apparent molecular weight of ∼90 kDa. Phosphorylation of AANAT facilitates the binding of enzyme to 14-3-3 proteins. Within the complex, AANAT is catalytically activated and protected from dephosphorylation and degradation. Light disrupts the AANAT/14-3-3 complex, leading to catalytic inactivation, dephosphorylation, and proteolytic degradation of the enzyme. In the presence of the proteasome inhibitor, lactacystin, light results in the formation of a high molecular weight complex (>150 kDa), which may represent an intermediate in the AANAT degradation process. These findings provide new insight into the roles of 14-3-3 proteins in Photoreceptor Cells and to the mechanisms controlling melatonin synthesis in the vertebrate retina.
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circadian rhythm and photic control of camp level in chick retinal cell cultures a mechanism for coupling the circadian oscillator to the melatonin synthesizing enzyme arylalkylamine n acetyltransferase in Photoreceptor Cells
Brain Research, 2003Co-Authors: Tamara N Ivanova, Michael P. IuvoneAbstract:Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in Photoreceptor Cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3',5'-monophosphate (cAMP) in cultured retinal Cells entrained to a daily light-dark (LD) cycle, as well as the role of Ca(2+) and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the nocturnal increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca(2+) influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca(2+) channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca(2+) influx, cAMP formation, and AANAT activity in retinal Cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in Photoreceptor Cells.
Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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muller glia phagocytose dead Photoreceptor Cells in a mouse model of retinal degenerative disease
The FASEB Journal, 2019Co-Authors: Sanae Sakami, Yoshikazu Imanishi, Krzysztof PalczewskiAbstract:Retinitis pigmentosa is a devastating, blinding disorder that affects 1 in 4000 people worldwide. During the progression of the disorder, phagocytic clearance of dead Photoreceptor cell bodies has a protective role by preventing additional retinal damage from accumulation of cellular debris. However, the Cells responsible for the clearance remain unidentified. Taking advantage of a mouse model of retinitis pigmentosa ( RhoP23H/P23H), we clarified the roles of Muller glia in the phagocytosis of rod Photoreceptor Cells. During the early stage of retinal degeneration, Muller glial Cells participated in the phagocytosis of dying or dead rod Photoreceptors throughout the outer nuclear layer. Nearly 50% of Muller glia engaged in phagocytosis. Among the Muller phagosomes, >90% matured into phagolysosomes. Those observations indicated that Muller glial Cells are the primary contributor to phagocytosis. In contrast, macrophages migrate to the inner part of the outer nuclear layer during Photoreceptor degeneration, participating in the phagocytosis of a limited population of dying or dead Photoreceptor Cells. In healthy retinas of wild-type mice, Muller glial Cells phagocytosed cell bodies of dead rod Photoreceptors albeit at a lower frequency. Taken together, the phagocytic function of Muller glia is responsible for retinal homeostasis and reorganization under normal and pathologic conditions.-Sakami, S., Imanishi, Y., Palczewski, K. Muller glia phagocytose dead Photoreceptor Cells in a mouse model of retinal degenerative disease.
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Photoreceptor Cells produce inflammatory products that contribute to retinal vascular permeability in a mouse model of diabetes
Diabetologia, 2017Co-Authors: Deoye Tonade, Timothy S Kern, Krzysztof Palczewski, Haitao LiuAbstract:Recent studies suggest that Photoreceptor Cells produce mediators or products that contribute to retinal capillary damage in diabetes. The purpose of this study was to determine if Photoreceptor Cells release soluble factors that contribute to retinal vascular permeability in diabetes. To assess retinal vascular leakage, a streptozotocin-induced mouse model of diabetes, with hyperglycaemia for 8 months, and age-matched control mice, were injected with FITC-BSA. Fluorescence microscopy was used to detect leakage of FITC-BSA from the retinal vasculature into the neural retina. Ex vivo and in vitro experiments were performed to determine if Photoreceptor Cells released products that directly increased retinal endothelial cell permeability or cell death. Effects of products released by Photoreceptors on tight junction and cell adhesion proteins were assessed by quantitative reverse transcription PCR (qRT-PCR). Inflammatory products released by Photoreceptors into media were measured using protein arrays. Eight months duration of diabetes increased retinal vascular permeability in wild-type mice, but this defect was inhibited in opsin-deficient diabetic mice in which Photoreceptor Cells had degenerated earlier. Photoreceptor Cells from diabetic wild-type mice released inflammatory products (e.g. IL-1α, IL-1β, IL-6, IL-12, chemokine C-X-C motif ligand 1 [CXCL1], monocyte chemoattractant protein 1 [MCP-1], CXCL12a, I-309, chemokine ligand 25 [CCL25] and TNF-α), which directly contributed to increased retinal endothelial cell permeability, at least in part via changes in claudin (tight junction) mRNA. Products released from Photoreceptor Cells from diabetic mice or under diabetes-like conditions did not directly kill retinal endothelial Cells in vitro. Photoreceptor Cells can produce inflammatory products that contribute to retinal vascular permeability in mouse models of diabetes.
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Photoreceptor Cells Influence Retinal Vascular Degeneration in Mouse Models of Retinal Degeneration and Diabetes.
Investigative ophthalmology & visual science, 2016Co-Authors: Haitao Liu, Krzysztof Palczewski, Deoye Tonade, Jie Tang, Aicha Saadane, Ivy S. Samuels, Alex Veenstra, Timothy S KernAbstract:PURPOSE Loss of Photoreceptor Cells is associated with retinal vascular degeneration in retinitis pigmentosa, whereas the presence of Photoreceptor Cells is implicated in vascular degeneration in diabetic retinopathy. To investigate how both the absence and presence of Photoreceptors could damage the retinal vasculature, we compared two mouse models of Photoreceptor degeneration (opsin-/- and RhoP23H/P23H ) and control C57Bl/5J mice, each with and without diabetes. METHODS Retinal thickness, superoxide, expression of inflammatory proteins, ERG and optokinetic responses, leukocyte cytotoxicity, and capillary degeneration were evaluated at 1 to 10 months of age using published methods. RESULTS Retinal Photoreceptor Cells degenerated completely in the opsin mutants by 2 to 4 months of age, and visual function subsided correspondingly. Retinal capillary degeneration was substantial while Photoreceptors were still present, but slowed after the Photoreceptors degenerated. Diabetes did not further exacerbate capillary degeneration in these models of Photoreceptor degeneration, but did cause capillary degeneration in wild-type animals. Photoreceptor Cells, however, did not degenerate in wild-type diabetic mice, presumably because the stress responses in these Cells were less than in the opsin mutants. Retinal superoxide and leukocyte damage to retinal endothelium contributed to the degeneration of retinal capillaries in diabetes, and leukocyte-mediated damage was increased in both opsin mutants during Photoreceptor cell degeneration. CONCLUSIONS Photoreceptor Cells affect the integrity of the retinal microvasculature. Deterioration of retinal capillaries in opsin mutants was appreciable while Photoreceptor Cells were present and stressed, but was less after Photoreceptors degenerated. This finding proves relevant to diabetes, where persistent stress in Photoreceptors likewise contributes to capillary degeneration.
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dicer1 is essential for survival of postmitotic rod Photoreceptor Cells in mice
The FASEB Journal, 2014Co-Authors: Thomas R Sundermeier, Ning Zhang, Frans Vinberg, Debarshi Mustafi, Hideo Kohno, Marcin Golczak, Xiaodong Bai, Akiko Maeda, Vladimir J Kefalov, Krzysztof PalczewskiAbstract:Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote Photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod Photoreceptor Cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in Photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod Photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod Photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.—Sundermeier, T. R., Zhang, N., Vinberg, F., Mustafi, D., Kohno, H., Golczak, M., Bai, X., Maeda, A., Kefalov, V. J., Palczewski, K. DICER1 is essential for survival of postmitotic rod Photoreceptor Cells in mice.