10 research outputs found
Phagocytosis-Dependent Ketogenesis in Retinal Pigment Epithelium
Daily, the retinal pigment epithelium (RPE) ingests a bolus of lipid and protein in the form of phagocytized photoreceptor outer segments (OS). The RPE, like the liver, expresses enzymes required for fatty acid oxidation and ketogenesis. This suggests that these pathways play a role in the disposal of lipids from ingested OS, as well as providing a mechanism for recycling metabolic intermediates back to the outer retina. In this study, we examined whether OS phagocytosis was linked to ketogenesis. We found increased levels of β-hydroxybutyrate (β-HB) in the apical medium following ingestion of OS by human fetal RPE and ARPE19 cells cultured on Transwell inserts. No increase in ketogenesis was observed following ingestion of oxidized OS or latex beads. Our studies further defined the connection between OS phagocytosis and ketogenesis in wild-type mice and mice with defects in phagosome maturation using a mouse RPE explant model. In explant studies, the levels of β-HB released were temporally correlated with OS phagocytic burst after light onset. In the Mreg-/- mouse where phagosome maturation is delayed, there was a temporal shift in the release of β-HB. An even more pronounced shift in maximal β-HB production was observed in the Abca4-/- RPE, in which loss of the ATP-binding cassette A4 transporter results in defective phagosome processing and accumulation of lipid debris. These studies suggest that FAO and ketogenesis are key to supporting the metabolism of the RPE and preventing the accumulation of lipids that lead to oxidative stress and mitochondrial dysfunction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc
Phagocytosis-Dependent Ketogenesis in Retinal Pigment Epithelium
Daily, the retinal pigment epithelium (RPE) ingests a bolus of lipid and protein in the form of phagocytized photoreceptor outer segments (OS). The RPE, like the liver, expresses enzymes required for fatty acid oxidation and ketogenesis. This suggests that these pathways play a role in the disposal of lipids from ingested OS, as well as providing a mechanism for recycling metabolic intermediates back to the outer retina. In this study, we examined whether OS phagocytosis was linked to ketogenesis. We found increased levels of β-hydroxybutyrate (β-HB) in the apical medium following ingestion of OS by human fetal RPE and ARPE19 cells cultured on Transwell inserts. No increase in ketogenesis was observed following ingestion of oxidized OS or latex beads. Our studies further defined the connection between OS phagocytosis and ketogenesis in wild-type mice and mice with defects in phagosome maturation using a mouse RPE explant model. In explant studies, the levels of β-HB released were temporally correlated with OS phagocytic burst after light onset. In the Mreg−/− mouse where phagosome maturation is delayed, there was a temporal shift in the release of β-HB. An even more pronounced shift in maximal β-HB production was observed in the Abca4−/− RPE, in which loss of the ATP-binding cassette A4 transporter results in defective phagosome processing and accumulation of lipid debris. These studies suggest that FAO and ketogenesis are key to supporting the metabolism of the RPE and preventing the accumulation of lipids that lead to oxidative stress and mitochondrial dysfunction
The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium
A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ™-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation. © 2014, Springer Science+Business Media New York
The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium
A main requisite in the phagocytosis of ingestedmaterial is a coordinated series of maturation steps which leadto the degradation of ingested cargo. Photoreceptor outersegment (POS) renewal involves phagocytosis of the distaldisk membranes by the retinal pigment epithelium (RPE).Previously, we identified melanoregulin (MREG) as an intra-cellular cargo-sorting protein required for the degradation ofPOS disks. Here, we provide evidence that MREG-dependentprocessing links both autophagic and phagocytic processes inLC3-associated phagocytosis (LAP). Ingested POSphagosomes are associated with endogenous LC3 andMREG. The LC3 association with POSs exhibited propertiesof LAP; it was independent of rapamycin pretreatment, butdependent on Atg5. Loss of MREG resulted in a decrease inthe extent of LC3-POS association. Studies using DQ™-BSAsuggest that loss of MREG does not compromise the associ-ation and fusion of LC3-positive phagosomes with lysosomes.Furthermore, the mechanism of MREG action is likelythrough a protein complex that includes LC3, as determinedby colocalization and immunoprecipitation in both RPE cellsand macrophages. We posit that MREG participates incoordinating the association of phagosomes with LC3 forcontent degradation with the loss of MREG leading tophagosome accumulation
Microtubule-Associated Protein 1 Light Chain 3B, (LC3B) is Necessary to Maintain Lipid-Mediated Homeostasis in the Retinal Pigment Epithelium
Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B), and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B-/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis. © 2018 Dhingra, Bell, Peachey, Daniele, Reyes-Reveles, Sharp, Jun, Bazan, Sparrow, Kim, Philp and Boesze-Battaglia
Microtubule-Associated Protein 1 Light Chain 3B, (LC3B) Is Necessary to Maintain Lipid-Mediated Homeostasis in the Retinal Pigment Epithelium
Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B), and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B-/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis
IV Foro Internacional de Ganadería Sustentable: conectividad ecosistémica y articulación territorial hacia la Agenda 2030
En este espacio plural de análisis y reflexiones, buscamos articular conceptos y posibilidades para los territorios de montaña del centro de México, dando importancia a medios de vida vinculados a la ganadería y su interacción ecosistémica, fundamentado en innovaciones, casos de éxito e iniciativas emblemáticas nacionales e internacionales. La socialización de experiencias es uno de los pilares para transitar hacia la sostenibilidad de los sistemas productivos ganaderos: compartir logros e iniciativas, crear sinergias e identificar vulnerabilidades desde distintos enfoques.GIZ, Agencia de Cooperación Aleman
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The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium
A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ™-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation
mPEG-PAMAM-G4 Nucleic Acid Nanocomplexes: Enhanced Stability, RNase Protection, and Activity of Splice Switching Oligomer and Poly I:C RNA
Dendrimer chemistries have virtually
exploded in recent years with
increasing interest in this class of polymers as gene delivery vehicles.
An effective nucleic acid delivery vehicle must efficiently bind its
cargo and form physically stable complexes. Most importantly, the
nucleic acid must be protected in biological fluids and tissues, as
RNA is extremely susceptible to nuclease degradation. Here, we characterized
the association of nucleic acids with generation 4 PEGylated poly(amidoamine)
dendrimer (mPEG-PAMAM-G4). We investigated the formation, size, and
stability over time of the nanoplexes at various <i>N</i>/<i>P</i> ratios by gel shift and dynamic light scatter
spectroscopy (DLS). Further characterization of the mPEG-PAMAM-G4/nucleic
acid association was provided by atomic force microscopy (AFM) and
by circular dichroism (CD). Importantly, mPEG-PAMAM-G4 complexation
protected RNA from treatment with RNase A, degradation in serum, and
various tissue homogenates. mPEG-PAMAM-G4 complexation also significantly
enhanced the functional delivery of RNA in a novel engineered human
melanoma cell line with splice-switching oligonucleotides (SSOs) targeting
a recombinant luciferase transcript. mPEG-PAMAM-G4 triconjugates formed
between gold nanoparticle (GNP) and particularly manganese oxide (MnO)
nanorods, poly IC, an anticancer RNA, showed enhanced cancer-killing
activity by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) cell viability assay