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

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

Loss of Melanoregulin (MREG) Enhances Cathepsin-D Secretion by the Retinal Pigment Epithelium

Cathepsin-D (Cat-D) is a major proteolytic enzyme in phagocytic cells. In the retinal pigment epithelium (RPE), it is responsible for the daily degradation of photoreceptor outer segments (POSs) to maintain retinal homeostasis. Melanoregulin (MREG)-mediated loss of phagocytic capacity has been linked to diminished intracellular Cat-D activity. Here, we demonstrate that loss of MREG enhances the secretion of intermediate Cat-D (48 kDa), resulting in a net enhancement of extracellular Cat-D activity. These results suggest that MREG is required to maintain Cat-D homeostasis in the RPE and likely plays a protective role in retinal health. In this regard, in the Mreg dsu/dsu mouse, we observe increased basal laminin. Loss of the Mreg dsu allele is not lethal and therefore leads to slow age-dependent changes in the RPE. Thus, we propose that this model will allow us to study potential dysregulatory functions of Cat-D in retinal disease

Loss of Melanoregulin (MREG) Enhances Cathepsin-D Secretion by the Retinal Pigment Epithelium

Abstract Cathepsin-D (Cat-D) is a major proteolytic enzyme in phagocytic cells. In the retinal pigment epithelium (RPE), it is responsible for the daily degradation of photoreceptor outer segments (POSs) to maintain retinal homeostasis. Melanoregulin (MREG)-mediated loss of phagocytic capacity has been linked to diminished intracellular Cat-D activity. Here, we demonstrate that loss of MREG enhances the secretion of intermediate Cat-D (48 kDa), resulting in a net enhancement of extracellular Cat-D activity. These results suggest that MREG is required to maintain Cat-D homeostasis in the RPE and likely plays a protective role in retinal health. In this regard, in the Mreg dsu/dsu mouse, we observe increased basal laminin. Loss of the Mreg dsu allele is not lethal and therefore leads to slow age-dependent changes in the RPE. Thus, we propose that this model will allow us to study potential dysregulatory functions of Cat-D in retinal disease. Copyright © Cambridge University Press, 2013

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

Aggregatibacter Actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

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