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 ß-hydroxybutyrate (ß-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 ß-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 ß-HB. An even more pronounced shift in maximal ß-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.

Microtubule-Associated Protein 1 Light Chain 3 (LC3) Isoforms in RPE and Retina.

Microtubule-associated protein 1 light chain 3 (MAP1LC3), a human homologue of yeast Atg8, is an essential component of autophagy. LC3 plays a critical role in hybrid degradation pathways in which some but not all components of autophagy are coupled with phagocytosis in a process known as LC3-associated phagocytosis (LAP). LC3 exists as three highly homologous isoforms in human (LC3A, LC3B, and LC3C) with two of these (LC3A and LC3B) in mouse. LC3B predominated in both fetal and adult human retinal pigment epithelium (RPE) relative to LC3A and LC3C, while in mouse RPE and neural retina, LC3A and LC3B were expressed at approximately equivalent levels. In situ hybridization studies localized LC3A and LC3B transcripts in the retina and RPE. LC3B protein was detected in C57Bl6/J RPE and retinal lysates and was absent in the LC3BKO mouse.

Cholesterol in the rod outer segment: A complex role in a "simple" system.

The rod outer segment (ROS) of retinal photoreceptor cells consists of disk membranes surrounded by the plasma membrane. It is a relatively uncomplicated system in which to investigate cholesterol distribution and its functional consequences in biologically relevant membranes. The light sensitive protein, rhodopsin is the major protein in both membranes, but the lipid compositions are significantly different in the disk and plasma membranes. Cholesterol is high in the ROS plasma membrane. Disk membranes are synthesized at the base of the ROS and are also high in cholesterol. However, cholesterol is rapidly depleted as the disks are apically displaced. During this apical displacement the disk phospholipid fatty acyl chains become progressively more unsaturated, which creates an environment unfavorable to cholesterol. Membrane cholesterol has functional consequences. The high cholesterol found in the plasma membrane and in newly synthesized disks inhibits the activation of rhodopsin. As disks are apically displaced and cholesterol is depleted rhodopsin becomes more responsive to light. This effect of cholesterol on rhodopsin activation has been shown in both native and reconstituted membranes. The modulation of activity can be at least partially explained by the effect of cholesterol on bulk lipid properties. Cholesterol decreases the partial free volume of the hydrocarbon region of the bilayer and thereby inhibits rhodopsin conformational changes required for activation. However, cholesterol binds to rhodopsin and may directly affect the protein also. Furthermore, cholesterol stabilizes rhodopsin to thermal denaturation. The membrane must provide an environment that allows rhodopsin conformational changes required for activation while also stabilizing the protein to thermal denaturation. Cholesterol thus plays a complex role in modulating the activity and stability of rhodopsin, which have implications for other G-protein coupled receptors.

A Journey of Cytolethal Distending Toxins through Cell Membranes.

The multifunctional role of lipids as structural components of membranes, signaling molecules, and metabolic substrates makes them an ideal partner for pathogens to hijack host cell processes for their own survival. The properties and composition of unique membrane micro-domains such as membrane rafts make these regions a natural target for pathogens as it affords them an opportunity to hijack cell signaling and intracellular trafficking pathways. Cytolethal distending toxins (Cdts), members of the AB2 family of toxins are comprised of three subunits, the active, CdtB unit, and the binding, CdtA-CdtC unit. Cdts are cyclomodulins leading to cell cycle arrest and apoptosis in a wide variety of cell types. Cdts from several species share a requirement for membrane rafts, and often cholesterol specifically for cell binding and CdtB mediated cytotoxicity. In this review we focus on how host-cell membrane bilayer organization contributes to the cell surface association, internalization, and action of bacteria derived cytolethal distending toxins (Cdts), with an emphasis on Aggregatibacter actinomycetemcomitans Cdt.