COLLABORATIONS
We can measure oxygen consumption rates (OCR), a marker of mitochondrial metabolism, continuously over weeks, inside a hypoxia chamber, to understand the effects of oxygen availability on RPE metabolism. OCR is measured by a novel device called Resipher (black rectangle above), enclosed within a hypoxia chamber in a cell culture incubator.
Fuel Preference and Fate in RPE Cultures
The Hurley lab at University of Washington is one of the preeminent retinal metabolism groups in the world, having pioneered the concept of an opposite but synergistic metabolic relationship between photoreceptors and RPE - a metabolic ecosystem. In specific collaboration with Hurley lab super-braniac research fellow Dan Hass, we are seeking to better understand when and if RPE utilizes fatty acids for mitochondrial metabolism (β-oxidation). The RPE takes in an enormous lipid load on a daily basis from both photoreceptor outer segments and from uptake of lipoprotein particles from the choroidal circulation. It has been assumed that the RPE utilizes at least some of this lipid as fuel. However, initial experiments in culture suggest that β-oxidation may be limited. We are seeking to understand what conditions promote β-oxidation, as we think that conditions which promote lipid degradation will decrease the RPE's lipid load and consequently decrease secretion of excess intracellular lipid that contributes to the buildup of pathologic deposits in AMD (drusen and reticular pseudodrusen). This project involves a combination of measuring oxygen consumption rates, C13 tracing of metabolites, and measuring metabolites by both enzymatic and mass spec methods.
The Role of Transcription Factor MYRF in RPE Homeostasis
Supported by a grant from the BrightFocus Foundation, this collaboration seeks to understand the role of the membrane associated transcription factor myelin regulatory factor (MYRF) in RPE homeostasis, and its ability to protect RPE against AMD-relevant insults. MYRF is one of the most highly and specifically expressed transcription factors in the RPE, but it's role in the RPE is largely unknown. While it is important for RPE development, its expression remains high in adulthood, and it is known to control processes in the RPE important for AMD pathogenesis, including the RPE cytoskeleton and complement regulation. This collaboration seeks to better understand MYRF's role in protecting the RPE against disease, using both cell culture and animal models.
The amazing Prasov lab (with photobombing Miller lab PI)
(Left) - Epithelial RPE. (Right) - RPE after EMT.
Vimentin is a marker for EMT. Phalloidin stains actin.
The Role of PKM2 in Regulating Metabolic Reprogramming in
RPE Undergoing Epithelial-Mesenchymal Transition (EMT)
Supported by a grant from Research to Prevent Blindness (RPB), this collaboration seeks to understand the role of the master glycolysis regulator pyruvate kinase (PKM2) in regulating RPE epithelial-mesenchymal (EMT) transition. RPE under stress undergoes a phenotypic switch from an epithelial state to a mesenchymal/fibroblastic state. In an extreme case, this phenotypic switch results in a devastating scarring condition in the retina called proliferative vitreoretinopathy (PVR). In more subtle forms, this phenotypic switch may play a role in the degeneration seen in diseases like macular degeneration (AMD). When the RPE undergoes EMT, it switches its metabolism from oxidative phosphorylation to glycolysis. This project seeks to understand whether reversing this metabolic reprogramming, via manipulation of PKM2 activity, can reverse the EMT switch, thereby providing a possible therapy for both PVR and possibly AMD.