Enhanced immunoprecipitation techniques for the identification of RNA-binding protein partners: IGF2BP1 interactions in mammary epithelial cells.

RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested "exclusion-based" purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.

Aerobic Glycolysis in Photoreceptors Supports Energy Demand in the Absence of Mitochondrial Coupling.

Metabolism is adapted to meet energetic needs. Based on the amount of ATP required to maintain plasma membrane potential, photoreceptor energy demands must be high. The available evidence suggests that photoreceptors primarily generate metabolic energy through aerobic glycolysis, though this evidence is based primarily on protein expression and not measurement of metabolic flux. Aerobic glycolysis can be validated by measuring flux of glucose to lactate. Aerobic glycolysis is also inefficient and thus an unexpected adaptation for photoreceptors to make. We measured metabolic rates to determine the energy-generating pathways that support photoreceptor metabolism. We found that photoreceptors indeed perform aerobic glycolysis and this is associated with mitochondrial uncoupling.

Monocarboxylate Transporter 1 (MCT1) Mediates Succinate Export in the Retina.

Purpose: Succinate is exported by the retina and imported by eyecup tissue. The transporters mediating this process have not yet been identified. Recent studies showed that monocarboxylate transporter 1 (MCT1) can transport succinate across plasma membranes in cardiac and skeletal muscle. Retina and retinal pigment epithelium (RPE) both express multiple MCT isoforms including MCT1. We tested the hypothesis that MCTs facilitate retinal succinate export and RPE succinate import. Methods: We assessed retinal succinate export and eyecup succinate import in short-term ex vivo culture using gas chromatography-mass spectrometry. We tested the dependence of succinate export and import on pH, proton ionophores, conventional MCT substrates, and the MCT inhibitors AZD3965, AR-C155858, and diclofenac. Results: Succinate exits retinal tissue through MCT1 but does not enter the RPE through MCT1 or any other MCT. Intracellular succinate levels are a contributing factor that determines if an MCT1-expressing tissue will export succinate. Conclusions: MCT1 facilitates export of succinate from retinas. An unidentified, non-MCT transporter facilitates import of succinate into RPE.

Succinate metabolism in the retinal pigment epithelium uncouples respiration from ATP synthesis.

Fumarate can be a surrogate for O2 as a terminal electron acceptor in the electron transport chain. Reduction of fumarate produces succinate, which can be exported. It is debated whether intact tissues can import and oxidize succinate produced by other tissues. In a previous report, we showed that mitochondria in retinal pigment epithelium (RPE)-choroid preparations can use succinate to reduce O2 to H2O. However, cells in that preparation could have been disrupted during tissue isolation. We now use multiple strategies to quantify intactness of the isolated RPE-choroid tissue. We find that exogenous 13C4-succinate is oxidized by intact cells then exported as fumarate or malate. Unexpectedly, we also find that oxidation of succinate is different from oxidation of other substrates because it uncouples electron transport from ATP synthesis. Retinas produce and export succinate. Our findings imply that retina succinate may substantially increase O2 consumption by uncoupling adjacent RPE mitochondria.

An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice.

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.

Succinate Can Shuttle Reducing Power from the Hypoxic Retina to the O2-Rich Pigment Epithelium.

When O2 is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives in a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by reversing the succinate dehydrogenase reaction to use fumarate to accept electrons instead of O2. Reverse succinate dehydrogenase activity produces succinate and is enhanced by hypoxia-induced downregulation of cytochrome oxidase. Retinas can export the succinate they produce to the neighboring O2-rich retinal pigment epithelium-choroid complex. There, succinate enhances O2 consumption by severalfold. Malate made from succinate in the pigment epithelium can then be imported into the retina, where it is converted to fumarate to again accept electrons in the reverse succinate dehydrogenase reaction. This malate-succinate shuttle can sustain these two tissues by transferring reducing power from an O2-poor tissue (retina) to an O2-rich one (retinal pigment epithelium-choroid).

Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca2+ uptake in photoreceptor mitochondria.

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.

Increasing Ca2+ in photoreceptor mitochondria alters metabolites, accelerates photoresponse recovery, and reveals adaptations to mitochondrial stress.

Photoreceptors are specialized neurons that rely on Ca2+ to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca2+ homeostasis is disrupted. Ca2+ homeostasis is maintained partly by mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), which can influence cytosolic Ca2+ signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca2+ overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca2+ and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca2+ uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca2+ stress by decreasing MICU3, an enhancer of MCU-mediated Ca2+ uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca2+ can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.