Biology subjects

Hass, D. T.

Publications and source records attributed to Hass, D. T..

12 recordsLinked to original sources

bioRxiv2026

Chronic Hypoxic Signalling Reprograms Metabolism and Alters Redox and Lipid Homeostasis in Rod Photoreceptors

Photoreceptors are among the most metabolically active cells in the retina and are therefore highly sensitive to fluctuations in oxygen availability. Age-related tissue changes in the eye affect oxygen delivery to the outer retina, which may result in hypoxic stress within photoreceptors and can contribute to disease development and retinal degeneration. To investigate how chronic hypoxic signalling affects photoreceptor metabolism, we examined a rod-specific Vhl knockout mouse (Rod{Delta}Vhl), in which constitutive HIF activation mimics the molecular response to hypoxia. Combining a cell-type-enriched multi-omics approach with metabolic flux analysis, we identified an early metabolic response in the retina of Rod{Delta}Vhl mice prior to degeneration. This response was characterized by a shift towards an oxidative redox environment indicated by a decrease in nucleotide precursors and an increased antioxidant response. While steady-state glycolytic flux remained unchanged, the dynamic 13C-glucose tracing revealed accelerated carbon flow through the three-carbon glycolytic intermediates, indicating a carbon rerouting. Outer segment lipidomics revealed selective remodelling of phosphatidylcholine and phosphatidylethanolamine species toward more oxidation-resistant and elongated acyl chains, supported by early gene upregulation of essential enzymes involved in fatty acid elongation, desaturation and oxidation. Together, these findings indicate a coordinated shift in metabolic and lipid pathways in photoreceptors under chronic hypoxic stress, consistent with an adaptive response that may help preserve outer segment integrity and improve stress resilience.

neuroscience↗

bioRxiv2026

Nutrient microenvironments reprogram RPE metabolism

Induced pluripotent stem cell-derived retinal pigment epithelium (iPSC RPE) has become a widely used model for studying the mechanisms of age-related macular degeneration (AMD). However, the nutrient composition of currently used RPE culture media is highly variable, posing a major challenge to reproducibility in RPE metabolism and phenotype. We systematically investigate how six distinct nutrient microenvironments shape RPE phenotype, function and metabolism in both iPSC RPE and fetal RPE (fRPE). These included MEM, DMEM-HG/F12 basal media, physiological human plasma-like medium (HPLM) supplemented with FBS or B27, and X-VIVO 10. Although canonical RPE markers were expressed across all conditions, B27 supplementation and X-VIVO 10 increased RPE cell size, hexagonality, and transepithelial resistance. Culture in HPLM+FBS induced accumulation of lipid droplets and sub-RPE deposits, whereas X-VIVO 10 resulted in the formation of large intracellular vacuoles. B27 supplementation enhanced basal respiration, while X-VIVO 10 increased glycolytic capacity. Amino acid consumption was broadly conserved across media types, including complete depletion of proline in all conditions by 48 hours; however, lipid and nucleotide metabolism varied substantially between conditions. Notably, B27 supplementation in specific media types reversed the net direction of several metabolites, with creatine, serine and taurine shifting from consumption to production, while riboflavin and guanine shifted from production to consumption. These findings establish the nutrient environment as a key determinant of RPE phenotype, function and metabolism. Our work provides a valuable resource for media selection and interpretation of cellular and metabolic phenotypes relevant to RPE disease modeling.

cell biology↗

bioRxiv2024

Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate ({beta}-HB). Prior work, based on detecting {beta}-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export {beta}-HB across the apical membrane. Here, we compare the accuracy of measuring {beta}-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of {beta}-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete {beta}-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming {beta}-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce {beta}-HB. Altogether, we substantiate {beta}-HB secretion in RPE but find that the secretion is equal apically and basally, RPE {beta}-HB can derive from ketogenic amino acids or fatty acids, and accurate {beta}-HB assessment requires mass spectrometric analysis.

cell biology↗

bioRxiv2023

Acetyl-CoA carboxylase inhibition increases RPE cell fatty acid oxidation and limits apolipoprotein efflux

PurposeIn age-related macular degeneration (AMD) and Sorsbys fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating {beta}-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate {beta}-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. MethodsWe probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13C6-glucose and 13C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). ResultsACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsbys fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. ConclusionsFirsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

biochemistry↗

bioRxiv2023

The Circulation of Metabolites in the Outer Retina

Photoreceptors in the retina of a vertebrates eye are supported by a tissue adjacent to the retina, the retinal pigment epithelium (RPE). The RPE delivers glucose to the outer retina, consumes photoreceptor outer segments discs, and regenerates 11-cis-retinal. Here we address the question of whether photoreceptors also provide metabolic support to the RPE. We use complementary approaches and animal models to show that glucose is the primary fuel for the retina, that photoreceptors are the primary cell type in the retina to consume glucose, and that lactate derived from photoreceptor glucose consumption is transported to and catabolized by the RPE. These data rigorously support and extend the concept of a metabolic ecosystem between photoreceptors and RPE.

biochemistry↗

bioRxiv2023

Medium depth influences O2 availability and metabolism in cultured RPE cells

Structured AbstractO_ST_ABSPurposeC_ST_ABSRPE oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism. MethodsWe altered the availability of O2 to human primary RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCR), glucose consumption, lactate production, 13C6-glucose flux, hypoxia inducible factor (HIF-1) stability, intracellular lipid droplets after a lipid challenge, trans-epithelial electrical resistance, cell morphology, and pigmentation. ResultsMedium volumes commonly employed during RPE culture limit diffusion of O2 to cells, triggering hypoxia, activating HIF-1, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2 availability decrease acetyl-CoA utilization, increase glycolysis, and alter the size and number of intracellular lipid droplets under lipid-rich conditions. ConclusionsDespite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood". As RPE-centric diseases like age-related macular degeneration (AMD) involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2 availability seen with smaller media volumes.

cell biology↗

bioRxiv2021

Monocarboxylate Transporter 1 (MCT1) mediates succinate export in the retina

PurposeSuccinate is exported by the retina and imported by eyecup tissue. The transporter(s) 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. MethodsWe assessed retinal succinate export and eyecup succinate import in short term ex vivo culture using gas chromatography-mass spectrometry. We test the dependence of succinate export and import on pH, proton ionophores, conventional MCT substrates, and the MCT inhibitors AZD3965, AR-C155858, and diclofenac. ResultsSuccinate exits retinal tissue through MCT1 but does not enter RPE through MCT1 or any other MCT. Intracellular succinate levels are a contributing factor that determines if an MCT1-expressing tissue will export succinate. ConclusionsMCT1 facilitates export of succinate from retinas. An unidentified, non-MCT transporter facilitates import of succinate into RPE.

biochemistry↗

bioRxiv2021

Metabolic function in aging retina and retinal pigment epithelium remains robust despite vision loss

PurposeCharacterize how metabolic function in the murine retina and retinal pigment epithelium-choroid-sclera (eyecup) complex is impacted by natural aging. MethodsWe examined scotopic and photopic visual function of young (3-6 months) and aged (23-26 months) C57Bl/6J mice using electroretinograms (ERGs). Metabolic changes in retina and eyecup explants were characterized by measuring uptake and usage of U-13C-glucose or U-13C-glutamine at different timepoints by gas chromatography-mass spectrometry (GC-MS), measuring oxygen consumption rate (OCR) using a perifusion apparatus, and determining ATP levels with a bioluminescence assay. ResultsScotopic and photopic ERG responses declined in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected by the age of the mouse. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. ConclusionsThe ex vivo approach in our study to examine aging glucose and glutamine metabolism in retina and RPE showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The surprising metabolic stability of these tissues ex vivo suggests age-related metabolic alterations in these tissues may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or other structural changes influence ocular metabolism in vivo.

biochemistry↗

bioRxiv2021

Succinate is Broadly Tissue-Permeant and Uncouples Mitochondrial Respiration from ATP Synthesis

Succinate is central in energy metabolism. It is thought to be made and consumed within mitochondria, and not to cross the plasma membrane. Contrary to the canonical role of succinate, it is produced in the retina and released into the extracellular milleau. It is unknown how this affects adjacent retinal pigment epithelium, within eyecup tissue. By measuring consumption of 13C4-succinate and O2 we probe metabolism of the eyecup, we find robust succinate consumption by eyecup but not retina tissue. Succinate-dependant stimulation of eyecup respiration is oligomycin resistant; indicating that in eyecup tissue succinate uncouples mitochondrial ATP synthesis from respiration.

biochemistry↗

bioRxiv2020

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 within a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by relying on the "reverse" succinate dehydrogenase reaction, where fumarate accepts electrons instead of O2. Reverse succinate dehydrogenase activity produces succinate and is enhanced by down-regulation of cytochrome oxidase subunits. Retinas can export the succinate to the neighboring retinal pigment epithelium/choroid complex. There, succinate stimulates O2 consumption several fold and enhances synthesis and release of malate. Malate released from the pigment epithelium can be imported into the retina, where it is converted to fumarate and again used to accept electrons in the reverse succinate dehydrogenase reaction. Our findings show how a 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).

biochemistry↗

bioRxiv2019

Cell Autonomous Neuroprotection by the Mitochondrial Uncoupling Protein 2 in a Mouse Model of Glaucoma

Glaucoma is a group of disorders associated with retinal ganglion cell (RGC) degeneration and death. There is a clear contribution of mitochondrial dysfunction and oxidative stress toward glaucomatous RGC death. Mitochondrial uncoupling protein 2 (Ucp2) is a well-known regulator of oxidative stress that increases cell survival in acute models of oxidative damage. The impact of Ucp2 on cell survival during sub-acute and chronic neurodegenerative conditions however is not yet clear. Herein, we test the hypothesis that increased Ucp2 expression will improve retinal ganglion cell survival in a mouse model of glaucoma. We show that increasing retinal ganglion cell but not glial Ucp2 expression in transgenic animals decreases glaucomatous RGC death, but also that the PPAR-{gamma} agonist rosiglitazone, an endogenous transcriptional activator of Ucp2, does not significantly alter RGC loss during glaucoma. Together, these data support a model whereby increased Ucp2 expression mediates neuroprotection during a long-term oxidative stressor, but that transcriptional activation alone is insufficient to elicit a neuroprotective effect, motivating further research in to the post-transcriptional regulation of Ucp2.

neuroscience↗

bioRxiv2018

Mitochondrial Uncoupling Protein 2 Knockout Promotes Mitophagy to Prevent Retinal Ganglion Cell Death in Glaucoma

Glaucoma is a neurodegenerative disorder characterized by mitochondrial dysfunction and an increase in oxidative damage, leading to retinal ganglion cell (RGC) death. The oxidative status of RGCs is regulated intrinsically and also extrinsically by retinal glia. The mitochondrial uncoupling protein 2 (UCP2) relieves oxidative and neuronal damage in a variety of neurodegenerative disease models. We hypothesized that deletion of Ucp2 in either RGCs or retinal glia would increase retinal damage and retinal ganglion cell death in a mouse model of glaucoma. Paradoxically, we found the reverse, and deletion of mitochondrial UCP2 decreased oxidative protein modification and reduced retinal ganglion cell death in male and female mice. This paradox was resolved after finding that Ucp2 deletion also increased levels of mitophagy in cell culture and retinal tissue. Our data suggest that Ucp2 deletion facilitates increased mitochondrial function by improving quality control. An increase in mitochondrial function explains the resistance of Ucp2-deleted retinas to glaucoma and may provide a therapeutic avenue for other chronic neurodegenerative conditions.\n\nSignificance StatementMany unsolved neurodegenerative conditions result from defects in mitochondrial function. Molecular tools that can manipulate mitochondria will therefore be central to developing neuroprotective therapies. Among the most potent regulators of mitochondrial function are the uncoupling proteins, particularly UCP2. In this manuscript we show that while loss of UCP2 does increase mitochondrial membrane potential and the production of reactive oxygen species, it also initiates an increase in mitophagy that is ultimately neuroprotective. This novel protective consequence of uncoupling protein inhibition may lead to new therapeutic approaches to combat neurodegenerative disease, particularly because pharmacological compounds do exist that can selectively inhibit UCP2.

neuroscience↗