Biology subjects

Eminhizer, M.

Publications and source records attributed to Eminhizer, M..

6 recordsLinked to original sources

bioRxiv2026

Mapping the chaperonin TRiC/CCT interactome in mouse photoreceptors reveals functional significance for energy metabolism

The eukaryotic chaperonin TRiC/CCT is essential for folding a diverse set of proteins, yet its interactome and functional roles in specialized neurons remain incompletely understood. To investigate TRiC-mediated folding in rod photoreceptors, we generated a transgenic mouse line expressing an epitope-tagged Tcp-1 subunit, enabling purification of intact TRiC complexes from retinal tissue. Mass spectrometry identified 226 TRiC-interacting proteins, including known TRiC substrates and co-chaperones as well as numerous novel candidates enriched in RNA processing, cytoskeletal organization, and cell-cycle regulation. Using a TRiC loss-of-function model in which expression of a short splice isoform of phosducin-like protein (PhLPs) competitively inhibits TRiC activity, we observed marked reductions in canonical TRiC substrates, including tubulins, transducin {beta} subunits, and triosephosphate isomerase, as well as secondary alterations in proteins involved in cytoskeletal stability, membrane trafficking, energy metabolism, and phototransduction. Quantitative metabolomic profiling revealed that TRiC deficiency induces a metabolic "energy crisis" characterized by reduced glycolytic- and tricarboxylic acid cycle intermediates, acylcarnitines, ATP, NAD, and NADH, implicating widespread impairment of glucose utilization, mitochondrial bioenergetics, and fatty acid oxidation. Integrative proteomic-metabolomic analysis identified a small subset of proteins, including Rab10 and Anxa1, as potential drivers of these metabolic disruptions, with defective Rab10-dependent GLUT4 trafficking emerging as a plausible mechanism underlying impaired glucose uptake in TRiC-deficient rods. Finally, experiments using a perpetually unfolded G{beta}1 mutant and G{gamma}1-knockout mice demonstrated that substrate overload sequesters TRiC and competitively displaces other clients, exacerbating proteostasis imbalance. Together, our study provides a comprehensive in vivo mapping of the TRiC interactome in mammalian rods, reveals a connection between TRiC-dependent proteostasis and energy metabolism in rods, and indicates a mechanism by which misfolded TRiC substrates exacerbate a proteostasis imbalance that ultimately results in neurodegeneration.

biochemistry↗

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↗

bioRxiv2025

Absolute quantification of TCA cycle intermediates in mouse ocular tissues reveals distinct tissue- and sex-specific mitochondrial metabolism

ObjectiveMitochondrial tricarboxylic acid (TCA) cycle is central to energy production and redox balance in the eye, which must sustain high metabolic activity to support vision. Retinal neurons, the retinal pigment epithelium (RPE), cornea, and lens each have distinct physiological roles and metabolic demands, yet the absolute concentrations of key TCA intermediates and their variation by tissue, sex, and time of day are not well-defined. MethodsTargeted gas chromatography-mass spectrometry was employed to quantify the absolute concentrations of TCA cycle metabolites in mouse ocular tissues collected at 10 AM and 2 PM to capture diurnal variations. Key metabolite ratios were subsequently calculated to provide insight into TCA cycle dynamics across eye tissues. ResultsThe retina showed the highest concentrations of TCA metabolites among all ocular tissues, particularly succinate, citrate, and malate, consistent with its high energy demands. The RPE/choroid demonstrated well-balanced intermediates with the highest -ketoglutarate (-KG)/Isocitrate ratio, reflecting its efficient mitochondrial oxidation and reductive carboxylation. Corneal metabolism was featured by dominant malate, especially in females, suggesting a metabolic adaptation for redox regulation and oxidative stress defense. The lens had uniformly low metabolite levels except for succinate, indicating minimal mitochondrial activity under physiologically low oxygen conditions. Notably, both the cornea and lens showed significant sex-dependent and diurnal variations in TCA cycle intermediates. ConclusionThis study demonstrates distinct tissue-specific mitochondrial metabolism in the eye, reflecting the unique functional and biochemical demands of each tissue. These metabolic signatures may underlie their susceptibility to mitochondrial dysfunction in various ocular diseases.

biochemistry↗

bioRxiv2025

Loss of PIKfyve in Rod Photoreceptors and RPE Cells Leads to Endolysosomal Dysfunction and Retinal Degeneration

Photoreceptor outer segment (OS) degradation is primarily mediated by retinal pigment epithelial (RPE) cells through daily phagocytosis of shed distal OS tips. In contrast, much less is understood about the cell-autonomous mechanisms photoreceptors use to clear mislocalized molecules caused by protein misfolding or trafficking defects. Mislocalized or excess rhodopsin that fails to reach the OS is retained in the inner segment or cell body, where it is presumably degraded via the endolysosomal system. We identify PIKfyve, a phosphoinositide kinase that generates PI(3,5)P2, as a key regulator of this pathway. Using Translating Ribosome Affinity Purification (TRAP), we find that PIKfyve is highly expressed in rod photoreceptors. Rod-specific PIKfyve deletion causes progressive retinal degeneration, marked by inner segment vacuolation, elevated LAMP1/2, thinning of the outer nuclear layer, and eventual loss of rod and cone function. Loss of one copy of PIKfyve in rod photoreceptors accelerates degeneration in P23H rhodopsin mutant mice. In RPE cells, PIKfyve loss disrupts phagocytosis and autophagy, leading to accumulation of rhodopsin, LAMP1, LC3A/B, and lipid droplets, along with metabolic disturbances. These findings demonstrate that PIKfyve is essential for photoreceptor and RPE health by regulating lysosomal function, phagocytosis, autophagy and metabolism, and suggest that enhancing PIKfyve activity could be a therapeutic strategy for retinal degenerative diseases.

neuroscience↗

bioRxiv2025

Beyond Ornithine Metabolism in Gyrate Atrophy: Tissue-Specific Proteomic Insights into Neonatal and Adult OAT Deficiency

Ornithine aminotransferase (OAT) links the urea cycle, TCA cycle, and amino acid metabolism by interconverting ornithine to pyrroline-5-carboxylate and glutamate. Mutations in OAT cause hyperornithinemia and predominantly affect the eye, leading to gyrate atrophy of the choroid and retina (GA), a rare inherited blinding disorder. To understand the early molecular changes that make the eye susceptible to damage, we performed quantitative proteomic and metabolomic profiling of liver, retina, and retinal pigment epithelium and choroid (RPE/Cho) from OAT-deficient (Oatrhg) mice prior to detectable vision impairment. In addition to reduced OAT expression and elevated ornithine, methylation-related metabolites such as N(6)-methyl-lysine were altered in all examined tissues of Oatrhgmice. In the liver, ornithine disposal through the urea cycle was enhanced, together with altered expression of detoxification enzymes and histone H2B proteins. In contrast, the retina had minimal proteomic changes but pronounced alterations in amino acid pathways supporting glutamate homeostasis. The RPE/Cho demonstrated the most extensive proteomic changes, particularly in mitochondrial metabolism, cytoskeleton, and extracellular matrix, along with reductions in metabolites involved energy metabolism and antioxidant capacity. Together, these findings highlight common and tissue-specific impacts of OAT on the liver and ocular tissues and provide insight into early molecular changes that contribute to the selective vulnerability of the eye in GA. Proteomics data are available via ProteomeXchange (PXD063614) and metabolomics data via MassIVE repository (MSV000101103).

biochemistry↗

bioRxiv2025

Dysregulated Proline Metabolism Contributes to Subretinal Fibrosis in Neovascular AMD: Therapeutic Potential of Prolyl-4-Hydroxylase Inhibition

Subretinal fibrosis, a major cause of irreversible vision loss in neovascular age-related macular degeneration (nAMD), is driven by excessive deposition of extracellular matrix such as collagens. While proline metabolism is known to play a critical role in collagen biosynthesis and fibrosis, its involvement in subretinal fibrosis remains unclear. Here, we characterized the progression of fibrovascular lesions in JR5558 mice, observing significant molecular alterations as early as 4 weeks of age and phenotypic changes by 8 weeks. Transcriptomic and metabolomic analyses revealed elevated levels of 4-hydroxyproline, an essential component of collagen, alongside significant alterations of other fibrosis-related pathways. P4HA1, a catalytic subunit of prolyl-4-hydroxylase essential for 4-hydroxyproline biosynthesis, was prominently expressed in fibrotic lesions in retinas of JR5558 and two-stage laser-induced murine models, as well as human eyes with nAMD. Targeting P4HA1 with the small-molecule inhibitor diethyl pythiDC significantly attenuated fibrovascular lesion growth in the JR5558 murine models and reduced collagen turnover in human retinal pigment epithelium cells. Combining diethyl pythiDC with aflibercept had a stronger antifibrotic effect than monotherapies in JR5558 mice. These findings suggest a key contribution of proline metabolism, particularly proline hydroxylation, in subretinal fibrosis. Inhibiting P4HA1 with diethyl pythiDC inhibited fibrosis in the models we studied, offering a novel therapeutic strategy. Further research is warranted to explore the potential benefits of combining existing anti-angiogenic therapies with drugs that inhibit proline metabolism for the management of nAMD-associated fibrosis.

neuroscience↗