bims-mideyd 2024-06-16 papers

J Vis Exp. 2024 May 24.

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice.

Alyssa Hubal, Amelia Pfaff, Sarah Vos, Mala Upadhyay, Vera Bonilha, Carlos S Subauste.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

DOI: https://doi.org/10.3791/66921

Results Chem. 2023 Dec;pii: 100995. [Epub ahead of print]6

Cysteinyl leukotriene-like metabolites are generated in retinal pigment epithelial cells through glutathionylation/reduction of an oxidatively truncated fragment of arachidonate.

Mikhail Linetsky, Anshula Mondal, Si-Yang Liu, Abby M Hite, Shravani Enduri, Yu-Shiuan Cheng, Beatriz Feijo, Graham Kang, Nana Arhin, Hong Zeng, Olivia R Laniak, John Denker, Robert G Salomon.

γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of arachidonyl phospholipids by radical-induced oxidative cleavage. Metabolism of HNE by Michael addition of glutathione (GSH) followed by reduction of the aldehyde carbonyl produces a GSH derivative of 1,4-dihydroxynonane (DHN)-GSH. Analogous biochemistry was anticipated to produce a GSH derivative of 5,8-dihydroxyoctanoic acid (DHOA-GSH) that has structural and functional similarity to the cysteinyl leukotriene (LT)C4. We now report that exposure of human retinal pigment epithelial cells to CoCl2, an in vitro model of hypoxia-induced oxidative stress, generates DHOA-GSH and two products of its peptidolysis, DHOA-CysGly and DHOA-Cys that resemble LTD4 and LTE4. Identification of these metabolites was confirmed by unambiguous chemical syntheses that also provided a heavy isotope labeled quantitative standard 13C2 15N-DHOA-GSH. The availability of pure samples of these arachidonate metabolites will enable assessment of their biological activities, and testing the hypothesis that øLTs promote pathological inflammation by serving as LT receptor agonists. Because LT biosynthetic enzymes, e.g., 5-lipoxygenase, are not involved in the generation of øLTs in vivo, inhibitors of LT biosynthesis, e.g., Zileuton, are not expected to prevent the generation of øLTs. On the other hand, if øLTs are leukotriene receptor agonists, then the therapeutic effects of leukotriene receptor antagonist drugs, e.g., Montelukast, may include inhibition not only of LT-induced but also øLT-induced LT receptor activation and signaling.

Keywords: Free radical-induced lipid oxidation; Hypoxia-inducible factor; Pseudo leukotrienes; Retinal Pigment Epithelial Cells

DOI: https://doi.org/10.1016/j.rechem.2023.100995

bioRxiv. 2024 Jun 01. pii: 2024.05.29.595206. [Epub ahead of print]

Targeting complement C3a receptor resolves mitochondrial hyperfusion and subretinal microglial activation in progranulin-deficient frontotemporal dementia.

Mutations in progranulin ( GRN ) cause frontotemporal dementia ( GRN -FTD) due to deficiency of the pleiotropic protein progranulin. GRN -FTD exhibits diverse pathologies including lysosome dysfunction, lipofuscinosis, microgliosis, and neuroinflammation. Yet, how progranulin loss causes disease remains unresolved. Here, we report that non-invasive retinal imaging of GRN -FTD patients revealed deficits in photoreceptors and the retinal pigment epithelium (RPE) that correlate with cognitive decline. Likewise, Grn -/- mice exhibit early RPE dysfunction, microglial activation, and subsequent photoreceptor loss. Super-resolution live imaging and transcriptomic analyses identified RPE mitochondria as an early driver of retinal dysfunction. Loss of mitochondrial fission protein 1 (MTFP1) in Grn -/- RPE causes mitochondrial hyperfusion and bioenergetic defects, leading to NF-kB-mediated activation of complement C3a-C3a receptor signaling, which drives further mitochondrial hyperfusion and retinal inflammation. C3aR antagonism restores RPE mitochondrial integrity and limits subretinal microglial activation. Our study identifies a previously unrecognized mechanism by which progranulin modulates mitochondrial integrity and complement-mediated neuroinflammation.

DOI: https://doi.org/10.1101/2024.05.29.595206

Exp Eye Res. 2024 Jun 08. pii: S0014-4835(24)00187-8. [Epub ahead of print]245 109966

Reassessing retinal pigment epithelial ketogenesis: Enzymatic assays for ketone body levels provide inaccurate results.

Gillian A Gulette, Daniel T Hass, Kriti Pandey, Qitao Zhang, John Y S Han, Abbi Engel, Jennifer R Chao, Nancy J Philp, James B Hurley, Jason M L Miller.

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 (β-HB). Prior work, based on detecting β-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export β-HB across the apical membrane. Here, we compare the accuracy of measuring β-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of β-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete β-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming β-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 β-HB. Altogether, we substantiate β-HB secretion in RPE but find that the secretion is equal apically and basally, RPE β-HB can derive from ketogenic amino acids or fatty acids, and accurate β-HB assessment requires mass spectrometric analysis.

Keywords: Beta-hydroxybutyrate; Ketogenesis; Retinal pigment epithelium

DOI: https://doi.org/10.1016/j.exer.2024.109966

Cell Death Dis. 2024 Jun 10. 15(6): 405

Aberrant ER-mitochondria communication is a common pathomechanism in mitochondrial disease.

Patricia Morcillo, Khushbu Kabra, Kevin Velasco, Hector Cordero, Sarah Jennings, Taekyung D Yun, Delfina Larrea, H Orhan Akman, Eric A Schon.

Genetic mutations causing primary mitochondrial disease (i.e those compromising oxidative phosphorylation [OxPhos]) resulting in reduced bioenergetic output display great variability in their clinical features, but the reason for this is unknown. We hypothesized that disruption of the communication between endoplasmic reticulum (ER) and mitochondria at mitochondria-associated ER membranes (MAM) might play a role in this variability. To test this, we assayed MAM function and ER-mitochondrial communication in OxPhos-deficient cells, including cybrids from patients with selected pathogenic mtDNA mutations. Our results show that each of the various mutations studied indeed altered MAM functions, but notably, each disorder presented with a different MAM "signature". We also found that mitochondrial membrane potential is a key driver of ER-mitochondrial connectivity. Moreover, our findings demonstrate that disruption in ER-mitochondrial communication has consequences for cell survivability that go well beyond that of reduced ATP output. The findings of a "MAM-OxPhos" axis, the role of mitochondrial membrane potential in controlling this process, and the contribution of MAM dysfunction to cell death, reveal a new relationship between mitochondria and the rest of the cell, as well as providing new insights into the diagnosis and treatment of these devastating disorders.

DOI: https://doi.org/10.1038/s41419-024-06781-9