bims-mideyd 2022-03-20 papers

Exp Eye Res. 2022 Mar 10. pii: S0014-4835(22)00094-X. [Epub ahead of print] 109013

Differential mitochondrial and cellular responses between H vs. J mtDNA haplogroup-containing human RPE transmitochondrial cybrid cells.

Ana Rubin Panvini, Anzor Gvritishvili, Hannah Galvan, Sonali Nashine, Shari R Atilano, M Cristina Kenney, Joyce Tombran-Tink.

Mitochondrial dysfunction is associated with several retinal degenerative diseases including Age-related Macular Degeneration (AMD). Human mitochondrial DNA (mtDNA) haplogroups are inherited from a common ancestral clan and are defined by specific sets of genetic differences. The purpose of this study was to determine and compare the effects of mtDNA haplogroups H and J on transcriptome regulation and cellular resilience to oxidative stress in human RPE cytoplasmic hybrid (cybrid) cell lines in vitro. ARPE-19 cybrid cell lines containing mtDNA haplogroups H and J were created by fusing platelets obtained from normal individuals containing H and J haplogroups with mitochondria-deficient (Rho0) ARPE-19 cell lines. These cybrids were exposed to oxidative stress using 300 μM hydrogen peroxide (H2O2), following which mitochondrial structural dynamics was studied at varying time points using the mitochondrial markers - TOMM20 (Translocase of Outer Mitochondrial Membrane 20) and Mitotracker. To evaluate mitochondrial function, levels of ROS, ΔΨm and [Ca2+]m were measured using flow cytometry, and ATP levels were measured using luminescence. The H and J cybrid cell transcriptomes were compared using RNAseq to determine how changes in mtDNA regulate gene expression. Inflammatory and angiogenic markers were measured using Luminex assay to understand how these mtDNAs influenced cellular response to oxidative stress. Actin filaments' morphology was examined using confocal microscopy. Following exposure to H2O2 stress, the J cybrids showed increased mitochondrial swelling and perinuclear localization, disturbed fission and fusion, increased calcium uptake (p < 0.05), and higher secreted levels of TNF-α and VEGF (p < 0.001), compared to the H cybrids. Calcium uptake by J cybrids was reduced using an IP3R inhibitor. Thirteen genes involved in mitochondrial complex I and V function, fusion/fission events cellular energy homeostasis, antioxidant defenses, and inflammatory responses, were significantly downregulated with log2 fold changes ranging between -1.5 and -5.1. Actin levels were also significantly reduced in stressed J cybrids (p ≤ 0.001) and disruption in actin filaments was observed. Thirty-eight genes involved in mitochondrial and cellular support functions, were upregulated with log2 fold changes of +1.5 to +5.9 in J cybrids compared to H cybrids. Our results demonstrate significant structural and functional differences between mtDNA haplogroups H and J -containing cybrid cells. Our study suggests that the J mtDNA haplogroup can alter the transcriptome to increase cellular susceptibility to stress and retinal degenerations.

Keywords: AMD; Age-related macular degeneration; Mitochondrial DNA haplogroups; mtDNA H haplogroup; mtDNA J haplogroup

DOI: https://doi.org/10.1016/j.exer.2022.109013

Bioengineered. 2022 Apr;13(4): 8240-8254

Astragaloside-IV alleviates high glucose-induced ferroptosis in retinal pigment epithelial cells by disrupting the expression of miR-138-5p/Sirt1/Nrf2.

Xuyuan Tang, Xiuyi Li, Dongyan Zhang, Wei Han.

Astragaloside-IV (AS-IV) (C41H68O14) is a high-purity natural product extracted from Astragalus, which has demonstrated biological activities. However, the effect of AS-IV on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. In this study, high glucose (HG) was shown to promote ARPE-19 RPE cell death, increase the contents of reactive oxygen species (ROS) and oxidized glutathione (GSSG), and enhance lipid peroxidation density of mitochondrial membrane. In contrast, AS-IV decreased glutathione (GSH) content, mitochondria size and ridge. Addition of iron death inhibitor Ferrostatin-1 (Fer-1) to RPE cells decreased cell dead rate, thus indicating that HG-induced mitochondrial damage occurred due to ferroptosis. AS-IV alleviated HG-induced RPE cell damage. Furthermore, HG decreased levels of silent information regulator 1 (Sirt1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the nucleus of RPE cells; AS-IV could alleviate these effects and increased expression of glutathione peroxidase 4 (GPX4), glutamate cysteine ligase (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC), which are Nrf2 downstream genes. Mechanistically, AS-IV was shown to alleviate the effects of HG by increasing mir-138-5p expression in RPE cells and promoting expression of Sirt1 and Nrf2 in the nucleus. Transfection of mir-138-5p agonist inhibited the regulatory effects of AS-IV on Sirt1 and Nrf2, accompanied by decreased GPX4, GCLM and GCLC levels, and restoration of ferroptosis-related changes. Collectively, HG increased ferroptosis rate in RPE cells. In addition, AS-IV inhibited miR-138-5p expression, subsequently increasing Sirt1/Nrf2 activity and cellular antioxidant capacity to alleviate ferroptosis, resulting decreased cell death, which potentially inhibits the DR pathological process.

Keywords: AS-IV; DR; RPE; astragaloside-IV; diabetic retinopathy; retinal pigment epithelial

DOI: https://doi.org/10.1080/21655979.2022.2049471

J Biol Chem. 2022 Mar 11. pii: S0021-9258(22)00264-2. [Epub ahead of print] 101824

Mitochondrial glutathione peroxidase 4 is indispensable for photoreceptor development and survival in mice.

Kunihiro Azuma, Tomoko Koumura, Ryo Iwamoto, Masaki Matsuoka, Ryo Terauchi, Shu Yasuda, Tomoyasu Shiraya, Sumiko Watanabe, Makoto Aihara, Hirotaka Imai, Takashi Ueta.

Glutathione peroxidase 4 (GPx4) is known for its unique function in the direct detoxification of lipid peroxides in the cell membrane and as a key regulator of ferroptosis, a form of lipid peroxidation-induced non-apoptotic cell death. However, while the cytosolic isoform of GPx4 is considered to play a major role in inhibiting ferroptosis in somatic cells, the roles of the mitochondrial isoform of GPx4 (mGPx4) in cell survival are not yet clear. In the present study we found that mGPx4 knockout (KO) mice exhibit a cone-rod dystrophy-like phenotype in which loss of cone photoreceptors precedes loss of rod photoreceptors. Specifically, in mGPx4 KO mice, cone photoreceptors disappeared prior to their maturation, while rod photoreceptors persisted through maturation but gradually degenerated afterwards. Mechanistically, we demonstrated that vitamin E supplementation significantly ameliorated photoreceptor loss in these mice. Furthermore, liquid chromatography-mass spectrometry showed a significant increase in peroxidized phosphatidylethanolamine (PE) esterified with docosahexaenoic acid (DHA) in the retina of mGPx4 KO mice. We also observed shrunken and uniformly condensed nuclei as well as caspase-3 activation in mGPx4 KO photoreceptors, suggesting that apoptosis was prevalent. Taken together, our findings indicate that mGPx4 is essential for the maturation of cone photoreceptors, but not for the maturation of rod photoreceptors, although it is still critical for the survival of rod photoreceptors after maturation. In conclusion, we reveal novel functions of mGPx4 in supporting development and survival of photoreceptors in vivo.

Keywords: eye; fatty acid; gene knockout; glutathione peroxidase; lipid; lipid peroxidation; mitochondria; oxidative stress; phospholipid; photoreceptor; polyunsaturated fatty acid (PUFA); retina; vitamin E

DOI: https://doi.org/10.1016/j.jbc.2022.101824