bims-mideyd 2022-07-03 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2022–07–03
seven papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

Cyanidin-3-glucoside improves the barrier function of retinal pigment epithelium cells by attenuating endoplasmic reticulum stress-induced apoptosis.
Phloretin inhibits glucose transport and reduces inflammation in human retinal pigment epithelial cells.
Sulforaphane alleviates LPS-induced inflammatory injury in ARPE-19 cells by repressing the PWRN2/NF-kB pathway.
Glucose uptake by GLUT1 in photoreceptors is essential for outer segment renewal and rod photoreceptor survival.
Arap1 loss causes retinal pigment epithelium phagocytic dysfunction and subsequent photoreceptor death.
Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer.
Differentiation and Maturation Effect of All-trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy.

Food Res Int. 2022 Jul;pii: S0963-9969(22)00370-2. [Epub ahead of print]157 111313

Cyanidin-3-glucoside improves the barrier function of retinal pigment epithelium cells by attenuating endoplasmic reticulum stress-induced apoptosis.

Wenting Peng, Yalin Wu, Zhenzhen Peng, Wentao Qi, Tingting Liu, Bo Yang, Danxue He, Yixiang Liu, Yong Wang.

  Excessive exposure to blue light from smartphones, computers, and other video equipment causes retinal degeneration. Cyanidin-3-glucoside (C3G) exerts protective effects on retinal cells. However, the mechanism by which C3G enhances the barrier function of retinal pigment epithelial (RPE) cells remains unclear. This study investigated the effects of C3G on blue light-irradiated A2E-containing RPE cells and explored whether or not the endoplasmic reticulum (ER) stress and downstream nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways are involved in the mechanism. Results showed that C3G (10 and 25 μM) observably increased the viability and inhibited the apoptosis of RPE cells. Furthermore, C3G enhanced the barrier function of RPE cells and upregulated the expression of tight junction proteins. Blue light irradiation triggered ER stress, but C3G significantly suppressed the PERK/eIF2α/ATF4/CHOP pathway and maintained normal ER morphology in RPE cells. C3G also activated the Nrf2 pathway to promote RPE survival, which was independent of ER stress modulating Nrf2 activity. This study suggests that C3G promotes the barrier function of RPE cells by regulating ER stress-induced apoptosis, thereby offering a new approach to preventing retinal diseases. Thus, C3G is a potential functional food ingredient to improve visual health.

Keywords:  Blood–retinal barrier function; Cyanidin-3-glucoside; Endoplasmic reticulum stress; Nrf2 pathway; Retinal pigment epithelial cells; Visual health

DOI:  https://doi.org/10.1016/j.foodres.2022.111313
Mol Cell Biochem. 2022 Jun 30.

Phloretin inhibits glucose transport and reduces inflammation in human retinal pigment epithelial cells.

Maria Hytti, Johanna Ruuth, Iiris Kanerva, Niina Bhattarai, Maria L Pedersen, Carsten U Nielsen, Anu Kauppinen.

  During age-related macular degeneration (AMD), chronic inflammatory processes, possibly fueled by high glucose levels, cause a breakdown of the retinal pigment epithelium (RPE), leading to vision loss. Phloretin, a natural dihydroxychalcone found in apples, targets several anti-inflammatory signaling pathways and effectively inhibits transporter-mediated glucose uptake. It could potentially prevent inflammation and cell death of RPE cells through either direct regulation of inflammatory signaling pathways or through amelioration of high glucose levels. To test this hypothesis, ARPE-19 cells were incubated with or without phloretin for 1 h before exposure to lipopolysaccharide (LPS). Cell viability and the release of pro-inflammatory cytokines interleukin 6 (IL-6), IL-8 and vascular endothelial growth factor (VEGF) were measured. Glucose uptake was studied using isotope uptake studies. The nuclear levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were determined alongside the phosphorylation levels of mitogen-activated protein kinases. Phloretin pretreatment reduced the LPS-induced release of IL-6 and IL-8 as well as VEGF. Phloretin increased intracellular levels of reactive oxygen species and nuclear translocation of Nrf2. It also inhibited glucose uptake into ARPE-19 cells and the phosphorylation of Jun-activated kinase (JNK). Subsequent studies revealed that Nrf2, but not the inhibition of glucose uptake or JNK phosphorylation, was the main pathway of phloretin's anti-inflammatory activities. Phloretin was robustly anti-inflammatory in RPE cells and reduced IL-8 secretion via activation of Nrf2 but the evaluation of its potential in the treatment or prevention of AMD requires further studies.

Keywords:  Glucose transport; Inflammation; Nuclear factor erythroid 2-related factor 2 (Nrf2); Phloretin; Retinal degeneration

DOI:  https://doi.org/10.1007/s11010-022-04504-2
Immunopharmacol Immunotoxicol. 2022 Jun 29. 1-9

Sulforaphane alleviates LPS-induced inflammatory injury in ARPE-19 cells by repressing the PWRN2/NF-kB pathway.

Hui Song, Ying-Hao Wang, Hai-Yan Zhou, Kun-Ming Cui.

   BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population and its pathogenesis has been associated with inflammatory damage to retinal pigment epithelial (RPE) cells. Here, we explored the ability of sulforaphane to protect ARPE-19 cells from lipopolysaccharide (LPS)-induced inflammatory injury and elucidated the underlying molecular mechanism.
METHODS: Cell viability, apoptosis, inflammation, PWRN2 expression, nuclear transcription factor-kappa B (NF-kB) activity, and the interaction between PWRN2 and the IkBa protein were assessed in RPE cells under- or over-expressing PWRN2 that had been treated with LPS and sulforaphane.
RESULTS: Overexpression of PWRN2 in LPS-treated cells promoted NF-kB activation by interacting with IkBa, thus reducing cell viability. In contrast, PWRN2 downregulation repressed LPS-induced NF-kB activation and apoptosis in RPE cells. Similarly, sulforaphane downregulated PWRN2 and inhibited NF-kB activation in a concentration-dependent manner. Conversely, PWRN2 overexpression or NF-kB upregulation weakened the anti-inflammatory effects of sulforaphane.
CONCLUSION: Our results suggest that sulforaphane protects RPE cells from LPS-induced inflammatory injury by suppressing the PWRN2/NF-kB pathway.

Keywords:  NF-kB; PWRN2; Sulforaphane; age-related macular degeneration; inflammation; retinal pigment epithelial cells

DOI:  https://doi.org/10.1080/08923973.2022.2090954
FASEB J. 2022 Aug;36(8): e22428

Glucose uptake by GLUT1 in photoreceptors is essential for outer segment renewal and rod photoreceptor survival.

Lauren L Daniele, John Y S Han, Ivy S Samuels, Ravikiran Komirisetty, Nikhil Mehta, Jessica L McCord, Minzhong Yu, Yekai Wang, Kathleen Boesze-Battaglia, Brent A Bell, Jianhai Du, Neal S Peachey, Nancy J Philp.

  Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or rod-specific manner to better understand how glucose is utilized in the retina. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Slc2a1 from retinal neurons and Müller glia results in reduced OS growth and progressive rod but not cone photoreceptor cell death. Rhodopsin levels were severely decreased even at postnatal day 20 when OS length was relatively normal. Arrestin levels were not changed suggesting that glucose uptake is required to synthesize membrane glycoproteins. Rod-specific deletion of Slc2a1 resulted in similar changes in OS length and rod photoreceptor cell death. These studies demonstrate that glucose is an essential carbon source for rod photoreceptor cell OS maintenance and viability.

Keywords:   Slc2a1 ; GLUT1; glucose depervation; photoreceptors; retina; rhodopsin

DOI:  https://doi.org/10.1096/fj.202200369R
Dis Model Mech. 2022 Jun 27. pii: dmm.049343. [Epub ahead of print]

Arap1 loss causes retinal pigment epithelium phagocytic dysfunction and subsequent photoreceptor death.

Andy Shao, Antonio Jacobo Lopez, JiaJia Chen, Addy Tham, Seanne Javier, Alejandra Quiroz, Sonia Frick, Edward M Levine, K C Kent Lloyd, Brian C Leonard, Christopher J Murphy, Thomas M Glaser, Ala Moshiri.

  Retinitis Pigmentosa (RP), a retinal degenerative disease, is the leading cause of heritable blindness. Previously, we described that Arap1-/- mice develop a similar pattern of photoreceptor degeneration. Arap1 is an Arf-directed GTPase-activating protein (GAP) shown to modulate actin cytoskeletal dynamics. Curiously, Arap1 expression was detected in Müller Glia and retinal pigment epithelium (RPE), not the photoreceptors themselves. In this study, we generated conditional knockout (cKO) mice for Müller Glia/RPE, Müller Glia, and RPE via targeting Rlbp1, Glast, and Vmd2 promoters, respectively, to drive Cre recombinase expression to knock out Arap1. Vmd2-Cre Arap1tm1c/tm1c and Rlbp1-Cre Arap1tm1c/tm1c mice, but not Glast-Cre Arap1tm1c/tm1c mice, recapitulated the phenotype originally observed in germline Arap1-/- mice. Mass spectrometry analysis of ARAP1 co-immunoprecipitation identified candidate binding partners of ARAP1, revealing potential interactants involved in phagocytosis, cytoskeletal composition, intracellular trafficking, and endocytosis. Quantification of outer segment (OS) phagocytosis in vivo demonstrated a clear phagocytic defect in Arap1-/- mice compared to Arap1+/+ controls. We conclude that Arap1 expression in RPE is necessary for photoreceptor survival due to its indispensable function in RPE phagocytosis.

Keywords:  Arap1; Degeneration; Phagocytosis; RPE; Retina

DOI:  https://doi.org/10.1242/dmm.049343
Methods Mol Biol. 2022 ;2497 185-206

Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer.

Jessica Pfleger.

  Energy homeostasis is critical for cellular function. Significant increases in energy demand or reduced energy supply, however, often result in cellular dysfunction and death. Since mitochondria are the primary cellular energy source, their impairment is often pathogenic. Accordingly, quantitative measurements of cellular and mitochondrial energy utilization and production are crucial for understanding disease development and progression. In the final step of cellular respiration, specifically, oxidative phosphorylation within the mitochondria, oxygen is consumed and drives ATP production. Herein, we provide the complete protocols for measuring oxygen consumption rates and their coupling to ATP production in intact and permeabilized cells, as well as in mitochondria isolated from tissue using the Seahorse XF Extracellular Flux Analyzer (Agilent Technologies).

Keywords:  Bioenergetics; Cellular respiration; Mitochondrial respiration; Permeabilized cells; Seahorse XF Extracellular Flux Analyzer

DOI:  https://doi.org/10.1007/978-1-0716-2309-1_12
Curr Eye Res. 2022 Jun 28. 1-34

Differentiation and Maturation Effect of All-trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy.

Tingyu Yan, Na Yang, Wei Hu, Xinxin Zhang, Xuedong Li, Youjin Wang, Jun Kong.

   PURPOSE: Clinical trials using fetal retinal pigment epithelium (fRPE), human embryonic stem cell (hESC)-derived RPE, or human induced pluripotent stem cell (hiPSC)-derived RPE for cell-based therapy for degenerative retinal diseases have been carried out. We investigated the culture-induced changes in passaged fRPE, hESC-RPE and hiPSC-RPE cells and explored the differentiation and maturation effect of all-trans retinoic acid (ATRA) on cells for manufacturing and screening high quality RPE cells for clinical transplantation.
METHODS: RPE cell lines were set up and the culture-induced changes in subsequent passages caused by manipulating plating density, dissociation method and repeated passaging were studied by microscope, real-time quantitative PCR, western blot and immunofluorescent assays. Gene and protein expression and functional characteristics of RPE cells incubated with ATRA were evaluated.
RESULTS: Compared with fRPE, hESC-RPE and hiPSC-RPE showed decreased gene and protein expression of RPE markers. RPE cells underwent mesenchymal changes showing increased expression of mesenchymal markers including a-SMA, N-cadherin, fibronectin and decreased expression of RPE markers including RPE65, E-cadherin and ZO-1, as a subsequence of low plating density, inappropriate dissociated method, and repeated passaging. RPE cells treated by ATRA showed increased expression of RPE markers and increased expression of negative complement regulatory proteins (CRPs), and increased transepithelial resistance as well.
CONCLUSIONS: Differences in protein and gene expression among three RPE types exist. ATRA can increase RPE markers, CRPs gene expression in fRPE and stem cell-derived RPE. These can be used to guide the standard of screening RPE cells for clinical translational cell therapy.

Keywords:  all-trans retinoic acid; epithelial mesenchymal transition; human embryonic stem cells; human induced pluripotent stem cells; retinal pigment epithelium

DOI:  https://doi.org/10.1080/02713683.2022.2079144