bims-mideyd 2022-09-18 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2022–09–18
seven papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

The GPCR Antagonistic Drug CM-20 Stimulates Mitochondrial Activity in Human RPE Cells.
Electrohydrodynamic Jet-Printed Ultrathin Polycaprolactone Scaffolds Mimicking Bruch's Membrane for Retinal Pigment Epithelial Tissue Engineering.
Olaparib, a PARP-1 inhibitor, protects retinal cells from ocular hypertension-associated oxidative damage.
Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells.
Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources.
Tongqiao Mingmu formula alleviates retinal ganglion cell autophagy through PI3K/AKT/mTOR pathway.
Interplay between aging and other factors of the pathogenesis of age-related macular degeneration.

Open Biochem J. 2022 ;pii: e1874091X2206270. [Epub ahead of print]16

The GPCR Antagonistic Drug CM-20 Stimulates Mitochondrial Activity in Human RPE Cells.

Qing Chang, Siquan Chen, Tahua Yang.

   Background: Mitochondrial dysfunction in retinal pigment epithelium (RPE) is a pathogenic factor in age-related macular degeneration (AMD). Improvement of mitochondrial function may ameliorate RPE bioenergetics status, which may in turn nourish the retinal photoreceptors against degenerative loss.
Objective: The purpose of this study is to examine the G-protein coupled receptor (GPCR) antagonistic drug CM-20 in modulating mitochondrial function in RPE cells.
Methods: Human-derived ARPE-19 cell line was differentiated to improve RPE morphology. Dose response of CM-20 was performed to examine mitochondrial membrane potential (MMP). Secondary validation with multiplexed live-cell mitochondrial imaging was performed. Protection of CM-20 to mitochondria against oxidative stress was detected under co-treatment with hydrogen peroxide.
Results: Treatment with CM-20 elicited a dose-dependent increase of MMP. Multiplexed live-cell mitochondrial imaging showed consistent increase of MMP at an optimal concentration of CM-20 (12.5 μM). MMP was significantly reduced under hydrogen peroxide-induced oxidative stress and treatment with CM-20 showed rescue effects to MMP.
Conclusion: CM-20 increases mitochondrial function and protects mitochondria under oxidative stress. As both GPCRs and mitochondria are potential drug targets, retinal neuroprotective testing of CM-20 is warranted in animal models of retinal degeneration.

Keywords:  Age-related macular degeneration (AMD); GPCR; Mitochondria; Multi-target drug; Oxidative stress; Polypharmacology; RPE

DOI:  https://doi.org/10.2174/1874091X-v16-e2206270
Int J Bioprint. 2022 ;8(3): 550

Electrohydrodynamic Jet-Printed Ultrathin Polycaprolactone Scaffolds Mimicking Bruch's Membrane for Retinal Pigment Epithelial Tissue Engineering.

Hang Liu, Fan Wu, Renwei Chen, Yanan Chen, Kai Yao, Zengping Liu, Bhav Harshad Parikh, Linzhi Jing, Tiange Liu, Xinyi Su, Jie Sun, Dejian Huang.

  Age-related macular degeneration (AMD) is the leading cause of visual loss and affects millions of people worldwide. Dysfunction of the retinal pigment epithelium (RPE) is associated with the pathogenesis of AMD. The purpose of this work is to build and evaluate the performance of ultrathin scaffolds with an electrohydrodynamic jet (EHDJ) printing method for RPE cell culture. We printed two types of ultrathin (around 7 μm) polycaprolactone scaffolds with 20 μm and 50 μm pores, which possess mechanical properties resembling that of native human Bruch's membrane and are biodegradable. Light microscopy and cell proliferation assay showed that adult human retinal pigment epithelial (ARPE-19) cells adhered and proliferated to form a monolayer on the scaffolds. The progress of culture matured on the scaffolds was demonstrated by immunofluorescence (actin, ZO-1, and Na+/K+-ATPase) and Western blot analysis of the respective proteins. The RPE cells cultured on EHDJ-printed scaffolds with 20 μm pores presented higher permeability, higher transepithelial potential difference, and higher expression level of Na+/K+-ATPase than those cultured on Transwell inserts. These findings suggest that the EHDJ printing can fabricate scaffolds that mimic Bruch's membrane by promoting maturation of RPE cells to form a polarized and functional monolayered epithelium with potential as an in vitro model for studying retinal diseases and treatment methods.

Keywords:  Electrohydrodynamic jet printing; Polycaprolactone; Retinal pigment epithelium; Ultrathin scaffolds

DOI:  https://doi.org/10.18063/ijb.v8i3.550
Front Cell Dev Biol. 2022 ;10 925835

Olaparib, a PARP-1 inhibitor, protects retinal cells from ocular hypertension-associated oxidative damage.

Yuting Yang, Jihong Wu, Wei Lu, Yiqin Dai, Youjia Zhang, Xinghuai Sun.

  Glaucoma is the most common cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) and relative hypoxia in the retina stimulate the production of reactive oxygen species (ROS), which, in turn, puts the retina and optic nerve under chronic oxidative stress. Emerging evidence has shown that oxidative stress can trigger PARP-1 overactivation, mitochondrial-associated endoplasmic reticulum membrane (MAM) dysregulation, and NLRP3 activation. Oxidative damage can trigger inflammasome activation, and NLRP3 is the only inflammasome associated with MAM dysregulation. In addition, multiple transcription factors are located on the MAM. This study aimed to investigate the protective effects and underlying mechanisms of a PARP-1 inhibitor (olaparib) against chronic ocular hypertension-associated retinal cell damage. We also mimicked hypoxic stimulation of a retinal precursor cell line by exposing the cells to 0.2% O2 in vitro. We discovered that chronic ocular hypertension (COH) induces oxidative damage and MAM dysregulation in the retinal ganglion cells (RGCs). The protein levels of cleaved-PARP and NLRP3 were upregulated in the retinas of the COH rats. Olaparib, a PARP-1 inhibitor, alleviated COH-induced RGC loss, retinal morphological alterations, and photopic negative response amplitude reduction. Olaparib also relieved hypoxic stimulation-induced loss of cell viability and MAM dysregulation. Additionally, some indicators of mitochondrial performance, such as reactive oxygen species accumulation, mitochondrial Ca2+ influx, and mitochondrial membrane potential collapse, decreased after olaparib treatment. Olaparib attenuated the hypoxia-induced upregulation of NLRP3 protein levels as well as the phosphorylation of ERK1/2 and histone H2A.X. These results suggest that olaparib protects RGCs from chronic intraocular pressure elevation in vivo and alleviates the abnormal MAM dysregulation and mitochondrial dysfunction caused by hypoxia in vitro. This protection may be achieved by inhibiting PARP-1 overactivation, NLRP3 upregulation, and phosphorylation of ERK1/2.

Keywords:  RGCs; chronic ocular hypertension; mitochondrial-associated endoplasmic reticulum membrane; olaparib; oxidative damage

DOI:  https://doi.org/10.3389/fcell.2022.925835
Front Cell Dev Biol. 2022 ;10 910040

Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells.

Elke K Markert, Holger Klein, Coralie Viollet, Werner Rust, Benjamin Strobel, Stefan G Kauschke, Bar Makovoz, Heike Neubauer, Remko A Bakker, Timothy A Blenkinsop.

  The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE-derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting "immature" gene regulatory networks.

Keywords:  ARPE-19; RNA-sequencing; age-related macular degeneration; epithelial to mesenchymal transition; iPSC-RPE; proliferative vitreoretinopathy; retinal pigment epithelium; transplantation

DOI:  https://doi.org/10.3389/fcell.2022.910040
Sci Rep. 2022 Sep 16. 12(1): 15563

Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources.

Kakkad Regha, Mayuri Bhargava, Abdurrahmaan Al-Mubaarak, Chou Chai, Bhav Harshad Parikh, Zengping Liu, Claudine See Wei Wong, Walter Hunziker, Kah Leong Lim, Xinyi Su.

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.

DOI: https://doi.org/10.1038/s41598-022-19777-2
Anat Rec (Hoboken). 2022 Sep 13.

Tongqiao Mingmu formula alleviates retinal ganglion cell autophagy through PI3K/AKT/mTOR pathway.

Liyuan Wang, Tianyang Yu, Feixue Dong, Jiayu Xu, Jin Fu, He Sun.

  Glaucoma is a severe blindness-causing optic nerve disease characterized by a loss of retinal ganglion cells (RGCs). Previous studies have shown that the Tongqiao Mingmu (TQMM) formula can reduce retinal and optic nerve damage, but its mechanism of action requires further elucidation. In this study, an RGC injury model was prepared using glutamate and then treated with serum-containing drug from the TQMM formula (hereafter called "TQMM formula serum"). In the glutamate-induced RGC injury model, cell viability decreased with an increase in glutamate concentration, whereas the expression of autophagy-related biomarkers LC3 and Belicin-1 increased. An adenovirus transfection experiment revealed that glutamate markedly promoted autophagic flux in RGCs. Notably, TQMM formula serum inhibited the expression of autophagy-related biomarkers, reduced autophagy flux, and reversed the damage caused by glutamate to RGCs. Furthermore, the PI3K inhibitor LY294002 was used to intervene in the RGC autophagy model and was found to suppress the PI3K/AKT/mTOR pathway and enhance RGC autophagy. However, TQMM formula serum could generate an opposite effect and upregulate the expressions of the PI3K/AKT/mTOR pathway genes and proteins. In conclusion, the TQMM formula can prevent glutamate-induced autophagy in RGCs, possibly by activating the PI3K/AKT/mTOR pathway and reducing the expression of autophagy-related biomarkers LC3 and Belicin-1 to attenuate autophagy and maintain RGC viability.

Keywords:  PI3K/AKT/mTOR; Tongqiao Mingmu formula; autophagy; glaucoma; retinal ganglion cell; 青光眼, 通窍明目方, 视网膜节细胞, 自噬, PI3K/AKT/mTOR

DOI:  https://doi.org/10.1002/ar.25060
Ageing Res Rev. 2022 Sep 13. pii: S1568-1637(22)00177-5. [Epub ahead of print] 101735

Interplay between aging and other factors of the pathogenesis of age-related macular degeneration.

Janusz Blasiak, Piotr Sobczuk, Elzbieta Pawlowska, Kai Kaarniranta.

  Age-related macular degeneration is a complex eye disease with the retina as the target tissue and aging as per definition the most serious risk factor. However, the retina contains over 60 kinds of cells that form different structures, including the neuroretina and retinal pigment epithelium (RPE) which can age at different rates. Other established or putative AMD risk factors can differentially affect the neuroretina and RPE and can differently interplay with aging of these structures. The occurrence of β-amyloid plaques and increased levels of cholesterol in AMD retinas suggest that AMD may be a syndrome of accelerated brain aging. Therefore, the question about the real meaning of age in AMD is justified. In this review we present and update information on how aging may interplay with some aspects of AMD pathogenesis, such as oxidative stress, amyloid beta formation, circadian rhythm, metabolic aging and cellular senescence. Also, we show how this interplay can be specific for photoreceptors, microglia cells and REP cells as well as in Bruch's membrane and the choroid. Therefore, the process of aging may differentially affect different retinal structures. As an accurate quantiﬁcation of biological aging is important for risk stratiﬁcation and early intervention for age-related diseases, the determination how photoreceptors, microglial and RPE cells age in AMD may be helpful for a precise diagnosis and treatment of this largely untreatable disease.

Keywords:  Age-related macular degeneration; biological aging; cellular senescence; chronological aging; circadian rhythm; metabolic aging; microglia; neural retina; retinal pigment epithelium

DOI:  https://doi.org/10.1016/j.arr.2022.101735