bims-mideyd 2023-07-09 papers

Issue of 2023‒07‒09
five papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

Cells. 2023 May 10. pii: 1358. [Epub ahead of print]12(10):

Mitochondrial Dysfunction and Impaired Antioxidant Responses in Retinal Pigment Epithelial Cells Derived from a Patient with RCBTB1-Associated Retinopathy.

Zhiqin Huang, Dan Zhang, Shang-Chih Chen, Di Huang, David Mackey, Fred K Chen, Samuel McLenachan.

Mutations in the RCBTB1 gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with RCBTB1-associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated RCBTB1 and NFE2L2 expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that RCBTB1 deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response.

Keywords: RCBTB1; inherited retinal disease; mitochondria; oxidative stress; retinal pigment epithelium

DOI: https://doi.org/10.3390/cells12101358

Curr Neuropharmacol. 2023 Jul 05.

Curcumin as a Perspective Protection for Retinal Pigment Epithelium during Autophagy Inhibition in the Course of Retinal Degeneration.

Roberto Pinelli, Michela Ferrucci, Francesca Biagioni, Violet Bumah, Elena Scaffidi, Stefano Puglisi-Allegra, Francesco Fornai.

Defective autophagy in the retinal pigment epithelium (RPE) is involved in retinal degeneration, mostly in the course of age-related macular degeneration (AMD), which is an increasingly prevalent retinal disorder, eventually leading to blindness. However, most autophagy activators own serious adverse effects when administered systemically. Curcumin is a phytochemical, which induces autophagy with a wide dose-response curve, which brings minimal side effects. Recent studies indicating defective autophagy in AMD were analyzed. Accordingly, in this perspective, we discuss and provide some evidence about the protective effects of curcumin in preventing RPE cell damage induced by the autophagy inhibitor 3-methyladenine (3-MA). Cells from human RPE were administered the autophagy inhibitor 3-MA. The cell damage induced by 3-MA was assessed at light microscopy by hematoxylin eosin, Fluoro Jade-B, and ZO1 immunohistochemistry along with electron microscopy. The autophagy inhibitor 3-MA produces cell loss and cell degeneration of RPE cells. These effects are counteracted dose-dependently by curcumin. In line with the hypothesis that the autophagy machinery is key in sustaining the integrity of the RPE, here we provide evidence that the powerful autophagy inhibitor 3-MA produces dose-dependently cell loss and cell degeneration in cultured RPE cells, while inhibiting autophagy as shown by LC3-II/LC3-I ratio and gold-standard assessment of autophagy through LC3-positive autophagy vacuoles. These effects are prevented dose-dependently by curcumin, which activates autophagy. These data shed the perspective of validating the role of phytochemicals as safe autophagy activators to treat AMD.

Keywords: 3-methyladenine; Phytochemicals; age-related macular degeneration.; autophagy; neurodegeneration; retinal degeneration; retinal pigment epithelium

DOI: https://doi.org/10.2174/1570159X21666230705103839

Invest Ophthalmol Vis Sci. 2023 07 03. 64(10): 1

TBK1 Knockdown Alleviates Axonal Transport Deficits in Retinal Ganglion Cells Via mTORC1 Activation in a Retinal Damage Mouse Model.

Meng Ye, Yuanyuan Hu, Bowen Zhao, Qianxue Mou, Yueqi Ni, Jing Luo, Lu Li, Hong Zhang, Yin Zhao.

Purpose: Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by progressive retinal ganglion cell (RGC) death and optic nerve degeneration. Axonal transport deficits are the earliest crucial pathophysiological changes in glaucoma. Genetic variation in the TANK-binding kinase 1 gene (TBK1) plays a role in the pathogenesis of glaucoma. This study was designed to investigate intrinsic factors underlying RGCs' damage and to explore the molecular mechanism of TBK1 involvement in glaucomatous pathogenesis.Methods: We established a mouse model of acute ocular hypertension and used TBK1 conditional knockdown mice to investigate the role of TBK1 in glaucoma. CTB-Alexa 555 was utilized to evaluate axonal transport in mice. To observe the efficiency of gene knockdown, we performed immunofluorescence staining. Immunoblotting and immunoprecipitation assays were performed to examine protein‒protein colocalization. RT‒qPCR was performed to measure the mRNA levels of Tbk1.
Results: In this study, we found that conditional TBK1 knockdown in RGCs resulted in increased axonal transport and protection against axonal degeneration. Through mechanistic studies, we found that TBK1 inhibited mTORC1 pathway activation by phosphorylating RAPTOR at Ser1189. Phosphorylation of RAPTOR at Ser1189 abrogated the interaction of RAPTOR with the deubiquitinase USP9X, leading to an increase in RAPTOR ubiquitination and a subsequent decline in protein stabilization.
Conclusions: Our study identified a novel mechanism involving an interaction between the glaucoma risk gene TBK1 and the pivotal mTORC1 pathway, which may provide new therapeutic targets in glaucoma and other neurodegenerative diseases.

DOI: https://doi.org/10.1167/iovs.64.10.1

Cells. 2023 May 17. pii: 1408. [Epub ahead of print]12(10):

Red Wine Extract Prevents Oxidative Stress and Inflammation in ARPE-19 Retinal Cells.

Clarisse Cornebise, Maude Perus, François Hermetet, Josep Valls-Fonayet, Tristan Richard, Virginie Aires, Dominique Delmas.

Age-related macular degeneration (AMD) is one of the most commonly occurring ocular diseases worldwide. This degenerative condition affects the retina and leads to the loss of central vision. The current treatments are focused on the late stage of the disease, but recent studies have highlighted the importance and benefits of preventive treatments and how good dietary habits can reduce the risk of progression to an advanced form of the disease. In this context, we studied whether resveratrol (RSV) or a polyphenolic cocktail, red wine extract (RWE), are able to prevent the initiating events of AMD (i.e., oxidative stress and inflammation) in human ARPE-19 retinal pigment epithelial (RPE) cells and macrophages. This study highlights that RWE and RSV can prevent hydrogen peroxide (H2O2) or 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress and can subsequently prevent DNA damage via the inhibition of the ATM (ataxia telangiectasia-mutated)/Chk2 (checkpoint kinase 2) or Chk1 signaling pathways, respectively. Moreover, ELISA assays show that RWE and RSV can prevent the secretion of proinflammatory cytokines in RPE cells and in human macrophages. Interestingly, RWE exhibits a greater protective impact compared to RSV alone, even though RSV was more concentrated when used alone than in the red wine extract. Our results suggest that RWE and RSV may have potential interest as preventive nutritional supplementations against AMD.

Keywords: AMD; inflammation; oxidative stress; polyphenol; prevention; red wine extract; resveratrol

DOI: https://doi.org/10.3390/cells12101408

Biochim Biophys Acta Mol Cell Res. 2023 Jun 29. pii: S0167-4889(23)00102-7. [Epub ahead of print]1870(7): 119530

Inhibiting autophagy by miR-19a-3p/PTEN regulation protected retinal pigment epithelial cells from hyperglycemic damage.

Qiaoyun Gong, Dawei Luo, Haiyan Wang, Xun Xu, Ying Fan, Zhi Zheng, Tianwei Qian.

OBJECTIVE: The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear.METHODS: An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition.
RESULTS: Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition.
CONCLUSIONS: Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR.

Keywords: Autophagy; Hyperglycemia; PTEN; Retinal pigment epithelium cell; microRNA

DOI: https://doi.org/10.1016/j.bbamcr.2023.119530