bims-mideyd 2021-12-05 papers

Cutan Ocul Toxicol. 2021 Nov 30. 1-6

MicroRNA-124 ameliorates autophagic dysregulation in glaucoma via regulation of P2X7-mediated Akt/mTOR signaling.

Glaucoma is a neurodegenerative disease that leads to irrevocable blindness. In glaucoma, even though axonal damage and function deficit culminates in retinal ganglion cell (RGC) degeneration, our knowledge on the autophagic mechanisms and the role of specific microRNAs is still limited. In this study, we investigated the role of microRNA-124 (MiR-124) in surgically induced glaucomatous neurodegeneration using a mouse model. Animals were segregated into four cohorts of 10 each: (i) sham-operated (n = 10); (ii) surgically induced glaucoma (SIG; n = 10); (iii) SIG + miR-124 mimic; (iv) SIG + miR-NC. Chronic elevation of intraocular pressure (IOP) is a critical risk factor for glaucoma. In our study, chronically elevated IOP caused anterograde axonal transport (AAT) defect, increased the autophagic activity (manifested by significantly (p < 0.05) increased LC3-II/LC3-I ratio, beclin-1 and Atg7 protein expressions) and also downmodulated the protein expression of p-Akt and p-mTOR, mediated by the purinergic P2 receptor subtype 7 (P2X7) upmodulation-leading to retinal degeneration. However, administration of miR-124 mimic improved the retinal integrity and function, as indicated by the improved AAT function, normalized the autophagic dysfunction, modulated the protein expression of P2X7-mediated p-Akt and p-mTOR. Hence, we propose that development of miR-124-based advanced therapies might be a potential avenue in the treatment of glaucomatous neurodegeneration.

Keywords: Akt-mTOR signalling; anterograde transport; autophagy; glaucoma; microRNA-124; retinal ganglion cell

DOI: https://doi.org/10.1080/15569527.2021.2003378

Exp Eye Res. 2021 Nov 29. pii: S0014-4835(21)00423-1. [Epub ahead of print] 108857

Effects of fluoroquinolones and tetracyclines on mitochondria of human retinal MIO-M1 cells.

Nasim Salimiaghdam, Lata Singh, Kevin Schneider, Marilyn Chwa, Shari R Atilano, Angele Nalbandian, G Astrid Limb, M Cristina Kenney.

Our goal was to explore the detrimental impacts of ciprofloxacin (CPFX) and tetracycline (TETRA) on human retinal Müller (MIO-M1) cells in vitro. Cells were exposed to 30, 60 and 120 μg/ml of CPFX and TETRA. The cellular metabolism was measured with the MTT assay. The JC-1 and CM-H2DCFDA assays were used to evaluate the levels of mitochondrial membrane potential (MMP) and ROS (reactive oxygen species), respectively. Mitochondrial DNA (mtDNA) copy number, along with gene expression levels associated with apoptotic (BAX, BCL2-L13, BCL2, CASP-3 and CASP-9), inflammatory (IL-6, IL-1β, TGF-α, TGF-β1 and TGF-β2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4) were analyzed via Quantitative Real-Time PCR (qRT-PCR). Bioenergetic profiles were measured using the Seahorse® XF Flux Analyzer. Cells exposed 24 h to 120 μg/ml TETRA demonstrated higher cellular metabolism compared to vehicle-treated cells. At each time points, (i) all TETRA concentrations reduced MMP levels and (ii) ROS levels were reduced by TETRA 120 μg/ml treatment. TETRA caused (i) higher expression of CASP-3, CASP-9, TGF-α, IL-1B, GPX3 and SOD3 but (ii) decreased levels of TGF-B2 and SOD2. ATP production and spare respiratory capacity declined with TETRA treatment. Cellular metabolism was reduced with CPFX 120 μg/ml in all cultures and 60 μg/ml after 72 h. The CPFX 120 μg/ml reduced MMP in all cultures and ROS levels (72 h). CPFX treatment (i) increased expression of CASP-3, CASP-9, and BCL2-L13, (ii) elevated the basal oxygen consumption rate, and (iii) lowered the mtDNA copy numbers and expression levels of TGF-B2, IL-6 and IL-1B compared to vehicle-control cells. We conclude that clinically relevant dosages of bactericidal and bacteriostatic antibiotics can have negative effects on the cellular metabolism and mitochondrial membrane potential of the retinal MIO-M1 cells in vitro. It is noteworthy to mention that apoptotic and inflammatory pathways in exposed cells were affected significantly.

Keywords: Antibiotics; Bactericide; Bacteriostatic; MIO-M1 cells

DOI: https://doi.org/10.1016/j.exer.2021.108857

Oxid Med Cell Longev. 2021 ;2021 4053276

Temporary Upregulation of Nrf2 by Naringenin Alleviates Oxidative Damage in the Retina and ARPE-19 Cells.

Wenpei Chen, Yuxin Ye, Zhongrui Wu, Junli Lin, Yiting Wang, Qi Ding, Xinrong Yang, Wei Yang, Bingqing Lin, Baoqin Lin.

Dry age-related macular degeneration (dAMD) is a chronic degenerative ophthalmopathy that leads to serious burden of visual impairment. Antioxidation in retinal pigment epithelium (RPE) cells is considered as a potential treatment for dAMD. Our previous studies have showed that naringenin (NAR) protects RPE cells from oxidative damage partly through SIRT1-mediated antioxidation. In this study, we tested the hypothesis that the Nrf2 signaling is another protective mechanism of NAR on dAMD. NaIO3-induced mouse retinopathy and ARPE-19 cell injury models were established. Immunochemical staining, immunofluorescence, and western blotting were performed to detect the protein expressions of Nrf2 and HO-1. In addition, ML385 (activity inhibitor of Nrf2) and zinc protoporphyrin (ZnPP, activity inhibitor of HO-1) were applied to explore the effect of NaIO3 or NAR. The results showed that NAR increased the protein expressions of Nrf2 and HO-1 in the retinas in mice exposed to NaIO3 at the early stage. NAR treatment also resulted in a stronger activation of Nrf2 at the early stage in NaIO3-treated ARPE-19 cells. Moreover, inhibition of HO-1 by ZnPP weakened the cytoprotective effect of NAR. The constitutive accumulation and activation of Nrf2 induced by NaIO3 led to the death of RPE cells. However, NAR decreased the protein expressions of Nrf2 and HO-1 towards normal level in the mouse retinas and ARPE-19 cells exposed to NaIO3 at the late stage. Our findings indicate that NAR protects RPE cells from oxidative damage via activating the Nrf2 signaling pathway.

DOI: https://doi.org/10.1155/2021/4053276

Ocul Surf. 2021 Nov 24. pii: S1542-0124(21)00137-3. [Epub ahead of print]23 60-70

Expression of Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2) by human and rabbit meibomian glands and meibocytes.

Chang Rae Rho, Sun Woong Kim, Shelley Lane, Fangyuna Gao, Jinseor Kim, Yilu Xie, Donald J Brown, Dorota Skowronska-Krawczyk, James V Jester.

PURPOSE: Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes.
METHODS: Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70-80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein.
RESULTS: Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a -16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein.
CONCLUSIONS: AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Keywords: AWAT2; Cell culture; Meibocyte; Meibomian gland; Rabbit

DOI: https://doi.org/10.1016/j.jtos.2021.11.010