Potential Therapeutic Functions of PU-91 and Quercetin in Personalized Cybrids Derived from Patients with Age-Related Macular Degeneration, Keratoconus, and Glaucoma.

The aim of this study is to investigate the therapeutic potential of higher doses of PU-91, quercetin, or in combination on transmitochondrial cybrid cell lines with various mtDNA haplogroups derived from patients with age-related macular degeneration (AMD), glaucoma (Glc), keratoconus (KC), and normal (NL) individuals. Cybrids were treated with PU-91 (P) (200 µM) alone, quercetin (Q) (20 µM) alone, or a combination of PU-91 and quercetin (P+Q) for 48 h. Cellular metabolism and the intracellular levels of reactive oxygen species (ROS) were measured by MTT and H2DCFDA assays, respectively. Quantitative real-time PCR was performed to measure the expression levels of genes associated with mitochondrial biogenesis, antioxidant enzymes, inflammation, apoptosis, and senescence pathways. PU-91(P) (i) improves cellular metabolism in AMD cybrids, (ii) decreases ROS production in AMD cybrids, and (iii) downregulates the expression of LMNB1 in AMD cybrids. Combination treatment of PU-91 plus quercetin (P+Q) (i) improves cellular metabolism in AMD, (ii) induces higher expression levels of TFAM, SOD2, IL6, and BAX in AMD cybrids, and (iii) upregulates CDKN1A genes expression in all disease cybrids. Our study demonstrated that the P+Q combination improves cellular metabolism and mitochondrial biogenesis in AMD cybrids, but senescence is greatly exacerbated in all cybrids regardless of disease type by the P+Q combined treatment.

Mitochondrial Open Reading Frame of the 12S rRNA Type-c: Potential Therapeutic Candidate in Retinal Diseases.

Mitochondrial open reading frame of the 12S rRNA type-c (MOTS-c) is the most unearthed peptide encoded by mitochondrial DNA (mtDNA). It is an important regulator of the nuclear genome during times of stress because it promotes an adaptive stress response to maintain cellular homeostasis. Identifying MOTS-c specific binding partners may aid in deciphering the complex web of mitochondrial and nuclear-encoded signals. Mitochondrial damage and dysfunction have been linked to aging and the accelerated cell death associated with many types of retinal degenerations. Furthermore, research on MOTS-c ability to revive oxidatively stressed RPE cells has revealed a significant protective role for the molecule. Evidence suggests that senescent cells play a role in the development of age-related retinal disorders. This review examines the links between MOTS-c, mitochondria, and age-related diseases of the retina. Moreover, the untapped potential of MOTS-c as a treatment for glaucoma, diabetic retinopathy, and age-related macular degeneration is reviewed.

MOTS-c, the Most Recent Mitochondrial Derived Peptide in Human Aging and Age-Related Diseases.

MOTS-c, a 16 amino acid mitochondrial derived peptide, is encoded from the 12S rRNA region of the mitochondrial genome. Under stress conditions, MOTS-c translocates to the nucleus where it regulates a wide range of genes in response to metabolic dysfunction. It is colocalized to mitochondria in various tissues and is found in plasma, but the levels decline with age. Since MOTS-c has important cellular functions as well as a possible hormonal role, it has been shown to have beneficial effects on age-related diseases including Diabetes, Cardiovascular diseases, Osteoporosis, postmenopausal obesity and Alzheimer. Aging is characterized by gradual loss of (mitochondrial) metabolic balance, decreased muscle homeostasis and eventual diminished physical capability, which potentially can be reversed with MOTS-c treatment. This review examines the latest findings on biological effects of MOTS-c as a nuclear regulatory peptide and focuses on the role of MOTS-c in aging and age-related disorders, including mechanisms of action and therapeutic potential.

The impact of amphotericin B-fortified preservation media on donor rim cultures and posttransplant infection.

Purpose: To determine the impact of amphotericin B supplementation to donor cornea preservation solutions on the rates of positive donor rim fungal cultures and postkeratoplasty fungal infections. Methods: This was a retrospective analysis of cases undergoing corneal transplantations at a single tertiary referral center from 2016 to 2021. Patients undergoing corneal transplantations with and without amphotericin B supplementation to the storage media were reviewed for donor rim culture results and postoperative infection. The primary outcome measures were positive donor rim fungal culture results and postkeratoplasty fungal infection. Results: A total of 1238 corneal transplants were analyzed. Of these, 849 were stored in preservation solution without amphotericin B, while 389 had amphotericin B included. There was a lower incidence of positive donor rim fungal cultures in cases with amphotericin B supplementation (1.8%) compared to the cases without amphotericin B (2.9%), although this difference was not statistically significant (P = 0.24). Of the 389 cases with amphotericin B supplementation, one (0.25%) went on to develop clinically significant infection, while three of 849 (0.35%) cases without amphotericin B developed infection. The sample size was too small to determine the effect of amphotericin B on the incidence of postkeratoplasty fungal infection. Conclusion: The addition of amphotericin B to donor cornea preservation solution resulted in a downward trend of positive donor rim fungal cultures and postkeratoplasty fungal infections, although these differences did not reach statistical significance. Further studies with larger sample sizes are necessary to appropriately determine the impact of amphotericin B supplementation in the storage solution on positive donor rims and postkeratoplasty fungal infections.

Impacts of Bacteriostatic and Bactericidal Antibiotics on the Mitochondria of the Age-Related Macular Degeneration Cybrid Cell Lines.

We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. Reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔψM), cellular metabolism and ratio of apoptotic cells were measured using H2DCFDA, JC1, MTT and flow cytometry assays, respectively. Expression of genes of antioxidant enzymes, and pro-inflammatory and pro-apoptotic pathways were evaluated by quantitative real-time PCR (qRT-PCR). Higher ROS levels were found in U haplogroup cybrids when treated with CPFX 60 µg/mL concentrations, lower ΔψM of all haplogroups by CPFX 120 µg/mL, diminished cellular metabolism in all cybrids with CPFX 120 µg/mL, and higher ratio of dead cells in K and J cybrids. CPFX 120 µg/mL induced overexpression of IL-33, CASP-3 and CASP-9 in all cybrids, upregulation of TGF-ß1 and SOD2 in U and J cybrids, respectively, along with decreased expression of IL-6 in J cybrids. TETRA 120 µg/mL induced decreased ROS levels in U and J cybrids, increased cellular metabolism of treated U cybrids, higher ratio of dead cells in K and J cybrids and declined ΔψM via all TETRA concentrations in all haplogroups. TETRA 120 µg/mL caused upregulation of IL-6 and CASP-3 genes in all cybrids, higher CASP-7 gene expression in K and U cybrids and downregulation of the SOD3 gene in K and U cybrids. Clinically relevant dosages of ciprofloxacin and tetracycline have potential adverse impacts on AMD cybrids possessing K, J and U mtDNA haplogroups in vitro.

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

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-1ß, TGF-α, TGF-ß1 and TGF-ß2) 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 This is the first study showing the negative impact of fluoroquinolones and tetracyclines on mitochondrial behavior of human retinal MIO-M1 cells.

Potential adverse effects of ciprofloxacin and tetracycline on ARPE-19 cell lines.

BACKGROUND: We aim to determine the possible adverse effects of ciprofloxacin (CPFX) and tetracycline (TETRA), as examples of bactericidal and bacteriostatic agents, respectively, on cultured human retinal pigment epithelial cells (ARPE-19). METHODS: Cells were treated with 30, 60 and 120 µg/mL of CPFX and TETRA. Cell metabolism was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. JC-1 dye (5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) assay was conducted to measure the mitochondrial membrane potential (MMP). The level of reactive oxygen species (ROS) was measured using the -2',7'-dichlorodihydrofluorescein diacetate assay (H2DCFDA). Quantitative real-time PCR was performed to analyse the gene expression levels associated with apoptosis (BAX, BCL2-L13, BCL2, Caspase 3, Caspase 7 and Caspase 9), inflammatory (interleukin-1ß (IL-1ß), IL-6, IL-33, transforming growth factor-α (TGF-α), TGF-ß1 and TGF-ß2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4), along with the mitochondrial DNA (mtDNA) copy numbers. RESULTS: Results illustrated that while all three concentrations of CPFX decreased cellular viability of ARPE-19 during all incubation periods, the 120 µg/mL TETRA resulted in increased cellular viability. At 48 and 72 hours, levels of MMP and ROS decreased significantly with each antibiotic. BAX, BCL2-L13, CASP-7, CASP-9, SOD2 and GPX3 genes overexpressed by either antibiotics. There was higher expression of IL-6 and IL-1B with TETRA treatment. The level of mtDNA decreased using both treatments. CONCLUSIONS: Clinically relevant concentrations of CPFX and TETRA have detrimental impacts on ARPE-19 cell lines in vitro, including upregulation of genes related to apoptosis, inflammation and antioxidant pathways. Additional studies are warranted to investigate if these harmful effects might be seen in retinal degeneration models in vivo.