PEDF Prevents Mitochondrial Function Decay and ER Stress Induced by Rotenone in Aging RPE Cells

Background: Neurodegenerative diseases, including age-related macular degeneration (AMD), may be linked to mitochondrial dysfunction and endoplasmic reticulum (ER) stress. We examined whether Pigment epithelium-derived factor (PEDF) could prevent changes in the structure and function of these organelles by accelerating by rotenone (ROT), a mitochondrial inhibitor, in human retinal pigment epithelium (RPE) cells of chronological age. Methods: RPE cells from 9–20, 50–55, 60–70, and $>$ 70-year-old donors were isolated, grown as primary cultures, harvested, and treated with ROT and PEDF for electron microscope (EM), western blot analysis, and polymerase chain reaction (PCR). Reactive oxygen species (ROS) and cytoplasmic calcium [Ca ${}^{2+}$ ] ${}_{\text{c}}$ and mitochondrial calcium [Ca ${}^{2+}$ ] ${}_{\text{m}}$ levels were measured by flow cytometry using 2 ${{}^{\prime}}$ ,7 ${{}^{\prime}}$ -dichlorodihydrofluorescin diacetate (H2-DCF-DA), fluo-3/AM, and Rhod-2/AM, and ATP levels were measured using a luciferin/luciferase-based assay. Mitochondrial membrane potential ( $\Delta{}$ $\Psi{}$ m) was detected using 5,5 ${{}^{\prime}}$ ,6,6 ${{}^{\prime}}$ -tetrachloro1,1 ${{}^{\prime}}$ ,3,3 ${{}^{\prime}}$ -tetraethylbenzimid azolocarbocyanine iodide (JC-1), and susceptibility of the cells to ROT toxicity and PEDF-protective effect was determined by propidium iodide (PI) staining and lactate dehydrogenase (LDH) assay. The expression of ER stress-related genes was detected using real-time (RT)-PCR. Results: We observed decay in the mitochondria of aged RPE cells, including matrix abnormalities, elongation, loss of cristae, and disruption of membrane integrity after ROT treatment. We also observed lower [Ca ${}^{2+}$ ] ${}_{\text{c}}$ , higher ROS and [Ca ${}^{2+}$ ] ${}_{\text{m}}$ levels, decreased $\Delta{}$ $\Psi{}$ m after ROT treatment, and greater susceptibility to ROT toxicity in aged RPE cells. PEDF can protect the cristae and integrity of the mitochondrial membrane, increase ATP levels and $\Delta{}$ $\Psi{}$ m, and lower ROS, [Ca ${}^{2+}$ ] ${}_{\text{c}}$ , and [Ca ${}^{2+}$ ] ${}_{\text{m}}$ in aged RPE cells induced by ROT. In addition, there was an increase in RDH expression in RPE cells with increasing age after PEDF treatment. Similarly, PEDF decreased the expression of ROT-induced ER stress-related genes. Conclusions: Our study provides evidence that PEDF can reduce bioenergetic deficiencies, mitochondrial decay, and ER stress in aging RPE, a condition that may trigger the onset of retinal diseases such as AMD.

Keywords

PEDF

RPE cells

rotenone

mitochondria

ROS

Ca²⁺

Funding

82070964/National Natural Science Foundation of China

22KY0114/The fourth batch of school-level key disciplines of the Xi’an Medical University and Youth Project of the Second Affiliated Hospital of Xi ‘an Medical University

Figures

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Fig. 1.

Pigment epithelium-derived factor (PEDF) protects aging retinal pigment epithelium (RPE) cells from rotenone (ROT) toxicity. (A) Phase-contrast micrographs of primary cultures of human RPE cells, showing that RPE cells obtained from 9–20 and 50–55-year-old donors were round and more regularly shaped compared to the elongated features of those obtained from the 60–70 and $>$ 70 years age groups. After treatment with 5 µM ROT for 1 h, the aged RPE cells had a generally unhealthy and degenerative appearance. Fewer cells were observed in the aging group after ROT treatment, partly due to cell detachment and, in part, cell death. RPE cells from 9–20-year-old donors maintained a relatively healthy appearance, even in the presence of ROT. Pretreatment with PEDF (100 ng/mL) for 48 h blocked the effects of ROT on aged RPE cells (60–70 and $>$ 70 years). Scale bar = 30 µm. (B) PEDF prevents ROT-induced RPE cell death. The histogram illustrates the relative percentage of cell death in cultures after ROT treatment and protection by PEDF. (Upper) Relative amount of PI fluorescence intensity in RPE cultures determined by flow cytometry. Treatment with 1.25 µM ROT for 24 h caused an increase in PI levels in aged RPE cultures (50–55, 60–70, and $>$ 70 years) but had minimal effect on the cultures of the 9–20 years age group (1 $\times{}$ 10 ${}^{6}$ cells were used). Pretreatment of RPE cells with 100 ng/mL PEDF for 48 h prevented the increase in PI levels when the cells were treated with ROT, as was evident in cultures from donors aged 50–55, 60–70, and $>$ 70 years. (Lower) Increase in LDH release after 1 h of ROT treatment. The fold-changes in LDH release were 1.54 ( $\pm{}$ 0.24), 1.81 ( $\pm{}$ 0.26), and 2.23 ( $\pm{}$ 0.24) for the 50–55, 60–70, and $>$ 70 years age groups, respectively, indicating that aged RPE cells were more susceptible to death caused by ROT-induced toxicity. Pretreatment of cells with PEDF (100 ng/mL) for 48 h significantly reduced LDH release into the cytoplasm after ROT treatment compared with untreated cells. The fold-changes in LDH release were 1.12 ( $\pm{}$ 0.26), 1.23 ( $\pm{}$ 0.22), and 1.30 ( $\pm{}$ 0.31), for the 50–55, 60–70, and $>$ 70 years age groups, respectively. Data are presented as fold-changes in PI intensity or LDH release from treated RPE cells compared to untreated control cells. The results are expressed as the mean $\pm{}$ S.E. of three independent experiments, each performed in triplicate. * Indicates a significant difference from the untreated control RPE cells set at p $<$ 0.05.

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