Abstract
PurposePhotoreceptor degeneration occurs in various retinal diseases including age-related macular degeneration (AMD), Retinitis pigmentosa (RP), and diabetic retinopathy (DR). However, molecular mechanisms are not fully understood yet. The retinal pigment epithelium (RPE) forms the outer blood retinal barrier (oBRB) and supplies glucose, oxygen and nutrients from the fenestrated choriocapillaris to photoreceptors for visual function. Therefore, RPE dysfunction leads to photoreceptor injury/death and progression of blinding eye diseases. This study aims to understand the role of the thioredoxin (Trx) and its reductase (TrxR) redox signaling in human RPE dysfunction and cell death mechanism(s) in an in vitro system.MethodsA human RPE cell line (APRE-19) was cultured in DMEM/F12 medium and treated with auranofin (AF – 4 μM, an inhibitor of TrxR) for 4 and 24 h. Mitochondrial and lysosomal function, cellular oxidative stress and NLRP3 inflammasome activity were measured using cell assays, Western blotting, and confocal microscopy. Antioxidants and anti-inflammatory compounds were tested for blocking AF effects on RPE damage. Cell death mechanisms (LDH release to culture media) were determined using necroptosis, ferroptosis and pyroptosis inhibitors. P < 0.05 was considered significant in statistical analysis.ResultsAuranofin causes mitochondrial dysfunction (Δψm↓ and ATP↓), oxidative stress (H2O2↑) and mitophagic flux to lysosomes. Furthermore, the lysosomal enzyme (cathepsin L) activity is reduced while that of pro-inflammatory caspase-1 (NLRP3 inflammasome) is enhanced in ARPE-19. These effects of AF on ARPE-19 are inhibited by antioxidant N-acetylcysteine (5 mM, NAC) and significantly by a combination of SS31 (mitochondrial antioxidant) and anti-inflammatory drugs (amlexanox and tranilast). AF also causes cell death as measured by cytosolic LDH release/leakage, which is not inhibited by either ferrostatin-1 or necrostatin-1 (ferroptosis and necroptosis inhibitors, respectively). Conversely, AF-induced LDH release is significantly reduced by MCC950 and Ac-YVAD-cmk (NLRP3 and Caspase-1 inhibitors, respectively), suggesting a pro-inflammatory cell death by pyroptosis.ConclusionThe Trx/TrxR redox system is critical for RPE function and viability. We previously showed that thioredoxin-interacting protein (TXNIP) is strongly induced in DR inhibiting the Trx/TrxR system and RPE dysfunction. Therefore, our results suggest that the TXNIP-Trx-TrxR redox pathway may participate in RPE dysfunction in DR and other retinal neurodegenerative diseases.
Highlights
Retina is a window to the brain (Chiquita et al, 2019)
These results suggest that AF induces mitochondrial dysfunction and cellular oxidative stress in ARPE-19
We observed that cathepsin L activity is significantly reduced by AF (Figure 1D) and increases the activity of pro-inflammatory caspase-1 (Figure 1E) suggesting inflammasome activation by the AF treatment
Summary
Retina is a window to the brain (Chiquita et al, 2019). Being a part of the central nervous system, the retina consumes large amounts of glucose and oxygen for its bioenergetics (ATP production), light perception, and visual processing (Country, 2017). Breakdown of the outer BRB and RPE dysfunction is associated with age-related macular degeneration (AMD) (Handa et al, 2019; Jun et al, 2019) while gene mutation in RPE and photoreceptor cause photoreceptor degeneration and blindness including retinitis pigmentosa (RP) (Dias et al, 2018; Shu and Dunaief, 2018). In these various retinal diseases, RPE dysfunction, photoreceptor injury/death and retinal neurodegeneration leads to blindness. In addition to nutrient exchange, RPE involves in recycling of the visual pigment (retinoid) to photoreceptors, daily phagocytosis of the photoreceptor outer segment, and synthesis of melanosome (melanin) to absorb excess light in the retina (Biesemeier et al, 2015; Spencer et al, 2019)
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