Age-related diseases, such as Parkinson’s disease, Alzheimer’s disease, cardiovascular diseases, cancers, and age-related macular disease, have become increasingly prominent as the population ages. Oxygen is essential for living organisms, but it may also cause disease when it is transformed into reactive oxygen species via biological processes in cells. Most of the production of ROS occurs in mitochondrial complexes I and III. The accumulation of ROS in cells causes oxidative stress, which plays a crucial role in human ageing and many diseases. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key antioxidant transcription factor that plays a central role in many diseases and ageing in general. It regulates many downstream antioxidative enzymes when cells are exposed to oxidative stress. A basic-region leucine zipper (bZIP) transcription factor, MAF, specifically the small MAF subfamily (sMAFs), forms heterodimers with Nrf2, which bind with Maf-recognition elements (MAREs) in response to oxidative stress. The role of this complex in the human retina remains unclear. This review summarises the current knowledge about Nrf2 and its downstream signalling, especially its cofactor—MAF, in ageing and diseases, with a focus on the retina. Since Nrf2 is the master regulator of redox homeostasis in cells, we hypothesise that targeting Nrf2 is a promising therapeutic approach for many age-related diseases.

MAFF is a small MAF family transcription factor involved in antioxidant signaling and cytoprotective gene regulation. MAFF is differentially expressed across retinal regions, with particularly low expression in macular Müller cells, which play a central role in retinal redox balance. Reduced MAFF expression may weaken macular resilience to oxidative stress (OS), increasing susceptibility to age-related macular degeneration (AMD) and macular telangiectasia type 2. However, the function of MAFF in Müller cells remains unclear. Human primary Müller cells (huPMCs) from non-diseased human donor eyes were used to investigate MAFF modulation under stress. Cells were transfected with MAFF plasmid DNA to evaluate intracellular reactive oxygen species (ROS) following H₂O₂-induced stress. For functional and proteomic analyses, MAFF was overexpressed using mRNA or knocked down using siRNA and validated by Western blot. huPMCs were exposed to light stress and cell viability was assessed using the AlamarBlue assay. Molecular changes were characterized by liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based quantitative proteomics. MAFF upregulation significantly reduced intracellular ROS following H₂O₂-induced stress. MAFF protein expression decreased following siRNA-mediated knockdown (KD) and increased following mRNA-mediated overexpression (OE). MAFF OE improved Müller cell viability under light stress, whereas MAFF KD reduced viability. Exploratory proteomic profiling quantified 5907 proteins and identified candidate molecular changes associated with MAFF expression. Pathway analyses suggested that reduced MAFF expression was associated with oxidative-stress response signatures, whereas elevated MAFF expression was associated with changes in mitochondrial and immune/inflammatory pathways. KEAP1, BMP2K, VPS28, and PDCD4 were negatively associated with MAFF expression, suggesting potential regulation of antioxidant defense, vesicle trafficking, and apoptosis-related processes. Elevated MAFF expression was associated with reduced ROS accumulation and improved cell viability, while exploratory proteomic analysis identified changes in mitochondrial, oxidative-stress, and immune/inflammatory pathways. These findings support further investigation of MAFF as a contributor to macular stress susceptibility and as a candidate for future therapeutic studies.

Background: Regional heterogeneity of the macula and peripheral retinas contributes to the differences in retinal physiology, metabolic demand and susceptibility to macular diseases such as age-related macular degeneration (AMD), diabetic macular oedema (DME) and Macular telangiectasia type 2 (MacTel). Extracellular vesicles (EVs) released from the retinal pigment epithelium (RPE)-choroid are increasingly recognised as mediators of extracellular communication and correlate with the molecular biological states of their tissue of origin. However, regional variation in EV composition in human RPE-Choroid tissues remains poorly characterised. Methods: Human macular and peripheral RPE-choroid explants from non-diseased donor eyes (n = 4) were cultured ex vivo using a transwell system. EV and EV-depleted conditioned media were collected by differential ultracentrifugation, respectively. EVs were characterised by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteomic profiling was performed using LC-MS/MS followed by multivariate, pathway, and differential expression analyses. Results: TEM and NTA confirmed the presence of vesicle-like particles in a 119–140 nm size range, indicating small EVs in both regions. Proteomic analysis demonstrated a region-associated separation between the macular and peripheral samples in both EV proteome and EV-depleted soluble secretome datasets. GO enrichment analysis revealed that macular EV proteomes were enriched for wound healing, cell–substrate adhesion, and focal adhesion-related terms with a high abundance of integrin and annexin family members, whereas peripheral EV proteomes were enriched for retinoid- and vitamin-binding terms. In the EV-depleted soluble secretome, macular-enriched proteins were associated with actin binding and extracellular matrix-related terms, while peripheral-enriched proteins were correlated with RNA localisation and nuclear compartment terms. Comparative analysis identified EV-specific, secretome-specific, and shared extracellular protein pools. Several EV-specific markers and membrane proteins such as annexins and integrins, showed a relatively high enrichment in macular EV proteomes, whereas CD63 is more abundant in peripheral EV proteomes. In contrast, EV-depleted soluble secretome contains cytokine- and ligand-associated proteins, including MIF, SPP1, and IL6, which may suggest that EVs and soluble secreted proteins represent partially distinct extracellular signalling compartments. Conclusions: Human macular and peripheral RPE-choroid explants released secretory proteins in a regional differentiated manner, supported by PLS-DA and GO enrichment analyses of both EV proteomes and EV-depleted soluble secretome. Further analysis of the EV proteomes may show that such differences were also able to be reflected in protein categories such as EV-specific markers, mitochondrial and membrane proteins. These findings provide a foundation for future investigations into the role of EV-mediated communication in macular diseases and may support the development of region-specific extracellular biomarkers and therapeutic targets.