Potential Role of NUR77 in the Aging Retinal Pigment Epithelium and Age-Related Macular Degeneration

Abstract

The underlying mechanisms associated with age-related changes in the morphology and function of retinal pigmented epithelial (RPE) cells are poorly understood. The aging RPE progresses through several structural changes including loss of melanin granules, accumulation of lipofuscin, and cytoskeletal changes, among others. Extracellular to it, there is also thickening of Bruch’s membrane and changes in the integrity of the choroid. Recent studies have revealed that aging also affects the metabolic ecosystem of the RPE. Aged mitochondria exhibit decreased rates of oxidative phosphorylation, increased reactive oxygen species generation and increased number of mitochondrial mutations relative to baseline. These changes are also found in age-related macular degeneration (AMD), a late onset vision impairing disease, in which the RPE is particularly vulnerable. The orphan nuclear receptor NR4A1/NUR77 is an early response gene and regulator of various cellular processes during development, aging, and disease. Previously we observed decreased levels of Nur77/NUR77 in both mouse and human RPE as a function of age. Current knowledge on the function of this receptor in the RPE is limited. Herein we discuss the putative roles of NUR77 in the RPE during aging and disease.

XX.1. Introduction

Age-related macular degeneration (AMD) is an important cause of vision impairment in the elderly and is characterized by outer retinal functional and morphological changes. Of the retinal cells impacted, the RPE is particularly vulnerable (Choudhary and Malek 2023). RPE is a monolayer of cuboidal epithelial cells, with highly specialized functions essential for retinal homeostasis, including internalization and processing of shed photoreceptor outer segments, removal of free radicals from the highly photo-oxidative retinal milieu, creating an immunologically privileged ocular environment in its role as the outer blood retinal barrier, and acting as a metabolic gatekeeper of gas / nutrient exchange across the outer retina (Lakkaraju et al. 2020). RPE structure and function are essential to vision such that alterations results in impaired function and ocular pathologies. Though RPE dysfunction is a distinctive feature of all clinical sub-types of AMD, the causative processes have yet to be discovered.

XX.2. Age-related changes in RPE cell function and metabolism

RPE cellular homeostasis is maintained through the exchange of nutrients and metabolites between the RPE / choroid complex and the overlying photoreceptor layer. The neural retina is fueled by oxygen, glucose, and other nutrients that diffuse from the choroid and are filtered through the RPE monolayer. The RPE and retina work collaboratively forming a symbiotic metabolic ecosystem. Consequently, when RPE cells die, photoreceptor cell death often follows. RPE cells are subject to high metabolic demands as they support photoreceptor outer segment synthesis, phagocytosis, retinoid recycling, maintenance of the membrane potential, and ion flux through Na⁺ K⁺-ATPase channels (Hurley 2021). Aged RPE are characterized by accumulation of lipofuscin, loss of melanin granules on the apical surface, moderately decreased phagocytosis and overall changes in cellular architecture and homeostatic pathways (Inana et al. 2018). Additionally, accumulation of secondary lysosomes and residual bodies containing lipofuscin have been observed during aging (Baynes 2001; Boulton and Dayhaw-Barker 2001; Bonilha 2008). Finally, RPE secretome studies have revealed age-related changes in RPE soluble proteins and proteins secreted in extracellular vesicles which function as mediators of cell-to-cell signaling and modulate cell activities (Meyer et al. 2019; Jin et al. 2022).

Another cell structure affected during aging is the mitochondria. Mitochondria are powerhouses of cells and a major source of reactive oxygen species that if left uncontrolled may contribute to mitochondrial DNA damage, cell death, and initiation and progression of age-related diseases including AMD (Ferrington et al. 2021). Functionally, mitochondria undergo dynamic changes including fission, fusion, biogenesis, and mitophagy in response to cellular stress (Blasiak et al. 2020; La Cunza et al. 2021; Tong et al. 2022). Age- and disease-related alterations in RPE cell metabolism, mitochondrial integrity and function have been noted and are currently an area of intense investigation. Identifying factors that may regulate cell metabolism and mitochondrial function may reveal potential therapies for RPE cellular rejuvenation in aging and disease.

XX.3. Nuclear receptors and ocular health

Nuclear receptors (NRs) are the most abundant transcription factors in the human genome responsible for a wide spectrum of functions during development, reproduction, aging, and disease. NR ligands include but are not limited to steroids, hormones, toxins, metabolites of fatty acids, cholesterol, and bile acids (Malek and Lad 2014). NRs have become a strategic therapeutic target for many diseases due to their broad functional spectrum and their affinity for small molecule ligands. For example, dexamethasone, a common anti-inflammatory medication, binds to the glucocorticoid receptor and exerts a higher biological response than cortisol. Ligands for various NRs such as the estrogen receptor and peroxisome proliferator-activated receptor have been synthetized for pharmacological treatments of cancer and diabetes (Mo and He 2014; Safe et al. 2014). NRs have also been investigated in ocular presentations including but not limited to keratitis, cataracts, glaucoma, uveitis, retinopathy, and ophthalmic tumors (Chen et al. 1999; Kobayashi et al. 1999; Yang et al. 2014; Zhang et al. 2015; Choudhary and Malek 2016). As observed in other tissues, the role of NRs in ocular diseases is diverse, complicated, and receptor specific. Their regulatory role and potential as a therapeutic target may be better understood with the use of appropriate in vitro and in vivo models along with activating receptor ligands.

XX.4. The orphan nuclear receptor NR4A family

The orphan nuclear receptor NR4A family includes three members: Nur77/NR4A1 (human homologue TR3, mouse homologue Nur77, and rat homologue NGFI-B), Nurr1/NR4A2, and Nor1/ NR4A3. These receptors share structural domain similarities with other NRs; however, their endogenous ligands are unknown (Safe et al. 2016). They also exhibit a high degree of homology to each other in genomic structure, and interactions with common cis-genomic elements. However, they exhibit unique activities that are cell and tissue specific. Although their endogenous ligands have not been identified, there is increasing evidence that structurally diverse synthetic molecules can directly interact with the ligand binding domain of NR4A1 and act as agonists and antagonists, and ligands for NR4A2 and NR4A3 (Safe et al. 2016). Functionally, NR4A receptors play an important role in maintaining cellular homeostasis, immune processes, and metabolism. They have also been linked to cancer, cardiovascular, neurologic, and inflammatory diseases. NR4A1/NUR77 is the most extensively studied member of the NR4A family and there is an overlapping and compensatory biological function between NR4A1 and the other two members because of their structural homology. Previously we reported on the role of NR4A2/Nurr1 in the RPE as a regulator of inflammation and epithelial to mesenchymal transition (Yao et al. 2022). Herein we discuss potential functions of NUR77 in the RPE as they may be related to AMD.

XX.5. Functions of NUR77

The orphan nuclear receptor NUR77 is an early response gene with pleotropic functions that are rapidly and transiently induced by a diverse range of signals, including growth factors, cytokines, glucose, fatty acids, lipopolysaccharides and many small molecular compounds (Zhang et al. 2018; Safe et al. 2021). Accordingly, NUR77 regulates various cellular processes including glucose and lipid metabolism, insulin synthesis, apoptosis, vascular stability, inflammation, immunity, and carcinogenesis (Xu et al. 2022). Differential activity of NUR77 has also been linked to several diseases including cardiovascular disease, neurodegenerative diseases, atherosclerosis, obesity, diabetes, arthritis, inflammatory bowel disease, multiple sclerosis, acute liver inflammation, neuroinflammation, tumor inflammation, respiratory diseases and oncogenesis (Koenis et al. 2018; Qin et al. 2019; Odagiu et al. 2020; Lith and de Vries 2021; Safe et al. 2021; Xu et al. 2022). Additionally, NUR77 is frequently overexpressed in a variety of cancer types, such as colon cancer and pancreatic cancer, and drives cancer development and progression.

NUR77 is expressed in several metabolically active tissues including skeletal muscle, liver, pancreas and adipose. It is also present in the RPE and choroid (Dwyer et al. 2011; Peavey et al. 2022). Nur77 null mice are viable and when fed a high fat diet present with decreased energy usage, insulin resistance, lowered blood glucose clearance, and increased lipid content in skeletal muscle, indicating its regulation of cell metabolism (Xu et al. 2022). Susceptibility to inflammation is also increased in Nur77 null mice indicating a role for immune regulation (Li et al. 2015; Crean and Murphy 2021). In the metabolically active pancreatic β cells, Nur77 was found to be protective, able to attenuate endoplasmic reticulum stress and oxidative stress induced apoptosis (Yu et al. 2015). NUR77 has also been shown to decrease intracellular steroid production by inhibiting the formation of reactive oxygen species in aged mice.

In our preliminary results we have discovered that NUR77 is present in freshly isolated retina, RPE and choroid tissues from human donor eyes. We have also found that the expression of this receptor in RPE cells decreases with age in both human RPE cells and in RPE / choroid tissues isolated from C57BL/6J mice, indicating an age-associated decrease in functionality of processes regulated by NUR77. The NUR77 molecular signature to date dictates a need to investigate its role in RPE biology further.

XX.6. Future experimental approach

Further investigation into the role of NUR77 is possible using in vitro and in vivo model systems as well as treatment with activating ligands and selective modulators (as they become available). These studies are integral to understanding the role of this receptor in the eye as a function of age and disease, and in the diverse signaling pathways that may also play a role in AMD development (Figure 1).

Fig XX.1:

Potential roles of Nur77 in RPE and age-related ocular diseases