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Page 47 Plössl et al. J Transl Genet Genom 2022;6:46-62 https://dx.doi.org/10.20517/jtgg.2021.39

oxidative stress response mediated through the NRF2 pathway.

Conclusion: Delineating the NRF2-mediated oxidative stress response was sought in iPSC-RPE cell lines with
maximally divergent genetic AMD risk profiles. Under the specific stress conditions chosen, our data indicate that
genetic predisposition to AMD may not exert a major influence on the NRF2 signaling pathway.

Keywords: Age-related macular degeneration, induced pluripotent stem cells, retinal pigment epithelium, genetic
risk score, oxidative stress, NRF2 pathway

INTRODUCTION
Age-related macular degeneration (AMD) is a degenerative disease of the central retina and is the leading
[1]
cause of vision loss in developed countries , with prevalence rates in European individuals ≥ 70 years of age
as high as 13.2% for early AMD and 3.0% for the late forms . The molecular mechanisms underlying AMD
[2]
pathology are not fully understood, in part due to its nature as a complex disease entity where an individual
risk to develop AMD is determined by a unique combination of genetic as well as environmental risk
[3,4]
factors . Our understanding of AMD genetics has greatly benefitted from large genome wide association
studies, the latest of which identified 52 single nucleotide polymorphisms (SNPs) at 34 independent loci to
[5]
be significantly associated with the late-stage form of AMD . Environmental risk factors for AMD include
aging, cigarette smoking, sunlight exposure, hypertension, cardiovascular disease, and diet, although some
of these data are still controversial .
[6]

Almost all of the environmental risk factors for AMD considered so far have an underlying theme in
common that points to increased oxidative stress as an important trigger of disease pathology (reviewed
in ). A particularly high oxidative stress burden can be observed in the retinal pigment epithelium (RPE),
[7-9]
the retinal cell layer between the choriocapillaris and the layer of cone and rod photoreceptor cells, thought
to represent the primary site of AMD pathology . Specifically, oxidative stress in the RPE results from an
[10]
exceptionally high metabolic rate of this tissue, an oxygen tension between the retina and the oxygen-rich
choriocapillaris, photooxidative stress caused by intense light exposure, and a continuous phagocytosis of
shed photoreceptor outer segments (POS) [9,11-13] .

To cope with an unfavorable oxidative stress condition, RPE cells use an efficient machinery to neutralize
reactive oxygen species (ROS). One pathway playing a crucial role in the defense system is the route via
nuclear factor erythroid 2-related factor 2 (NRF2). In its function as a transcription factor, activated NRF2
binds to antioxidant response elements (ARE) to initiate transcription of target genes such as heme
oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1) [9,14-17] . In healthy RPE cells, the
NRF2 signaling pathway is responsible for neutralizing oxidative stress and preserving cellular redox
homeostasis [9,18] . For example, in ARPE-19, a widely used cell line with a number of features characteristic of
RPE cells, cigarette smoke extracts increase the NRF2 signaling response and lead to complement
activation . Further, Nrf2 deficiency increases the complement response, which suggests that Nrf2 is not
[16]
only involved in oxidative damage but, in absentia, also promotes a pro-inflammatory environment . Of
[16]
note, Nrf2-deficient mice develop age-related retinal pathology similar to an AMD-like degeneration of the
RPE, thickening of Bruch’s membrane, development of drusen, lipofuscin accumulation, and inflammatory
protein deposition in the sub-RPE area .
[19]

To gain a better understanding of the molecular pathology of AMD and the contribution of oxidative stress
responses to disease-associated changes, we aimed to generate a cellular model which allows us to combine
genetic and environmental risk factors in an in vitro cell culture system. To reflect the individual genetic risk