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Page 59 Plössl et al. J Transl Genet Genom 2022;6:46-62 https://dx.doi.org/10.20517/jtgg.2021.39

of intracellular ROS more recently leading to a growing interest in the context of AMD pathogenesis [55-57] .
Interestingly, NRF2 signaling has been shown to also be involved in mitochondrial homeostasis and
[58]
metabolism (reviewed in ). Even though it is widely appreciated that SI is cytotoxic and causes RPE cell
death, the exact mechanism of cellular damage induced by SI remains elusive. A recent study performed in
ARPE-19 cells suggests that SI triggers cell death via ferroptosis rather than apoptosis or necrosis . In
[59]
contrast, another study also using ARPE-19 cells (48 h treatment with SI) showed an increase of NLRP3,
Caspase-1, and IL1β, which points more towards pyroptosis as the mechanism of cell death induced by SI
[60]
treatment . Given the plethora of oxidative stress response mechanisms and the complex interplay of
distinct processes suspected to play a role in AMD pathology, drawing finite conclusions from in vitro data
to the in vivo situation in AMD patients is a major challenge and needs careful consideration in future
research. Additionally, the variety of stressors and treatment durations used in different studies makes it
rather difficult to compare in vitro data from different research groups. Several of the studies [39,41-45] that used
iPSC-RPE disagree with our results, as those studies did indeed observe differences between AMD patient-
derived iPSC-RPE cells and control cells. It should be noted, however, that in the present study a major
selection criterion focused on the AMD-related genotypes rather than on phenotypes. Additionally, baseline
culture conditions of iPSC-RPE cells are frequently different between different laboratories, making it
inherently difficult to compare data from different research groups.

Based on the data presented, we suggest that an extreme GRS for AMD development (high or low) does not
correlate with impaired NRF2-mediated oxidative stress response, at least for the short-term treatment
conditions chosen, and that HR RPE cells are not more prone to oxidative damage than LR cells or vice
versa. The fact that the present study only used rather short stress induction times of 24 and 72 h as well as a
single chemical stressor only allows drawing conclusions for this particular in vitro setting. Extending
treatment periods towards a more chronic treatment regimen as performed in or using aged RPE cells
[49]
would reflect a more natural situation and needs to be considered for future studies. Nevertheless, as
oxidative stress is regarded as a major risk factor for AMD, a key response mechanism such as the NRF2
pathway may still represent a promising target for therapeutic approaches. Consequently, several NRF2
activators/stabilizers promoting antioxidant gene expression have been proposed as potential therapeutic
agents in AMD therapy [49,51,61-63] . Our data fail to reveal differences in NRF2-mediated oxidative stress
defense in HR vs. LR cell lines, suggesting that individuals independent of their genetic profile could equally
benefit from boosting the oxidative stress response machinery even before first signs of AMD emerge.

DECLARATIONS
Acknowledgments
We thank Dr. Ulrike Friedrich (Institute of Human Genetics, University of Regensburg, Germany) for help
with the FV3000 confocal microscope, Nico Hertel for technical help with iPSC cultures and RPE
differentiations, Ardita Ramadani for support with immunocytochemistry, Sandra Rast for help with
iPSC/RPE maintenance and Peter Schönberger for genotyping the AMD patients (all Institute of Human
Genetics, University of Regensburg, Germany).

Authors’ contributions
Made substantial contributions to conception, design and supervision of the study and performed data
analysis and interpretation; wrote the manuscript: Plössl K, Weber BHF
Contacted and examined AMD patients, obtained biological samples and helped with establishing iPSC
cultures: Brandl C
Performed data acquisition and evaluation; provided tables and figures: Webster E, Kiel C, Grassmann F