The AMD-associated genetic polymorphism CFH Y402H confers vulnerability to Hydroquinone-induced stress in iPSC-RPE cells

Abstract

Introduction: Age-related macular degeneration (AMD), a degenerative disease of the macula, is caused by an interplay of diverse risk factors (genetic predisposition, age and lifestyle habits). One of the main genetic risks includes the Y402H polymorphism in complement Factor H (FH), an inhibitor of complement system activation. There has been, and continues to be, much discussion around the functional consequences of this Y402H polymorphism, whether the soluble FH protein confers its risk association, or if the cells expressing the protein themselves are affected by the genetic alteration. In our study, we examined the cell characteristics of the retinal pigment epithelium (RPE) cells, which play a major role in retinal homeostasis and stability and which are synonymously linked to AMD.

Methods: Here, we employ RPE cells derived from induced pluripotent stem cells (iPSC) generated from donors, carrying either homozygous 402Y (low risk) or 402H (high risk) variants of the CFH gene. RPE cells were treated with Hydroquinone (HQ), a component of cigarette smoke, to induce oxidative damage.

Results: Intriguingly, RPE cells carrying high genetic risk proved more vulnerable to oxidative insult when exposed to HQ, as demonstrated by increased cytotoxicity and caspase activation, compared to the low-risk RPE cells. The exposure of RPE cells to RPE conditioned medium, normal human serum (NHS) and inactivated NHS (iNHS) had minimal impact on cell cytotoxicity and caspase activation, nor did the presence of purified soluble FH rescue the observed effects. Considering the known connection of oxidative stress to proteotoxic stress and degrading processes, we investigated the unfolded protein response (UPR) and autophagy. When exposed to HQ, RPE cells showed an increase in autophagy markers; however, iPSC-RPE cells carrying high genetic risk showed an overall reduced autophagic flux.

Discussion: Our findings suggest that the degree of cellular susceptibility to oxidative stress is not conferred by soluble FH protein and other complement sources, but intercellularly because of the corresponding genetic risk predisposition. Our data support the hypothesis that RPE cells carrying high genetic risk are less resilient to oxidative stress.

Keywords: age-related macular degeneration (AMD); autophagy; complement factor H (CFH); oxidative stress; retinal pigment epithelium (RPE) cells.

Figure 1

Generation of CFH low risk (LR) and high risk (HR) iPSC-RPE. (A–D) Representative light microscopy images of iPSC-RPE cells derived from LR donors iPSC-RPE2 (A), iPSC-PRE3 (B) and HR donors iPSC-RPE5 (C), iPSC-PRE8 (D). Images show proper RPE cuboidal shape and visible pigmentation. Scale bar = 100 µM. (E–H) Representative immunostaining images of RPE marker ZO-1 in LR donors iPSC-RPE1 (E), iPSC-PRE3 (F), and HR donors iPSC-RPE6 (G), iPSC-PRE7 (H). Scale bar = 50 µM. (I, J) Transmission electron micrographs semithin sections of LR donors iPSC-RPE3 (I) and HR donors iPSC-RPE8 (J). Images show an RPE monolayer and microvilli typical of RPE cells. Scale bar = 20 µM. (K) Pigment epithelium‐derived factor (PEDF) ELISA of apical and basal supernatant. Data collected from iPSC-RPE3, 5, 6, 7, 8. (L) Trans‐epithelial resistance (TER) measured in iPSC-RPE3, 5, 8. (M) Representative WB images of FH levels in iPSC-RPE1 and iPSC-RPE5 cellular lysates. n=3. Tubulin was used as housekeeping control.

Figure 2

Hydroquinone (HQ) induces cellular damage and apoptosis in CFH high risk (HR) iPSC-RPE. (A) Schematic representation of the experimental set up. (B, C) Membrane damage assessed by cytotoxicity assay GF-AFC (B) and Caspase-3 activity (C) in LR and HR iPSC-RPE. Data points were collected from LR iPSC-RPE1, 2, 3 (n=3 biological replicates) and from HR iPSC-RPE4, 5, 6, 7, 8 (n=5 biological replicates). HQ-treated relative values are normalized to the respective untreated controls (dotted line) in each individual experiment for each cell line. HQ effects in each group were assessed with paired Student’s t-test (#) compared to controls (dotted line). Differences between LR and HR groups were determined with unpaired Student’s t-test (*). (D, E) Representative WB images (D) of Bax and Bcl-2 levels LR and HR iPSC-RPE cells treated with HQ. Data points were collected from LR iPSC-RPE1, 2, 3 (n=3 biological replicates) and from HR iPSC-RPE4, 5, 6, 7, 8 (n=5 biological replicates). Tubulin was used as housekeeping control. Quantification of Bax/Bcl-2 ratio is shown in (E) Differences between LR and HR groups were assessed with one-way ANOVA (*). Data are shown as mean ± SEM. ** p < 0.01. ^## p < 0.01 ^#### p < 0.0001.

Figure 3

CFH high risk (HR) iPSC-RPE cells show impaired mitochondria homeostasis. (A) ATP levels assessed by cell titer assay in LR and HR iPSC-RPE. HQ-treated relative values are normalized to the respective untreated controls (dotted line) in each individual experiment for each cell line. HQ effects in each group were determined with paired Student’s t-test (#) compared to controls (dotted line). Differences between LR and HR groups were determined with unpaired Student T-test (*). (B) JC-1 fluorescence measurements were recorded, and ratio was determined. HQ effects in each group were assessed with paired Student’s t-test (#) compared to untreated controls. (C–H) Representative EM images of LR iPSC-RPE3 and HR iPSC-RPE8 showing mitochondria abnormalities. (C, D) show the mitochondria localization (highlighted in green) in the apical compartment in HR (D) compared to basal localization (C). scale bar = 1µm. (E–H) show mitochondria morphology. White arrowheads in (E, F) mark healthy mitochondria in LR iPSC-RPE cells. In (G, H) white arrowheads mark polymorphic mitochondria, black arrowheads mark swollen mitochondria, asterisks mark bent mitochondria. Scale bar = 500nm. (I, J) Gene expression levels of PINK (I), PARKIN (K) and PPARG1A (J) analyzed via RT-qPCR in LR and HR iPSC-RPE treated with HQ. Significance was determined with one-way ANOVA (*). Data are shown as mean ± SEM. Data points were collected from LR iPSC-RPE1, 2, 3 (n=3 biological replicates) and from HR iPSC-RPE4, 5, 6, 7, 8 (n=5 biological replicates). ^## p < 0.01 ^#### p < 0.0001.

Figure 4

Addition of extracellular FH and complement sources does not affect the vulnerability to HQ-mediated stress in HR iPCS-RPE. (A) Schematic representation of the experimental set up of FH supplementation. (B, C) Membrane damage assessed by cytotoxicity assay GF-AFC (B) and Caspase-3 activity (C) in HQ-treated HR iPSC-RPE with supplementation of purified FH. Values are normalized to the respective controls (dotted line) in each individual experiment for each cell line. Data are shown as mean ± SEM. (D) Schematic representation of the LR and HR iPSC-RPE conditioned medium supplementation experimental set up. (E, F) Membrane damage assessed by cytotoxicity assay GF-AFC (B) and caspase-3 activity (C) in HQ-treated HR iPSC-RPE with supplementation of LR and HR conditioned medium. Values are normalized to the respective controls (dotted line) in each individual experiment for each cell line. Significance was derived with 2-way ANOVA. (H) Schematic representation of the experimental set up of HQ-treated HR iPSC-RPE supplemented with NHS and iNHS. (I, J) Membrane damage assessed by cytotoxicity assay GF-AFC (B) and caspase-3 activity (C) in HQ-treated HR iPSC-RPE with supplementation of LR and HR conditioned medium. Values are normalized to the respective controls (dotted line) in each individual experiment for each cell line. Data points were collected from HR iPSC-RPE5, 6, 7, 8 (n=4 biological replicates). Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 5

HR iPSC-RPE shows impaired autophagic flux. (A-C) Representative WB images (A) of LC3 and SQSTM1 in LR and HR iPSC-RPE cells treated with HQ. Tubulin was used as housekeeping. Quantification is shown for LC3 (B) and SQSTM1 (C). Data points were collected from LR iPSC-RPE1, 2, 3 (n=3 biological replicates) and from HR iPSC-RPE4, 5, 6, 7, 8 (n=5 biological replicates). (D, E) Representative WB images (D) of LC3 LR and HR iPSC-RPE cells treated with Bafilomycin. Tubulin was used as housekeeping. Quantification are shown for LC3 (E). Data points were collected from LR iPSC-RPE1, 3 (n=2 biological replicates) and from HR iPSC-RPE6, 8 (n=2 biological replicates). Differences between groups were determined with one-way ANOVA (*). Data are shown as mean ± SEM. **** p < 0.0001.